A mixed-strain fermented Poria cocos fungus bag and its preparation method and application

Abstract

The present invention belongs to the technical field of the cultivation of medicinal and edible fungi, and specifically relates to a fermented Poria cocos strain package with a mixed strain and its preparation method and application. The fermented Poria cocos strain package with a mixed strain provided by the present invention comprises the following components and their mass percentages: 70-96% of a mixed strain fermentation composition, 2-6% of sucrose, and 0.5-2.0% of gypsum powder; the mixed strain fermentation composition is a combination of a mixed strain fermentation of pine needles and pine branches, a mixed strain fermentation of wheat bran, and a mixed strain fermentation of pine sawdust. The fermented Poria cocos strain package with a mixed strain provided by the present invention can effectively improve the growth rate and quality of Poria cocos mycelium in the fermented strain package with a mixed strain by using different strains in combination and fermenting different raw materials, and improve the yield and quality of Poria cocos in the later underground cultivation.

Description

Technical Field

[0001] The present invention belongs to the technical field of the cultivation of medicinal and edible fungi, and particularly relates to a mixed-strain fermented Poria cocos mushroom bag and its preparation method and application. Background Art

[0002] Poria cocos (Schw.) Wolf of the family Polyporaceae is a commonly used medicine for promoting diuresis and excreting dampness. Poria cocos is sweet, light, and has a neutral nature, and belongs to the lung, spleen, and kidney meridians. It has the effects of promoting diuresis and excreting dampness, strengthening the spleen and stomach, and nourishing the heart and calming the mind. Clinically, it is used for oliguria, edema, cough due to fluid retention, poor appetite due to spleen deficiency, diarrhea, palpitation, and insomnia. Modern pharmacological studies have shown that Poria cocos has pharmacological effects such as protecting the liver, sedation and hypnosis, and enhancing immunity. Poria cocos mainly contains triterpenoids and polysaccharide compounds. Yu Juntong et al. have shown that the intestinal flora metabolites of fermented Poria cocos polysaccharides can alleviate the symptoms of chronic non-bacterial prostatitis in rats. Poria cocos extract can improve functional dyspepsia by regulating brain-gut peptides, immune function, and repairing gastrointestinal mucosa, and can also play a beneficial role in immune regulation activities, and enhance non-specific immunity by promoting the secretion of interferon-γ (IFN-γ). Poria cocos is increasingly widely used in the modern medical field. There is a saying that "nine prescriptions contain Poria cocos" in traditional Chinese medicine prescriptions, which makes the market demand for Poria cocos expand day by day. The traditional cultivation methods of Poria cocos are pine log cultivation and pine stump cultivation, which require cutting down a large number of pine trees. This cultivation method consumes a large amount of forestry resources and is not conducive to ecological balance, making the contradiction between fungi and forests increasingly acute. At the same time, the growth process of Poria cocos is easily interfered by external factors such as soil quality, climate, and pests and diseases, and has disadvantages such as a long growth cycle, low biological effect, excessive pesticide residues, and poor product quality stability. After the country implemented a strict pine tree cutting policy, the Poria cocos cultivation industry is facing a severe test. Therefore, finding a more environmentally friendly Poria cocos cultivation method is the key to solving the current problem, and the key to Poria cocos cultivation lies in the quality of the Poria cocos mushroom bag. At present, the mushroom bags of Poria cocos on the market mainly use unfermented pine sawdust supplemented with wheat bran, rice bran, sucrose, and gypsum as the culture medium. Pine wood is obtained by cutting down pine trees. The present invention uses different mixed bacteria to ferment pine needles and pine branches as the culture medium for the Poria cocos mushroom bag, and pine needles and pine branches do not require cutting down pine trees, so as to alleviate the increasingly serious contradiction between fungi and forests.

[0003] At present, the cultivation of commercial Poria cocos strains generally adopts asexual reproduction, including four methods: meat inoculation method, wood inoculation method, pulp inoculation method, and mushroom inoculation method, and the mushroom inoculation method is widely used. Microbial fermentation is a process of providing a suitable living environment for microorganisms, changing the properties of materials through microorganisms, and finally obtaining the target product. The key to Poria cocos cultivation technology lies in the quality of the Poria cocos mushroom bag and whether the Poria cocos mushroom bag is suitable for the growth of Poria cocos mycelium, which are the key factors affecting the yield and quality of Poria cocos, and provide conditions for the subsequent underground cultivation of Poria cocos.

[0004] At present, there are few reports on preparing Poria cocos fungus bags through microbial fermentation to prepare high-quality and high-yield Poria cocos. Therefore, developing a more environmentally friendly and efficient cultivation method for Poria cocos is an urgent technical problem to be solved in the field of Poria cocos strain cultivation. Summary of the Invention

[0005] To solve the problems existing in the prior art, the purpose of the present invention is to provide a mixed-strain fermented Poria cocos fungus bag and its preparation method and application. The mixed-strain fermented Poria cocos fungus bag provided by the present invention, by using Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium lactis, etc. in combination according to a ratio, ferments different substrates. As a Poria cocos fungus bag, it can effectively improve the growth rate of Poria cocos mycelium after mixed-strain fermentation, as well as the yield and quality of Poria cocos in later underground cultivation, laying a foundation for the high-yield and high-quality cultivation technology of Poria cocos.

[0006] The technical solution of the present invention is as follows:

[0007] A mixed-strain fermented Poria cocos fungus bag, including the following components and their mass percentages:

[0008] Mixed-strain fermentation composition 70-96%, sucrose 2-6%, gypsum powder 0.5-2.0%;

[0009] The mixed-strain fermentation composition is a combination of mixed-strain fermented pine needles and pine branches, mixed-strain fermented wheat bran, and mixed-strain fermented pine sawdust;

[0010] The mixed strains are a combination of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis.

[0011] Furthermore, the mixed-strain fermented Poria cocos fungus bag consists of the following components and their mass percentages:

[0012] Mixed-strain fermentation composition 95%, sucrose 4%, gypsum powder 1%;

[0013] Further, the preparation methods of the mixed-strain fermented pine needles and pine branches, the mixed-strain fermented wheat bran, and the mixed-strain fermented pine sawdust include the following steps:

[0014] S1. Mix the bacterial suspensions of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis evenly according to a volume ratio of 1:1:1:1:1 to obtain a mixed-strain bacterial suspension, inoculate it into a liquid medium, and culture for 24 hours to obtain a mixed-strain seed liquid;

[0015] The liquid medium is composed of MRS medium and TPY medium mixed according to a volume ratio of 4:1.

[0016] The MRS medium consists of the following components: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract powder, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 1.0 g of Tween 80, 2.0 g of ammonium citrate, and 1000 mL of distilled water.

[0017] The TPY medium consists of the following components: 10.0 g of hydrolyzed casein, 5.0 g of peptone from plants, 2.0 g of yeast powder, 5.0 g of glucose, 0.5 g of L-cysteine, 2.0 g of dipotassium hydrogen phosphate, 0.5 g of magnesium chloride, 0.25 g of zinc sulfate, 0.15 g of calcium chloride, 0.0001 g of ferric chloride, 1.0 g of Tween 80, and 1000 mL of distilled water.

[0018] S2. Pour the mixed strain seed liquid obtained in step S1 into a fermenter and mix it evenly with the pine needle and pine branch mixture, wheat bran, and pine sawdust respectively. Place it indoors and ferment for 70 days. After 70 days, take it out for autoclaving to terminate the fermentation, and thus obtain the pine needle and pine branch fermented by the mixed strain, the wheat bran fermented by the mixed strain, and the pine sawdust fermented by the mixed strain.

[0019] Further, the pine needle and pine branch mixture is composed of pine needles and pine branches in a weight ratio of 2:3.

[0020] Further, the inoculation amount of the mixed strain suspension in step S1 into the liquid medium is 10%, and the addition amount of the mixed strain seed liquid in step S2 is calculated according to 2.0×10 10 CFU per 1000 g of fermentation raw material, and the fermentation raw materials are the pine needle and pine branch mixture, wheat bran, or pine sawdust.

[0021] Further, before preparing the mixed strain seed liquid, it may further include the activation of the mixed strain and the preparation of the mixed strain suspension.

[0022] The activation process of the mixed strain is as follows:

[0023] Dissolve the freeze-dried powders of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis in sterile water respectively, blow and mix evenly, and perform gradient dilution. Then inoculate them into a solid medium and culture them in a constant temperature incubator at 37°C. Bifidobacterium lactis is cultured for 72 h, and the other bacteria are cultured for 24 h. Then use an inoculation loop to inoculate the evenly distributed single colonies onto the solid medium and culture them in a constant temperature incubator at 37°C. Put the cultured solid medium into the refrigerator for cold storage and reserve for use.

[0024] The preparation process of the bacterial suspension is as follows: Take out the above-mentioned inoculated solid medium from the refrigerator, and use an inoculation loop to inoculate the colonies into the liquid medium respectively, and place them in a constant temperature water bath oscillator at 37°C for 24 hours of culture; after 24 hours, shake well, centrifuge at 2000 r / min for 5 minutes, discard the supernatant, take the bacterial precipitate at the bottom, and add sterile water to the bacterial precipitate according to the volume ratio of the liquid medium to sterile water of 4:1 to prepare a bacterial suspension, and measure the concentration of the bacterial suspension by the plate counting method.

[0025] The concentrations of the Lactobacillus casei bacterial suspension, Lactobacillus rhamnosus bacterial suspension, Lactobacillus acidophilus bacterial suspension, Lactobacillus plantarum bacterial suspension, and Bifidobacterium lactis bacterial suspension are all 7.5×10 9 CFU / mL.

[0026] Furthermore, in the preparation process of the strain activation and the mixed-strain bacterial suspension, the solid medium and liquid medium used for Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Lactobacillus casei are MRS medium; the solid medium and liquid medium used for Bifidobacterium bifidum are TPY medium.

[0027] Furthermore, the MRS liquid medium is composed of the following components: peptone 10.0 g, beef extract 10.0 g, yeast extract powder 5.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, Tween 80 1.0 g, ammonium citrate 2.0 g, distilled water 1000 mL.

[0028] The TPY liquid medium is composed of the following components: hydrolyzed casein 10.0 g, peptone 5.0 g, yeast powder 2.0 g, glucose 5.0 g, L-cysteine 0.5 g, dipotassium hydrogen phosphate 2.0 g, magnesium chloride 0.5 g, zinc sulfate 0.25 g, calcium chloride 0.15 g, ferric chloride 0.0001 g, Tween 80 1.0 g, distilled water 1000 mL.

[0029] The formulations of the MRS solid medium and the TPY solid medium are respectively adding 20 g of agar powder to the MRS liquid medium and the TPY liquid medium.

[0030] The present invention also provides a preparation method of the mixed-strain fermented Poria cocos fungus bag, including the following steps:

[0031] Dissolve sucrose in water, put the mixed-strain fermented pine sawdust into it and boil, fish it out after fully absorbing the sugar solution; then mix the mixed-strain fermented pine needles and twigs, the mixed-strain fermented wheat bran, the gypsum powder with the mixed-strain fermented pine sawdust after absorbing the sugar solution, sterilize by high-pressure steam, and cool to obtain the mixed-strain fermented Poria cocos fungus bag;

[0032] The parameter settings for high-pressure steam sterilization are as follows: pressure: 0.1 MPa, temperature: 121 °C, time: 120 min.

[0033] Further, in the method for preparing the mixed-strain fermented Poria cocos fungus bag, the water addition amount is 1 to 1.5 times that of sucrose. After adding the mixed strains to ferment the pine sawdust, the boiling time is 30 min. The water content of the finally obtained mixed-strain fermented Poria cocos fungus bag after thorough mixing is 63%.

[0034] Furthermore, the mixed-strain fermentation composition is composed of mixed-strain fermented pine needles and twigs, mixed-strain fermented pine sawdust, and mixed-strain fermented wheat bran in a weight ratio of 11:4:4.

[0035] The Poria cocos fungus bag fermented by the mixed strains prepared in the present invention uses five specific strains such as Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis for mixing, and respectively ferments substrates such as pine needle and twig mixtures, pine sawdust, and wheat bran. By fermenting the substrate with specific mixed microorganisms and using the principle of microbial phagocytosis to break the cell walls of plant cells, the components and nutrients inside the cells are released more thoroughly, providing a carbon source, nitrogen source, and various mineral elements for the growth of Poria cocos mycelium. After fermenting various substrates and mixing them for use, the finally obtained Poria cocos fungus bag can provide a better growth environment for Poria cocos mycelium. At the same time, after the substrate is fermented, the macromolecular substances therein can be effectively transformed into small-molecular substances, providing richer conditions for the production of secondary metabolites (i.e., medicinal components) of Poria cocos, thereby promoting the production of polysaccharide components and triterpenoid components during the growth of Poria cocos mycelium and improving the yield and quality of Poria cocos cultivation.

[0036] The present invention also provides an application of the above-mentioned mixed-strain fermented Poria cocos fungus bag in cultivating Poria cocos strains.

[0037] In addition, the present invention also provides a method for cultivating Poria cocos secondary strains, including the following steps:

[0038] Inoculate the Poria cocos primary strain into the above-prepared mixed-strain fermented Poria cocos fungus bag and place it in an incubator at 25 °C for 20 to 30 days.

[0039] Further, the method for cultivating the Poria cocos primary strain includes the following steps: Inoculate the Poria cocos strain into a potato medium. The inoculation amount is a small piece of Poria cocos mother strain with a length × width of 2 cm × 1 cm, and make the mycelium surface closely adhere to the potato medium. Place it in an incubator at 25 °C for 5 to 7 days. When the mycelium covers the medium, the Poria cocos primary strain is obtained.

[0040] Further, the potato culture medium is composed of the following components: 1 L of 20% potato juice, 20 g of glucose, 3.0 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 8 mg of vitamin B 1 8, 20 g of agar, and pH 6 - 7.

[0041] Compared with the prior art, the mixed - strain fermented Poria cocos mushroom bag provided by the present invention has the following advantages:

[0042] (1) Using the mixed - strain fermented Poria cocos mushroom bag prepared by the preparation method of the present invention as the culture medium for Poria cocos mushroom seeds, mainly through the low - cost fermentation treatment of pine needles, pine branches, pine sawdust, and wheat bran by microorganisms, it can not only break the cell wall but also produce fiberase, protease, amylase, pectinase, etc. that can rupture cells, thereby reducing the resistance of the cell interstitium and cell wall, increasing the cell gap, and promoting the release of a large amount of active substances. The fermented substrate is more nutritious, more conducive to the growth of Poria cocos mycelium, and can better improve the utilization rate of the mushroom bag. It can be seen from the experimental results that when using the Poria cocos mushroom bag prepared by the present invention to culture the secondary Poria cocos mushroom seeds, the growth of the Poria cocos mushroom seeds in it is significantly better than that in the naturally fermented and unfermented mushroom bags.

[0043] (2) In the prior art, the main component of the Poria cocos mushroom bag is pine sawdust. Using the fermented pine needles, pine branches, pine sawdust, and wheat bran provided by the present invention in a specific proportion as the main components of the secondary Poria cocos mushroom bag can effectively reduce the amount of pine sawdust in the existing secondary Poria cocos mushroom bag, avoid a large amount of pine tree felling, and be more environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Surface properties of different mixed - strain bacterial suspensions and naturally fermented pine needle - pine branch mixtures after 70 days.

[0045] Figure 2 Surface properties of different mixed - strain bacterial suspensions and naturally fermented pine bark after 70 days.

[0046] Figure 3 Surface properties of different mixed - strain bacterial suspensions and naturally fermented pine sawdust after 70 days.

[0047] Figure 4 Surface properties of different mixed - strain bacterial suspensions and naturally fermented rice straw after 70 days.

[0048] Figure 5 Surface properties of different mixed - strain bacterial suspensions and naturally fermented wheat bran after 70 days.

[0049] Figure 6 Growth curves of primary Poria cocos strains CGMCC 5.0078 and GDMCC 5.219.

[0050] Figure 7 It is the microscopic morphology (10×100) of the first-grade Wolfiporia cocos mycelium CGMCC 5.0078.

[0051] Figure 8 It is the microscopic morphology (10×100) of the first-grade Wolfiporia cocos mycelium GDMCC 5.219.

[0052] Figure 9 It is the growth situation of the second-grade Wolfiporia cocos strain CGMCC 5.0078 in the mushroom bags prepared in different examples and comparative examples.

[0053] Figure 10 The growth situation of the second-grade Wolfiporia cocos strain GDMCC 5.219 in the mushroom bags prepared in different examples and comparative examples.

[0054] Figure 11 The growth situation of the second-grade Wolfiporia cocos strain CGMCC 5.0078 in the mushroom bag prepared in Example 3 at different time periods.

[0055] Figure 12 It is the growth situation of the second-grade Wolfiporia cocos mycelium CGMCC 5.0078 in the mushroom bags prepared in different examples and comparative examples.

[0056] Figure 13 It is the microscopic morphology (10×100) of the second-grade Wolfiporia cocos mycelium CGMCC 5.0078 in the Wolfiporia cocos mushroom bag of Example 3.

[0057] Figure 14 It is the microscopic morphology (10×100) of the second-grade Wolfiporia cocos mycelium CGMCC 5.0078 in the Wolfiporia cocos mushroom bag of Comparative Example 13.

[0058] Figure 15 It is the microscopic morphology (10×100) of the second-grade Wolfiporia cocos mycelium CGMCC 5.0078 in the Wolfiporia cocos mushroom bag of Comparative Example 14.

[0059] Figure 16 It is the microscopic morphology (10×100) of the second-grade Wolfiporia cocos mycelium CGMCC 5.0078 in the Wolfiporia cocos mushroom bag of Comparative Example 15.

[0060] Figure 17 It is the microscopic morphology (10×100) of the second-grade Wolfiporia cocos mycelium CGMCC 5.0078 in the Wolfiporia cocos mushroom bag of Comparative Example 16.

[0061] Figure 18 It is the microscopic morphology (10×100) of the second-grade Wolfiporia cocos mycelium CGMCC 5.0078 in the Wolfiporia cocos mushroom bag of Comparative Example 17. Detailed implementation manners

[0062] The present invention will be further described below through specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention. As long as they do not depart from the basic idea of the present invention, they are within the protection scope of the present invention.

[0063] In the following examples and comparative examples, reagents not specifically described are conventional reagents and can be purchased from conventional reagent production and sales companies.

[0064] Preparation method of mixed strain bacterial suspension in Example 1

[0065] The preparation method of the mixed strain bacterial suspension includes the following steps:

[0066] S1. Activation of strains: Weigh 1.00 g of freeze-dried powder of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis, add 10 ml of sterile water, mix well by pipetting, take 0.5 ml and transfer it to a centrifuge tube containing 4.5 ml of sterile water, and perform gradient dilution in sequence. Take 0.2 ml of the bacterial suspensions at four concentrations of 10 -5 , 10 -6 , 10 -7 , 10 -8 and spread them on a solid medium. Do 3 replicates for each concentration. Incubate in a constant temperature incubator at 37 °C. Count Bifidobacterium lactis after 72 h, and count the other bacteria after 24 h of growth. After counting, use an inoculation loop to inoculate the evenly distributed single colonies on the petri dish into a slant solid medium, incubate in a constant temperature incubator at 37 °C, and put the cultured slant solid medium into the refrigerator for storage for later use.

[0067] S2. Preparation of mixed strain bacterial suspension: Take out the inoculated slant solid medium from the refrigerator, use an inoculation loop to inoculate the colonies into a liquid medium, and place it in a constant temperature water bath oscillator at 37 °C for 24 h of incubation; after 24 h, shake well and centrifuge at 2000 r / min for 5 min. Discard the supernatant, take the bacterial precipitate at the bottom, and add sterile water to the bacterial precipitate according to the volume ratio of the liquid medium to sterile water of 4:1 to make a bacterial suspension. Measure the concentration of the bacterial suspension by the plate counting method.

[0068] The concentrations of the Lactobacillus casei bacterial suspension, Lactobacillus rhamnosus bacterial suspension, Lactobacillus acidophilus bacterial suspension, Lactobacillus plantarum bacterial suspension, and Bifidobacterium lactis bacterial suspension are all 7.5×10 9 CFU / mL. The mixed strain bacterial suspension is a mixture of Lactobacillus casei bacterial suspension, Lactobacillus rhamnosus bacterial suspension, Lactobacillus acidophilus bacterial suspension, Lactobacillus plantarum bacterial suspension, and Bifidobacterium lactis bacterial suspension in a volume ratio of 1:1:1:1:1.

[0069] In the steps S1 and S2, the solid medium and liquid medium used for Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Lactobacillus casei are MRS medium; the solid medium and liquid medium used for Bifidobacterium lactis are TPY medium.

[0070] The MRS liquid medium is composed of the following components: peptone 10.0 g, beef extract 10.0 g, yeast extract powder 5.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, Tween 80 1.0 g, ammonium citrate tribasic 2.0 g, distilled water 1000 mL.

[0071] The TPY liquid medium is composed of the following components: hydrolyzed casein 10.0 g, peptone from plants 5.0 g, yeast powder 2.0 g, glucose 5.0 g, L-cysteine 0.5 g, dipotassium hydrogen phosphate 2.0 g, magnesium chloride 0.5 g, zinc sulfate 0.25 g, calcium chloride 0.15 g, ferric chloride 0.0001 g, Tween 80 1.0 g, distilled water 1000 mL.

[0072] The formulations of the MRS solid medium and TPY solid medium are obtained by adding 20 g of agar powder to the MRS liquid medium and TPY liquid medium, respectively.

[0073] Preparation method of the fermentation matrix of the mixed strain in Example 2

[0074] The preparation method of the mixed strain fermentation composition includes the following steps:

[0075] (1) Mix the Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis bacterial suspensions obtained in Example 1 evenly at a volume ratio of 1:1:1:1:1 to obtain a mixed strain bacterial suspension, inoculate it into a liquid medium, with an inoculation amount of 10%, and culture for 24 h to obtain a mixed strain seed liquid;

[0076] The liquid medium is composed of MRS liquid medium and TPY liquid medium mixed at a volume ratio of 4:1. The components of the MRS liquid medium and TPY liquid medium are the same as those in Example 1.

[0077] (2) Take 201 ml of the mixed strain seed liquid obtained in step (1) and pour it into a fermentation tank respectively, mix it evenly with 500 g of pine needle and pine branch mixture, 500 g of wheat bran, and 500 g of pine sawdust, place it indoors, ferment for 70 days, take it out after 70 days for high-pressure sterilization, and terminate the fermentation to obtain the fermented pine needle and pine branch with mixed strain, the fermented wheat bran with mixed strain, and the fermented pine sawdust with mixed strain.

[0078] The pine needle and pine branch mixture is composed of pine needles and pine branches at a weight ratio of 2:3.

[0079] Example 3 A fermented Poria cocos mushroom bag with a mixed strain and its preparation method

[0080] The fermented Poria cocos mushroom bag with the mixed strain is composed of the following components and their weight percentages:

[0081] Fermented pine needles and pine branches with the mixed strain in Example 2: 55%, fermented pine sawdust with the mixed strain in Example 2: 20%, fermented wheat bran with the mixed strain in Example 2: 20%, sucrose: 4%, gypsum powder: 1%.

[0082] The preparation method of the fermented Poria cocos mushroom bag with the mixed strain is as follows:

[0083] Dissolve sucrose in water (1 - 1.5 times), put the fermented pine sawdust with the mixed strain into it and boil for 30 min, fish it out after fully absorbing the sugar solution; then mix the fermented pine needles and pine branches with the mixed strain, the fermented wheat bran with the mixed strain, the gypsum powder and the fermented pine sawdust with the absorbed sugar solution evenly, and sub-pack them into 500 ml wide-mouth bottles, with the filling amount being 4 / 5 of the bottle, leaving a small hole (about 1 cm) in the center, sterilize by high-pressure steam at 121 °C for 120 min, and obtain it after cooling.

[0084] Comparative Example 1 Preparation method of Pediococcus pentosaceus bacterial suspension

[0085] The preparation method of the Pediococcus pentosaceus bacterial suspension includes the following steps:

[0086] S1. Activation of the strain: Weigh 1.00 g of freeze-dried powder of Pediococcus pentosaceus, add 10 ml of sterile water, mix well by pipetting, take 0.5 ml and put it into a centrifuge tube containing 4.5 ml of sterile water, perform gradient dilution in sequence, take 0.2 ml of the bacterial suspensions with the four concentrations of 10 -5 , 10 -6 , 10 -7 , 10 -8 and spread them on the MRS solid medium, do 3 parallels for each concentration, culture them in a constant temperature incubator at 37 °C, and count after 24 h. After counting, use an inoculation loop to inoculate the evenly distributed single colonies on the culture dish to the slant solid medium, culture them in a constant temperature incubator at 37 °C, and put the cultured slant solid medium into the refrigerator for cold storage for later use.

[0087] S2. Preparation of Pediococcus pentosaceus bacterial suspension: Take out the inoculated slant MRS solid medium from the refrigerator, inoculate the colonies into the MRS liquid medium with an inoculation loop, and place it in a constant temperature water bath oscillator at 37°C for 24 h of incubation; after 24 h, shake well and centrifuge at 2000 r / min for 5 min. Discard the supernatant, take the bacterial precipitate at the bottom, and add sterile water to the bacterial precipitate according to the volume ratio of liquid medium to sterile water of 4:1 to prepare a bacterial suspension. The concentration of the bacterial suspension is measured by the plate counting method, and the concentration of the Pediococcus pentosaceus bacterial suspension is 7.5×10 9 CFU / mL.

[0088] Preparation method of Bacillus subtilis bacterial suspension in Comparative Example 2

[0089] Compared with the comparative example, the difference in Comparative Example 2 is that the Bacillus subtilis bacterial suspension is prepared separately. In steps S1 and S2, the solid medium of Bacillus subtilis is nutrient agar medium, and the liquid medium is LB broth medium; in step S2, Bacillus subtilis is centrifuged at 4000 r / min for 10 min; the concentration of the Bacillus subtilis bacterial suspension obtained by the plate counting method is 7.5×10 9 CFU / mL.

[0090] The nutrient agar medium is composed of the following components: 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, 20.0 g of agar powder, and 1000 mL of distilled water.

[0091] The LB broth medium is composed of the following components: 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of glucose, 10.0 g of sodium chloride, 20.0 g of agar powder, and 1000 mL of distilled water.

[0092] Other parameters and operations are the same as those in Comparative Example 1.

[0093] Preparation method of a mixed strain bacterial suspension in Comparative Example 3

[0094] Compared with Example 1, the difference in Comparative Example 3 is that the Pediococcus pentosaceus bacterial suspension prepared in Comparative Example 1 is used to replace the Lactobacillus plantarum bacterial suspension in Example 1. The mixed strain bacterial suspension prepared in Comparative Example 3 is a mixed suspension of Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Pediococcus pentosaceus, and Lactobacillus delbrueckii subsp. bulgaricus in a volume ratio of 1:1:1:1:1. Other operations and parameters are the same as those in Example 1.

[0095] Preparation method of a mixed strain bacterial suspension in Comparative Example 4

[0096] Compared with Example 1, the difference in Comparative Example 4 is that the Bacillus subtilis bacterial suspension prepared in Comparative Example 2 is used to replace the Lactobacillus rhamnosus bacterial suspension in Example 1. The mixed-strain bacterial suspension prepared in Comparative Example 4 is a mixed solution of Lactobacillus casei bacterial suspension, Bacillus subtilis bacterial suspension, Lactobacillus acidophilus bacterial suspension, Lactobacillus plantarum bacterial suspension, and Bifidobacterium lactis bacterial suspension in a volume ratio of 1:1:1:1:1. Other operations and parameters are the same as those in Example 1.

[0097] Preparation method of the mixed-strain bacterial suspension of Comparative Example 5

[0098] Compared with Example 1, the difference in Comparative Example 5 is that the Bacillus subtilis bacterial suspension prepared in Comparative Example 2 is used to replace the Lactobacillus casei bacterial suspension in Example 1. The mixed-strain bacterial suspension prepared in Comparative Example 5 is a mixed solution of Bacillus subtilis bacterial suspension, Lactobacillus rhamnosus bacterial suspension, Lactobacillus acidophilus bacterial suspension, Lactobacillus plantarum bacterial suspension, and Bifidobacterium lactis bacterial suspension in a volume ratio of 1:1:1:1:1. Other operations and parameters are the same as those in Example 1.

[0099] Preparation method of the mixed-strain fermentation substrate of Comparative Example 6

[0100] Compared with Example 2, the difference in Comparative Example 6 is that in step (1), the mixed-strain bacterial suspension prepared in Comparative Example 3 is used to ferment the pine needle and pine branch mixture, wheat bran, pine sawdust, pine bark, and rice straw respectively. Other parameters and operations are the same as those in Example 2.

[0101] Preparation method of the mixed-strain fermentation substrate of Comparative Example 7

[0102] Compared with Example 2, the difference in Comparative Example 7 is that in step (1), the mixed-strain bacterial suspension prepared in Comparative Example 4 is used to ferment the pine needle and pine branch mixture, wheat bran, pine sawdust, pine bark, and rice straw respectively. Other parameters and operations are the same as those in Example 2.

[0103] Preparation method of the mixed-strain fermentation substrate of Comparative Example 8

[0104] Compared with Example 2, the difference in Comparative Example 8 is that in step (1), the mixed-strain bacterial suspension prepared in Comparative Example 5 is used to ferment the pine needle and pine branch mixture, wheat bran, pine sawdust, pine bark, and rice straw respectively. Other parameters and operations are the same as those in Example 2.

[0105] Preparation method of the natural fermentation substrate of Comparative Example 9

[0106] The preparation methods of the naturally fermented pine needles and pine branches, naturally fermented pine bark, naturally fermented pine sawdust, naturally fermented rice straw, and naturally fermented wheat bran are as follows:

[0107] Pour 200 ml of water into a fermentation tank respectively, and mix it evenly with 500 g of pine needle and pine branch mixture, 500 g of pine bark, 500 g of rice straw, 500 g of wheat bran, and 500 g of pine sawdust. Seal it well and place it indoors for 70 days of fermentation. After 70 days, take it out for high-pressure sterilization to terminate the fermentation, thus obtaining naturally fermented pine needles and pine branches, naturally fermented pine bark, naturally fermented rice straw, naturally fermented wheat bran, and naturally fermented pine sawdust.

[0108] The pine needle and pine branch mixture is composed of pine needles and pine branches in a weight ratio of 2:3.

[0109] Preparation method of the fermentation substrate of the mixed strains in Comparative Example 10

[0110] Compared with Example 2, the difference in Comparative Example 10 is that in step (2), 500 g of pine bark and 500 g of rice straw are fermented respectively to obtain the fermentation substrate of the mixed strains of pine bark and the fermentation substrate of the mixed strains of rice straw, and other parameters and operations are the same as those in Example 2.

[0111] Comparative Example 11 A fermented Poria cocos strain package with mixed strains and its preparation method

[0112] The fermented Poria cocos strain package with mixed strains is composed of the following components and their weight percentages:

[0113] 55% of the fermentation substrate of the mixed strains of pine bark in Comparative Example 10, 20% of the fermentation substrate of the mixed strains of pine sawdust in Example 2, 20% of the fermentation substrate of the mixed strains of wheat bran in Example 2, 4% of sucrose, and 1% of gypsum powder. The preparation method of the fermented Poria cocos strain package with mixed strains is the same as that in Example 3.

[0114] That is, the fermentation substrate of the mixed strains of pine bark prepared in Comparative Example 10 is used to replace the fermentation substrate of the mixed strains of pine needles and pine branches prepared in Example 2 in the components of Example 3.

[0115] Comparative Example 12 A fermented Poria cocos strain package with mixed strains and its preparation method

[0116] The fermented Poria cocos strain package with mixed strains is composed of the following components and their weight percentages:

[0117] 55% of the fermentation substrate of the mixed strains of rice straw in Comparative Example 10, 20% of the fermentation substrate of the mixed strains of pine sawdust in Example 2, 20% of the fermentation substrate of the mixed strains of wheat bran in Example 2, 4% of sucrose, and 1% of gypsum powder. The preparation method of the fermented Poria cocos strain package with mixed strains is the same as that in Example 3.

[0118] That is, the fermentation substrate of the mixed strains of rice straw prepared in Comparative Example 10 is used to replace the fermentation substrate of the mixed strains of pine needles and pine branches prepared in Example 2 in the components of Example 3.

[0119] Comparative Example 13 A fermented Poria cocos strain package with mixed strains and its preparation method

[0120] The mixed-strain fermented Poria cocos mushroom bag is composed of the following components and their weight percentages:

[0121] The mixed-strain fermented pine needles and pine branches of Comparative Example 6 is 55%, the mixed-strain fermented pine sawdust of Comparative Example 6 is 20%, the mixed-strain fermented wheat bran of Comparative Example 6 is 20%, sucrose is 4%, and gypsum powder is 1%.

[0122] The preparation method of the mixed-strain fermented Poria cocos mushroom bag is the same as that of Example 3.

[0123] Comparative Example 14 A mixed-strain fermented Poria cocos mushroom bag and its preparation method

[0124] The mixed-strain fermented Poria cocos mushroom bag is composed of the following components and their weight percentages:

[0125] The mixed-strain fermented pine needles and pine branches of Comparative Example 7 is 55%, the mixed-strain fermented pine sawdust of Comparative Example 7 is 20%, the mixed-strain fermented wheat bran of Comparative Example 7 is 20%, sucrose is 4%, and gypsum powder is 1%.

[0126] The preparation method of the mixed-strain fermented Poria cocos mushroom bag is the same as that of Example 3.

[0127] Comparative Example 15 A mixed-strain fermented Poria cocos mushroom bag and its preparation method

[0128] The mixed-strain fermented Poria cocos mushroom bag is composed of the following components and their weight percentages:

[0129] The mixed-strain fermented pine needles and pine branches of Comparative Example 8 is 55%, the mixed-strain fermented pine sawdust of Comparative Example 8 is 20%, the mixed-strain fermented wheat bran of Comparative Example 8 is 20%, sucrose is 4%, and gypsum powder is 1%.

[0130] The preparation method of the mixed-strain fermented Poria cocos mushroom bag is the same as that of Example 3.

[0131] Comparative Example 16 Naturally fermented Poria cocos mushroom bag and its preparation method

[0132] The Poria cocos mushroom bag is composed of the following components and their weight percentages:

[0133] The naturally fermented pine needles and pine branches mixture of Comparative Example 9 is 55%, the naturally fermented pine sawdust of Comparative Example 9 is 20%, the naturally fermented wheat bran of Comparative Example 9 is 20%, sucrose is 4%, and gypsum powder is 1%.

[0134] The preparation method of the Poria cocos mushroom bag is the same as that of Example 3.

[0135] Comparative Example 17 Unfermented Poria cocos mushroom bag and its preparation method

[0136] The Poria cocos mushroom bag is composed of the following components and their weight percentages:

[0137] 55% of unfermented pine needle and pine branch mixture, 20% of unfermented pine sawdust, 20% of unfermented wheat bran, 4% of sucrose, 1% of gypsum powder. The unfermented pine needle and pine branch mixture is composed of pine needles and pine branches in a weight ratio of 2:3.

[0138] The preparation process of the unfermented bacterial bag is as follows: First, mix the pine sawdust, wheat bran, and gypsum powder evenly. Dissolve glucose in 1.5 times water, adjust the pH value to 5 - 6, put the pine sawdust into it and boil for 30 minutes. After the pine sawdust fully absorbs the sugar solution, take out the pine sawdust. Then add the pine needle sawdust and wheat bran into the sugar solution, mix well to make the water content 60% - 65%, and then mix in the pine sawdust. Divide it into 500 mL wide-mouth bottles, with the filling amount being 4 / 5 of the bottle, leaving a small hole (about 1 cm) in the center, and sterilize it at 121 °C for 120 minutes by high-pressure steam. After cooling, inoculate.

[0139] Experimental Example 1: Fermentation Results and Analysis of the Raw Materials of the Bacterial Bag

[0140] Test method: Use the mixed strain bacterial suspension prepared in Example 1, the mixed strain bacterial suspensions prepared in Comparative Examples 3 - 5, and the natural fermentation method (Comparative Example 9) to ferment the pine branch and pine needle mixture, pine bark, pine sawdust, rice straw, and wheat bran respectively for 70 days, and conduct visual observation. The results are shown in Table 1 and Figures 1 - 5 as shown, Figures 1 - 5 They are the surface characteristics of the pine branch and pine needle mixture, pine bark, pine sawdust, rice straw, and wheat bran after 70 days of fermentation respectively. Among them, A is fermented with the mixed strain bacterial suspension of Example 1; B is fermented with the mixed strain bacterial suspension of Comparative Example 3; C is fermented with the mixed strain bacterial suspension of Comparative Example 4; D is fermented with the mixed strain bacterial suspension of Comparative Example 5; E is the fermentation substrate of natural fermentation in Comparative Example 9.

[0141] Table 1 Fermentation Results of Different Examples and Comparative Examples on the Substrate

[0142]

[0143]

[0144] From Table 1 and Figures 1 - 5It can be seen that after 70 days of fermentation, white mycelia grew on the surfaces of the pine needle and pine branch mixture, pine bark, pine sawdust, and rice straw fermented with the mixed bacterial suspension of the strains prepared in Example 1 of the present invention. The color of the wheat bran was lighter than that in Comparative Example 5 and Comparative Example 6. There was a small amount of bacteria on the surfaces of the pine needle and pine branch mixture, pine bark, pine sawdust, and rice straw fermented in Comparative Examples 3 and 4, and the odor after fermentation was lighter than that in Example 1, indicating that the fermentation degree was lower than that in Example 1. In Comparative Example 9, green bacteria grew on the surface of the naturally fermented pine needles and pine branches, there was a thin layer of white mycelia on the surface of the pine bark, black-green bacteria grew on the surface of the pine sawdust, some mycelia were observed on the surface of the rice straw, and the color of the wheat bran was darker, showing a brownish-yellow color.

[0145] Experimental Example 2: Observation on the growth status of Poria mycelia

[0146] 1. Test objects: Poria strain CGMCC5.0078 and Poria strain GDMCC 5.219.

[0147] 2. Test methods:

[0148] (1) Cultivation of primary spawn:

[0149] Poria strain CGMCC5.0078 and Poria strain GDMCC 5.219 were respectively selected and cultured on a potato medium for the cultivation of primary spawn. The specific operation method was as follows: A small piece of medium with Poria fungus was cut from the test tube containing the Poria strain with a fungal hook and inoculated in the center of the culture dish, with the mycelial surface closely attached to the medium. It was placed in an incubator at 25 °C and cultured for 5 - 7 days. When the mycelia covered the medium, the primary Poria spawn was obtained. The growth conditions of the two primary Poria spawns CGMCC5.0078 and GDMCC 5.219 were respectively recorded, as shown in Table 2; the growth curve slopes of the two primary Poria mycelia were as shown in Table 3; the growth curves of the two primary Poria mycelia were as Figure 6 shown; the microscopic morphology (10×100) of the two primary Poria mycelia was as Figure 7 (CGMCC5.0078) and Figure 8 (GDMCC 5.219) shown.

[0150] The potato medium (PDA) consisted of the following components: 1 L of 20% potato juice, 20 g of glucose, 3.0 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 8 mg of vitamin B1, 20 g of agar, and the pH was natural.

[0151] Table 2 Measurement results of the average diameter of primary Poria mycelia

[0152]

[0153] Table 3 Growth curve slopes of the two primary Poria mycelia

[0154] Wolfiporia strain types Growth curve formula Slope <![CDATA[R 2 > CGMCC 5.0078 y = 0.2401x + 3.8533 0.2401 0.8914 GDMCC 5.219 y = 0.2316x + 2.7853 0.2316 0.8575

[0155] From Figure 6 and Tables 2 and 3, it can be seen that at the same cultivation time, the Wolfiporia strain CGMCC5.0078 grows faster than the Wolfiporia strain GDMCC5.219, and the diameter of the Wolfiporia hyphae is also thicker than that of the Wolfiporia strain GDMCC5.219. The growth curve slope of the Wolfiporia strain CGMCC5.0078 is greater than that of the Wolfiporia strain GDMCC5.219, which also indicates that the Wolfiporia strain CGMCC5.0078 grows faster than the Wolfiporia strain GDMCC5.219.

[0156] From Figure 7 and Figure 8 it can be known that under microscopic observation, the primary Wolfiporia hyphae are colorless or light brown, slender tubular, with many branches and obvious diaphragms.

[0157] (2) Cultivation of secondary spawn

[0158] Inoculate the primary Wolfiporia spawn CGMCC5.0078 and the Wolfiporia spawn GDMCC5.219 into the Wolfiporia fungus bags prepared in Example 3 and Comparative Examples 11 - 17 respectively. The inoculation amount is a small piece of primary Wolfiporia spawn with a length × width of 2 cm × 1 cm. Place it in an incubator at 25°C and cultivate for 20 - 30 days to obtain the secondary spawn. The growth conditions of the Wolfiporia strain CGMCC5.0078 and the Wolfiporia spawn GDMCC5.219 in the secondary fungus bags with different substrates are as Figure 9 and Figure 10 shown.

[0159] According to Figure 9 it is known that the secondary Wolfiporia spawn CGMCC5.0078 has the best growth state and the most hyphal distribution in the fermented fungus bag prepared in Example 3; it is second in Comparative Examples 13, 14, and 15; the hyphal distribution is relatively scattered in the naturally fermented fungus bag of Comparative Example 16; the hyphal growth is very slow in the unfermented fungus bag of Comparative Example 17; while the hyphal distribution is very little in the fungus bag prepared in Comparative Example 11 (using a mixed spawn to ferment pine bark to replace pine branches and pine needles), and the hyphal distribution is less and the growth condition is poor and it is prone to mildew in the fungus bag prepared in Comparative Example 12 (using a mixed spawn to ferment rice straw to replace pine branches and pine needles).

[0160] According to Figure 10It is known that the secondary Poria cocos strain GDMCC 5.219 has the best growth state in the fermentation fungus bags prepared in Example 3, with the most mycelium distribution. Its growth state is the second best in the fermentation fungus bags prepared in Comparative Example 11, with more mycelium distribution. However, the mycelium distribution of Poria cocos mycelium in the Poria cocos fungus bags prepared in Comparative Examples 12 - 17 is less, and the growth condition is poor. Through comprehensive analysis, the growth of Poria cocos strain CGMCC 5.0078 is better than that of Poria cocos strain GDMCC 5.219.

[0161] It is Figure 9 and Figure 10 known that the growth state of Poria cocos mycelium in the secondary fungus bags with the pine branch and pine needle mixture fermented by the mixed bacteria in Example 3 as the substrate is the best. Therefore, it is preferred to use the Poria cocos fungus bags fermented by the mixed strains prepared in Example 3 for the secondary cultivation of Poria cocos strain CGMCC 5.0078, and record the growth conditions of Poria cocos mycelium at different time periods after making the secondary fungus bags. The specific results are as Figure 11 shown, where A: the fermentation fungus bag prepared in Example 3; B: the fermentation fungus bag prepared in Comparative Example 15; C: the fermentation fungus bag prepared in Comparative Example 16.

[0162] It is Figure 11 known that through naked - eye observation, on the 3rd day of cultivation, the Poria cocos mycelium CGMCC 5.0078 begins to expand around, and on the 7th day, the growth rate of the Poria cocos mycelium is relatively fast. The Poria cocos mycelium grows the fastest in the fungus bag fermented in Example 3, grows relatively fast in the fermented fungus bag prepared in Comparative Example 15, and grows slowly in the naturally fermented fungus bag in Comparative Example 16.

[0163] (3) Culture results and analysis of the secondary Poria cocos strain CGMCC 5.0078

[0164] Inoculate the primary Poria cocos strain CGMCC 5.0078 into the Poria cocos fungus bags prepared in Example 3 and Comparative Examples 13 - 17 respectively. The inoculation amount is a primary Poria cocos strain with a length × width of 2 cm × 1 cm, and place it in an incubator at 25°C for 20 - 30 days to obtain the secondary strain.

[0165] Record the growth conditions of the secondary Poria cocos strains at different time periods in different Examples 3 and Comparative Examples 13 - 17 respectively, as shown in Table 4. Through the data in Table 4, obtain the growth slopes and growth conditions of the secondary Poria cocos mycelium in various types of fungus bags, as shown in Table 5 and Figure 12 shown.

[0166] Table 4 Growth conditions of the secondary Poria cocos strains at different time periods in different examples and comparative examples

[0167]

[0168]

[0169] Table 5 Growth Slope of Wolfiporia cocos Secondary Mycelia in Various Types of Mushroom Bags

[0170] Spawn bag type Growth curve formula Slope <![CDATA[R 2 > Example 3 y = 0.6006x + 1.4380 0.6006 0.9984 Comparative Example 13 y = 0.4943x + 1.3267 0.4943 0.9483 Comparative Example 14 y = 0.4411x + 1.3993 0.4411 0.9633 Comparative Example 15 y = 0.5186x + 1.2200 0.5186 1.2200 Comparative Example 16 y = 0.3943x + 1.7067 0.3943 1.7067 Comparative Example 17 y = 0.4786x + 0.9233 0.4786 0.9233

[0171] From Figure 12 , Table 4, and Table 5, it can be seen that according to the slope of the growth of Wolfiporia cocos mycelia in Wolfiporia cocos mushroom bags, the larger the slope, the faster the Wolfiporia cocos mycelia grow, and the more beneficial the mushroom bag is to the growth of Wolfiporia cocos mycelia. The growth curve slope of Wolfiporia cocos mycelia in the mushroom bag fermented in Example 3 is 0.6006; while the growth curve slope in the mushroom bag in Comparative Example 13 is 0.4943; the growth curve slope in the mushroom bag in Comparative Example 14 is 0.4411; the growth curve slope in the mushroom bag in Comparative Example 15 is 0.5186; the growth curve slope in the naturally fermented mushroom bag in Comparative Example 16 is 0.3943; the growth curve slope in the unfermented mushroom bag in Comparative Example 17 is 0.4786; it can be seen that the secondary Wolfiporia cocos mycelia grow fastest in the mushroom bag fermented in Example 3 of the present invention.

[0172] From Figures 13 - 18 it can be seen that under the microscope, it is observed that Wolfiporia cocos mycelia CGMCC 5.0078 grow better in the mushroom bag prepared in Example 3, with a thicker diameter.

[0173] The embodiments are only illustrative of the principles and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A fermented Poria cocos strain bag with a mixed bacterial strain, characterized in that, it comprises the following components and their mass percentages: 70 - 96% of a mixed bacterial strain fermentation composition, 2 - 6% of sucrose, 0.5 - 2.0% of gypsum powder; The mixed bacterial strain fermentation composition is a combination of a mixed bacterial strain fermented pine needles and twigs, a mixed bacterial strain fermented wheat bran, and a mixed bacterial strain fermented pine sawdust; the mixed bacterial strain is a combination of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis; The preparation methods of the mixed bacterial strain fermented pine needles and twigs, the mixed bacterial strain fermented wheat bran, and the mixed bacterial strain fermented pine sawdust include the following steps: S1. Mix the bacterial suspensions of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis evenly to obtain a mixed bacterial strain suspension, inoculate it into a liquid medium, and culture for 24 h to obtain a mixed bacterial strain seed liquid; S2. Pour the mixed bacterial strain seed liquid obtained in step S1 into a fermentation tank and mix it evenly with the pine needles and twigs mixture, wheat bran, and pine sawdust respectively, place it indoors, ferment for 70 days, and after 70 days, take it out for high - pressure sterilization to terminate fermentation, thus obtaining the mixed bacterial strain fermented pine needles and twigs, the mixed bacterial strain fermented wheat bran, and the mixed bacterial strain fermented pine sawdust respectively; The pine needles and twigs mixture is composed of pine needles and twigs in a weight ratio of 2:3; The preparation method of the fermented Poria cocos strain bag with a mixed bacterial strain comprises the following steps: Dissolve sucrose in water, put the mixed bacterial strain fermented pine sawdust into it and boil, fish it out after fully absorbing the sugar solution; then mix the mixed bacterial strain fermented pine needles and twigs, the mixed bacterial strain fermented wheat bran, gypsum powder with the mixed bacterial strain fermented pine sawdust after absorbing the sugar solution evenly, perform high - pressure steam sterilization, and cool to obtain the fermented Poria cocos strain bag with a mixed bacterial strain; The weight ratio of the mixed bacterial strain fermented pine needles and twigs, the mixed bacterial strain fermented pine sawdust, and the mixed bacterial strain fermented wheat bran is 11:4:

4.

2. The fermented Poria cocos strain bag with a mixed bacterial strain according to claim 1, characterized in that, In the step S1, the concentrations of the suspensions of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis are all 7.5×10 9 CFU / mL; the Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis are mixed evenly according to a volume ratio of 1:1:1:1:1 to obtain a mixed strain suspension.

3. The fermented Poria cocos strain bag with a mixed bacterial strain according to claim 1, characterized in that, The inoculation amount of the mixed strain bacterial suspension into the liquid medium in the step S1 is 10%; the addition amount of the mixed strain seed liquid in the step S2 is calculated according to 2.0×10 10 CFU per 1000 g of fermentation raw materials, and the fermentation raw materials are pine needle and pine branch mixture, wheat bran or pine sawdust.

4. The fermented Poria cocos strain bag with a mixed bacterial strain according to claim 1, characterized in that, The addition amount of water is 1 - 1.5 times the weight of sucrose, the boiling time after putting the mixed bacterial strain fermented pine sawdust is 30 min, and the water content of the finally obtained fermented Poria cocos strain bag with a mixed bacterial strain after mixing evenly is 63%.

5. The application of the fermented Poria cocos strain bag with a mixed bacterial strain according to any one of claims 1 - 4 in cultivating Poria cocos strains.

6. A method for cultivating Poria cocos strains, characterized in that, it comprises the following steps: inoculate the first - grade Poria cocos strain into the fermented Poria cocos strain bag with a mixed bacterial strain according to any one of claims 1 - 4, and place it in an incubator at 25°C for 20 - 30 days.

7. The method for cultivating Poria cocos strains according to claim 6, characterized in that, The cultivation method of the first - grade Poria cocos strain comprises the following steps: inoculate the Poria cocos strain into a potato medium, make the mycelium surface closely adhere to the potato medium, place it in an incubator at 25°C, and culture for 5 - 7 days. When the mycelium covers the medium, the first - grade Poria cocos strain is obtained.

8. The method for cultivating Poria cocos strains according to claim 7, characterized in that, The potato medium consists of the following components: 1 L of 20% potato juice, 20 g of glucose, 3.0 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 8 mg of vitamin B 1 8, 20 g of agar, pH 6 - 7.

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