Edible mushroom composite probiotic fermented beverage and preparation method and application thereof

Abstract

The present application relates to the field of food and biological fermentation technology, and discloses an edible mushroom compound probiotic fermented beverage as well as a preparation method and application thereof, which comprises edible mushrooms, deionized water, an outer enzyme combination, an inner enzyme combination, sucrose, a first-stage oxygen-tolerant probiotic group and a second-stage strict anaerobic probiotic group. After the mushroom is treated by pulping, the outer enzyme combination and the inner enzyme combination are sequentially added to carry out step-by-step constant-temperature enzymolysis, and time-limited ultrasonic treatment is assisted during the inner enzymolysis to destroy the fungal cell wall. After enzyme inactivation filtration and addition of a sucrose solution, the oxygen-tolerant probiotic group is inoculated to carry out fermentation to consume liquid-phase dissolved oxygen, and when the state changes to anoxic, the strict anaerobic probiotic group is inoculated to continue closed fermentation, and after the fermentation is completed, high-pressure homogenization treatment is carried out to obtain the product. The present application improves the dissolution rate of active substances by hierarchical targeted degradation, and uses a sequential fermentation microecological mechanism to ensure the viable bacterial titer, and is suitable for preparing a blood lipid-lowering auxiliary product.

Description

Technical Field

[0001] This invention relates to the field of food and bio-fermentation technology, specifically to an edible fungus compound probiotic fermented beverage, its preparation method, and its application. Background Technology

[0002] Edible fungi are rich in active substances such as fungal polysaccharides, small molecule polypeptides, and specific fungal sterols. Combining edible fungi with probiotics to prepare fermented beverages can improve the original earthy flavor of edible fungi. At the same time, the fermentation and metabolism of microorganisms produce short-chain fatty acids and other components, which increase the nutritional value of the product.

[0003] When preparing fermentation substrate for edible fungi, the cell wall structure of fungi is generally dense, characterized by an outer glycoprotein network wrapping an inner chitinous hard skeleton. Conventional mechanical crushing methods or single-component enzyme preparations cannot completely break down this complex physical barrier. Incomplete deconstruction results in a low dissolution rate of intracellular active polysaccharides and macromolecular proteins, and nutrients are locked inside the fungal cells. The subsequent inoculated probiotic population lacks sufficient metabolic substrates, resulting in insufficient fermentation and low accumulation of functional metabolites.

[0004] In existing technologies for mixed-strain fermentation, operators directly and simultaneously inoculate aerobic bacteria such as lactic acid bacteria with strict anaerobic bacteria such as bifidobacteria. The initial liquid phase of the fermentation substrate contains a high concentration of free oxygen. This microenvironment causes irreversible oxidative damage to the strict anaerobic bacteria, leading to a decrease in the number of viable anaerobic bacteria and disrupting the intended multi-strain symbiotic metabolic system. Some processes resort to introducing vacuum degassing equipment or adding physicochemical deoxygenating agents to forcibly establish an anaerobic environment. This intervention strips the original liquid of volatile flavor molecules and alters the original physicochemical state of the system. Furthermore, the wild-type probiotic strains used in conventional fermentation generally lack sufficient stress resistance. During the high-pressure homogenization process before product delivery and subsequent entry into the human stomach acid and intestinal bile salt environment, the living cells experience membrane rupture and mass death, resulting in a low concentration of effective viable bacteria reaching the intestines and establishing colonization, making it difficult to achieve the intended target for lipid metabolism intervention. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a fermented beverage containing edible fungi and probiotics, its preparation method, and its application. This invention solves the problems of limited dissolution of effective components from edible fungi in existing fermentation processes, low survival rate of strictly anaerobic bacteria due to direct mixed inoculation, and insufficient final effective live bacteria count due to poor resistance of conventional strains.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a fermented beverage containing edible fungi and probiotics. The fermented beverage is made from raw materials comprising the following parts by weight: 10 to 15 parts of edible fungi; 100 to 200 parts of deionized water; 0.7 to 1.0 parts of outer layer enzyme combination; 0.4 to 0.75 parts of inner layer enzyme combination; 4 to 8 parts of sucrose; 10 to 15 parts of a first-stage aerobic probiotic group; and 10 to 15 parts of a second-stage strictly anaerobic probiotic group.

[0008] By employing the provided technical solution, the combination of outer and inner enzyme combinations is used to deconstruct the fungal cell wall hierarchically to destroy the physical barrier, thereby releasing nutrients such as intracellular polysaccharides and proteins, thus providing substrates for microbial metabolism. By using a combination of first-stage aerobic probiotics and second-stage strictly anaerobic probiotics, the respiratory metabolism of aerobic strains during pre-fermentation consumes dissolved oxygen in the system and lowers the redox potential, causing the liquid phase environment to spontaneously transform into a strictly anaerobic state.

[0009] The strictly anaerobic environment then induces a large proliferation of strictly anaerobic probiotics in the second stage, forming a cross-generic feeding metabolic structure. The aerobic bacteria degrade sucrose and macromolecular polysaccharides to produce lactic acid and short-chain oligosaccharides, and convert some of the degradation products into direct carbon sources for the second-stage anaerobic bifidobacteria. Finally, a network of short-chain fatty acids such as acetic acid and propionic acid is accumulated in the fermentation broth, resulting in a microecological structure in which multiple metabolites coexist.

[0010] Preferably, the edible fungus is one or more of the following: Pleurotus eryngii, Pleurotus ostreatus, and Boletus sinensis.

[0011] By adopting the provided technical solution, the limited edible fungi are rich in fungal polysaccharides and specific fungal sterols. The polysaccharide macromolecules themselves contain a large number of β-1,3-glycosidic bonds and β-1,6-glycosidic bond branch structures, which are difficult to be excessively hydrolyzed into monosaccharides during subsequent enzymatic hydrolysis and fermentation. Objectively, they serve as exclusive prebiotic substrates for the proliferation of probiotic groups, ensuring the continuous operation of the acid production metabolic pathway in the later stage of fermentation.

[0012] Preferably, the outer enzyme assembly consists of β-glucanase, neutral protease, and mannanase; the inner enzyme assembly consists of chitinase, cellulase, and lysozyme.

[0013] By employing the provided technical solution, and utilizing a defined combination of enzyme systems, targeted degradation of the bilayered cell wall structure of edible fungi is achieved. The reaction mechanism mainly manifests in two levels: the outer enzyme combination first acts on the glycoproteins and mannan backbone around the cell wall, β-glucanase cleaves the side chains connecting proteins and polysaccharides, and neutral proteases hydrolyze matrix proteins, making the outer cell wall loose. On this basis, the inner enzyme combination directly contacts the exposed chitinous core layer, chitinase hydrolyzes β-1,4-polyN-acetylglucosamine polymers, and cellulase and lysozyme synergistically destroy the remaining peptidoglycan and cellulose microfibril network, leading to the rupture of cell protoplasts and the complete release of contents into the aqueous system.

[0014] Preferably, the first-stage aerobic probiotic group is composed of a mixture of Lactobacillus rhamnosus mutant strain, Pediococcus lactis mutant strain, and Lactobacillus paracasei mutant strain in equal weight ratios; the second-stage strictly anaerobic probiotic group is composed of a mixture of Bifidobacterium adolescentis mutant strain and Bifidobacterium infantis mutant strain in equal weight ratios; all mutant strains are obtained by screening after undergoing gradient acclimatization treatments of heat resistance, gastric acid resistance, bile salt resistance, osmotic pressure resistance, and oxidation resistance.

[0015] By adopting the provided technical solution, and by limiting the combination of specific bacterial genera and excluding wild-type strains, the selected mutant strains, after being acclimatized to environmental pressure, exhibit an increased proportion of unsaturated fatty acids in the phospholipid bilayer of their cell membranes. This objectively enhances the activity of the intracellular antioxidant enzyme system. During fermentation and human ingestion, the aerobic bacterial mutant strains are tolerant to the dissolved oxygen environment in the early stage of fermentation and the subsequent high-pressure homogenization heat treatment. The anaerobic Bifidobacterium mutant strains, relying on their enhanced resistance to gastric acid and bile salts, maintain a high survival rate when passing through the host's gastrointestinal tract and colonize the distal intestine, thereby completing the secretion of functional substances and the intervention of host metabolic targets.

[0016] Secondly, the present invention also provides a method for preparing a fermented beverage containing edible fungi and probiotics, the method comprising the following steps:

[0017] Edible fungi are mixed with deionized water and pulverized to obtain fungal slurry. The pH and temperature of the fungal slurry are adjusted, and an outer layer enzyme combination is added for isothermal stirring and enzymatic hydrolysis. Subsequently, the pH and temperature of the system are adjusted again, and an inner layer enzyme combination is added for isothermal stirring and enzymatic hydrolysis. During the inner layer enzymatic hydrolysis, time-limited ultrasonic treatment is applied to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is heated to inactivate enzymes and sterilize before fermentation. After cooling, it is filtered to obtain fungal juice, which is added to sucrose and stirred to dissolve, thus obtaining a fermentation base liquid. The first stage of aerobic probiotics is inoculated into the fermentation base liquid for isothermal fermentation to consume dissolved oxygen. After the system becomes oxygen-deficient, the second stage of strictly anaerobic probiotics is inoculated and isothermal closed fermentation continues until fermentation is complete, obtaining a fermented liquid. The fermented liquid is subjected to high-pressure homogenization, followed by aseptic filling and sealing to obtain a fermented beverage of edible fungi compound probiotics.

[0018] By adopting the provided technical solution, a multi-stage step-by-step control process is used to organically combine physical and mechanical cell disruption, two-stage gradient biological enzymatic hydrolysis, and sequential microecological establishment. This provides a basic contact area for pulverization and pulping. The two-step enzymatic hydrolysis, combined with temperature and acid control, achieves selective breakage of target bonds. The intervention of time-limited ultrasound generates cavitation effect to accelerate mass transfer. A biological deoxygenation mechanism is established during the fermentation stage, avoiding the loss of volatile flavor caused by the introduction of traditional physicochemical deoxygenating agents or degassing equipment. The subsequent high-pressure homogenization process micronizes the large molecular fragments and denatured proteins remaining in the liquid, establishing a stable suspended colloidal system, preventing precipitation and stratification, and obtaining a functional product with stable physicochemical state and containing live bacteria.

[0019] Preferably, the step of adding the outer layer enzyme combination for isothermal stirring enzymatic hydrolysis includes: adjusting the pH of the mushroom slurry to 5.0 to 5.5, raising the temperature to 38 to 42°C, adding the outer layer enzyme combination, and stirring and hydrolyzing under isothermal conditions for 1.5 to 2.0 hours; the step of adding the inner layer enzyme combination for isothermal stirring enzymatic hydrolysis includes: adjusting the pH of the system to 4.5 to 5.0 using inorganic acid, raising the temperature to 42 to 48°C, adding the inner layer enzyme combination, and stirring and hydrolyzing under isothermal conditions, so that the total enzymatic hydrolysis time of the inner layer reaches 1.5 to 2.0 hours.

[0020] By adopting the provided technical solution, independent reaction environments are set for two specific enzyme groups. The outer layer enzymatic hydrolysis is carried out at a slightly acidic and mild temperature to avoid excessive hydrolysis and blackening of polysaccharide chains. The inner layer enzymatic hydrolysis is carried out at a lower pH and a relatively higher temperature. This set parameter range activates the catalytic active center of chitinase and promotes the thermal motion of the inner layer macromolecular network, thereby improving the inner layer cell wall breaking efficiency and the extraction rate of functional peptides.

[0021] Preferably, the timed ultrasonic treatment step includes: starting an ultrasonic device with a power of 200 to 300W for ultrasonic-assisted treatment at 20 to 40 minutes after adding the inner layer enzyme combination, and turning off the ultrasonic device after 15 to 20 minutes of continuous treatment.

[0022] By adopting the provided technical solution, ultrasound of a specific power is introduced when the inner layer enzymatic hydrolysis has proceeded for 20 to 40 minutes. The reaction mechanism is as follows: at this time, the outer layer has been peeled off, and some binding sites of the inner layer chitin macromolecules are exposed. The cavitation bubbles formed by ultrasound in the liquid collapse on the surface of the cell wall fragments, generating microjets and local high pressure. This physical action physically tears apart the entangled chitin-polysaccharide complex, multiplying the internal enzymatic hydrolysis sites, thereby increasing the dissolution rate without increasing the amount of enzyme or the reaction time. The accompanying 15 to 20-minute time limit operation avoids the thermal effect of ultrasound causing thermal denaturation and inactivation of enzyme proteins.

[0023] Preferably, the steps of inoculating the first-stage aerobic probiotic group for isothermal fermentation include: inoculating the first-stage aerobic probiotic group and fermenting at a constant temperature of 35 to 40°C for 12 to 18 hours; and inoculating the second-stage strictly anaerobic probiotic group for continued isothermal closed fermentation include: after the fermentation environment is in an anaerobic state, inoculating the second-stage strictly anaerobic probiotic group and continuing isothermal closed fermentation for 24 to 36 hours until the pH value of the system drops to 3.8 to 4.2, at which point the fermentation ends.

[0024] By employing the provided technical solution, the defined temperature and time series are matched with the logarithmic growth phase of the selected bacterial community. In the initial inoculation stage, the lactic acid bacteria community is used for aerobic and facultative anaerobic metabolism, consuming liquid molecular oxygen and generating primary lactic acid, causing the pH to begin to decrease. As the environment transitions to an anaerobic state, Bifidobacteria are inoculated into the established highly anaerobic substrate rich in primary metabolites. Bifidobacteria metabolize carbon sources through the fructose-6-phosphate ketolase pathway, generating large amounts of acetic acid and lactic acid until the system acidity decreases to pH 3.8 to 4.2. The high concentration of hydrogen ions and undissociated organic acids produce feedback inhibition, terminating the growth of other bacteria and allowing the system to spontaneously enter a stable storage state.

[0025] Preferably, the step of high-pressure homogenization of the fermented liquid includes: directly feeding the fermented liquid into a homogenizer and performing high-pressure homogenization at a temperature of 55 to 65°C and a pressure of 15 to 20 MPa.

[0026] By adopting the provided technical solution, the homogenization conditions have the dual functions of physical homogenization and pasteurization to remove contaminants. Under this pressure and temperature window, coarse fiber residues and aggregated proteins are sheared into micron and nano-sized particles. At the same time, since the probiotics used in the process have all undergone multi-resistance gradient domestication and are equipped with heat and pressure stress screening mechanisms, probiotic mutants with corresponding resistance can survive through specific treatment ranges. Vegetative bacteria cells that do not have resistance mixed in the fermentation system will die due to cell membrane rupture under this temperature and pressure shearing, thus ensuring the microbial safety of the product and the targeted probiotic concentration.

[0027] Thirdly, the present invention also provides the application of edible fungi compound probiotic fermented beverage in the preparation of products that help lower blood lipids or regulate lipid deposition in organisms.

[0028] By employing the provided technical solution, fermented beverages, rich in specific short-chain fatty acid networks, free fungal sterols, and live Bifidobacteria and Lactobacillus, upon entering the host's intestines, allow propionic acid to enter the liver via the portal vein, participating in the inhibition of HMG-CoA reductase activity and reducing endogenous cholesterol synthesis. Free fungal sterols compete with free cholesterol for micro-matrix binding sites in the intestines, blocking the absorption of exogenous cholesterol. After colonization by the live microbiota, undigested dietary fiber in the intestines continues to ferment, continuously outputting lipid regulatory signaling factors. By relying on different intervention pathways to jointly influence the lipid metabolism network, the effects of assisting in lowering blood lipids and reducing lipid deposition are achieved.

[0029] This invention provides a fermented beverage made from edible fungi and probiotics, its preparation method, and its application. It has the following beneficial effects: 1. This invention utilizes a combination of outer and inner enzyme combinations for stepwise degradation, along with time-limited ultrasonic treatment, to deconstruct the bilayered cell wall of fungi. Matched acidity and temperature ranges are set for the outer glycoproteins and the inner chitin, respectively. While maintaining the original branched structure of polysaccharide macromolecules, the contents of the cell protoplast are effectively released. The targeted deconstruction operation improves the dissolution rate of effective substances and establishes a rich underlying matrix resource for subsequent microbial metabolism.

[0030] 2. This invention constructs a stepped fermentation process by employing a first-stage aerobic probiotic group and a second-stage strictly anaerobic probiotic group. During the pre-fermentation, the aerobic bacteria deplete free oxygen and lower the redox potential through respiratory metabolism, causing the liquid environment to spontaneously transform into a strictly anaerobic state. This removes environmental barriers for the large-scale proliferation of Bifidobacteria. The multi-stage microecological succession avoids flavor loss caused by the intervention of external degassing equipment. At the same time, it establishes a cross-generic metabolic network, accumulating abundant short-chain fatty acids and active metabolites in the fermentation system.

[0031] 3. This invention selects probiotic mutant strains obtained through multidimensional gradient stress acclimatization, and combines them with high-pressure homogenization within a limited temperature and pressure range to achieve dual protection of physical stability and microbial control. The homogenization operation within the limited parameter window cuts the residues in the fermentation liquid into fine particles, preventing the liquid product from settling and separating. The mutant strains with complex stress resistance can survive and pass through the set parameter range, while environmental bacteria without pressure and heat resistance will undergo cell rupture, objectively ensuring the live bacteria titer and storage safety of the fermented beverage. Attached Figure Description

[0032] Figure 1 This is a schematic diagram showing the change of free polysaccharide dissolution rate over time under different process conditions according to the present invention; Figure 2 This is a schematic diagram showing the change of cell wall breakage rate over time under different process conditions of the present invention; Figure 3 This is a schematic diagram showing the total viable cell count test results at the endpoint of different fermentation groups in this invention; Figure 4 This is a schematic diagram showing the results of the Bifidobacterium viable count test at the endpoint of different fermentation groups in this invention; Figure 5 This is a schematic diagram of the survival rate test results of the cross-protection acclimatization of the multiple stress system of the present invention; Figure 6 This is a schematic diagram showing the fold change in biochemical indicators of mice in each experimental group relative to the control group in this invention. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0035] Pleurotus eryngii, Pleurotus ostreatus, and Boletus sinensis are all prepared using fresh fruiting bodies; β-glucanase, CAS No. 9025-70-1, with a defined enzyme activity of ≥50000 U / g; Neutral protease, CAS No. 9068-59-1, with a defined enzyme activity of ≥100,000 U / g; Mannanase, CAS No. 37288-54-3, with a defined enzyme activity of ≥30000 U / g; Chitinase, CAS No. 9001-06-3, with a defined enzyme activity of ≥10000 U / g; Cellulase, CAS No. 9012-54-8, with a defined enzyme activity of ≥30000 U / g; Lysozyme, CAS No. 9001-63-2, with a defined enzyme activity of ≥20000 U / g; The standard strains of *Lactobacillus rhamnosus* (ATCC53103), *Pediococcus lactis* (ATCC8042), *Lactobacillus paracasei* (ATCC25302), *Bifidobacterium adolescentis* (ATCC15703), and *Bifidobacterium infantis* (ATCC15697) were all activated free bacterial suspensions, with an initial viable count of at least 1.0 × 10⁻⁶. 8 CFU / mL.

[0036] In this invention, the units of measurement for all materials and solvents are uniformly referred to as parts by weight.

[0037] Preparation Example 1: This preparation example provides a method for preparing multi-resistant probiotics, including the following steps: For Lactobacillus rhamnosus, Pediococcus lactis, Lactobacillus paracasei, Bifidobacterium adolescentis, and Bifidobacterium infantis, 10 parts by weight of each free bacterial suspension activated to the logarithmic growth phase were independently transferred to 100 parts by weight of fresh MRS liquid medium supplemented with 0.05 wt% L-cysteine ​​hydrochloride for parallel gradient acclimatization.

[0038] First, heat tolerance acclimatization was carried out, starting at 37℃, and then passaged three times each at 40℃, 42℃, 45℃, 48℃ and 50℃. Subsequently, four batches were subjected to heat stress acclimatization at 55℃ and 60℃, and heat-resistant strains that remained viable after heat treatment at 60℃ for 30 minutes were screened out.

[0039] The obtained heat-resistant strains were acclimatized to gastric acid and inoculated sequentially into MRS liquid medium with decreasing pH gradients. The strains were passaged three times each at pH 5.0, pH 4.0, and pH 3.5, and four times at pH 3.0. Acid-resistant strains that remained viable after 2.5 hours of treatment at pH 3.0 were selected.

[0040] Next, the acid-resistant strains were acclimated to bile salts by inoculating them into MRS liquid medium containing 0.05 wt%, 0.1 wt%, and 0.2 wt% ox bile salts, and then passaged them three times each. They were also passaged four times in MRS liquid medium containing 0.25 wt% ox bile salts. Bile salt-resistant strains that remained viable after being treated with 0.25 wt% ox bile salts for 4 hours were selected.

[0041] Bile salt-tolerant strains were acclimatized to osmotic pressure by being passaged three times each in MRS liquid medium containing 2.0 wt% and 4.0 wt% sodium chloride, and four times in MRS liquid medium containing 6.0 wt% sodium chloride. Hyperosmotic-tolerant strains with an OD600 value greater than or equal to 1.5 after culturing in a hyperosmotic environment of 6.0 wt% sodium chloride for 24 h were selected.

[0042] Next, the osmotic-tolerant strains were subjected to oxidation acclimatization. In an environment with an oxygen volume concentration of 5.0% to 10.0%, the strains were passaged three times each in MRS liquid medium containing 0.1 mM, 0.2 mM, and 0.5 mM hydrogen peroxide, and four times in MRS liquid medium containing 0.8 mM hydrogen peroxide. Oxidation-tolerant mutant strains that remained viable after 1 hour of treatment with 0.8 mM hydrogen peroxide were then screened.

[0043] Finally, the antioxidant mutant strains selected through sequential gradient acclimatization were inoculated into a combined stress MRS liquid medium containing 55℃, pH 3.0, 0.2wt% ox bile salt, 4.0wt% sodium chloride, and 0.5mM hydrogen peroxide for 2.5 h to verify the combined stress effect. The strains that could be inoculated into fresh liquid medium after combined stress treatment and cultured for 48 h with a viable count restored to 1.0 × 10⁻⁶ cells / year were screened. 8 For each mutant strain with a CFU / mL or higher, Lactobacillus rhamnosus, Pediococcus lactis, and Lactobacillus paracasei mutant strains were collected by centrifugation and then resuspended in equal weight ratios to obtain the first-stage aerobic probiotic group A1; for Bifidobacterium adolescentis and Bifidobacterium infantis mutant strains, they were collected by centrifugation and then resuspended in equal weight ratios to obtain the second-stage strictly anaerobic probiotic group A2.

[0044] Preparation Example 2: This preparation example provides a method for preparing multi-resistant probiotics, including the following steps: For Lactobacillus rhamnosus, Pediococcus lactis, Lactobacillus paracasei, Bifidobacterium adolescentis, and Bifidobacterium infantis, 8 parts by weight of each free bacterial suspension activated to the logarithmic growth phase were independently transferred to 90 parts by weight of fresh MRS liquid medium supplemented with 0.05 wt% L-cysteine ​​hydrochloride for parallel gradient acclimatization.

[0045] First, heat tolerance acclimatization was carried out, starting at 37℃, and then passaged three times each at 40℃, 42℃, 45℃, 48℃ and 50℃. Subsequently, four batches were subjected to heat stress acclimatization at 55℃, 60℃ and 65℃, and heat-resistant strains that remained viable after heat treatment at 65℃ for 30 minutes were screened out.

[0046] The obtained heat-resistant strains were acclimatized to gastric acid and inoculated sequentially into MRS liquid medium with decreasing pH gradients. The strains were passaged 3 times each at pH 5.0, pH 4.0 and pH 3.5, and 4 times each at pH 3.0 and pH 2.5. Acid-resistant strains that remained viable after 3.0 hours of treatment at pH 2.5 were selected.

[0047] Next, the acid-resistant strains were acclimated to bile salts by inoculating them into MRS liquid medium containing 0.05 wt%, 0.1 wt%, and 0.2 wt% ox bile salts, and then passaged three times each in MRS liquid medium containing 0.3 wt% and 0.4 wt% ox bile salts. The bile salt-resistant strains that remained viable after treatment with 0.4 wt% ox bile salts for 4 hours were then selected.

[0048] Bile salt-tolerant strains were subjected to osmotic acclimation by being passaged three times each in MRS liquid medium containing 2.0 wt% and 4.0 wt% sodium chloride, and four times each in MRS liquid medium containing 6.0 wt%, 8.0 wt%, and 10.0 wt% sodium chloride. Hyperosmotic-tolerant strains with an OD600 value greater than or equal to 1.2 after 24 h of culture in a hyperosmotic environment of 10.0 wt% sodium chloride were selected.

[0049] Next, the osmotic-tolerant strains were subjected to oxidation acclimatization. In an environment with an oxygen volume concentration of 5.0% to 10.0%, the strains were passaged three times each in MRS liquid medium containing 0.1 mM, 0.2 mM, and 0.5 mM hydrogen peroxide, and four times each in MRS liquid medium containing 1.0 mM and 2.0 mM hydrogen peroxide. Oxidation-tolerant mutant strains that remained viable after 1 hour of treatment with 2.0 mM hydrogen peroxide were then screened.

[0050] Finally, the antioxidant mutant strains selected through sequential gradient acclimatization were inoculated into a combined stress MRS liquid medium containing 60℃, pH 2.5, 0.3wt% ox bile salt, 6.0wt% sodium chloride, and 1.0mM hydrogen peroxide for 3.0 h to verify the combined stress effect. The strains that could be inoculated into fresh liquid medium after combined stress treatment and cultured for 48 h with a viable count restored to 5.0 × 10⁻⁶ cells / year were screened. 7 For each mutant strain with a CFU / mL or higher, Lactobacillus rhamnosus, Pediococcus lactis, and Lactobacillus paracasei mutant strains were collected by centrifugation and then resuspended in equal weight ratios to obtain the first-stage aerobic probiotic group B1; for Bifidobacterium adolescentis and Bifidobacterium infantis mutant strains, they were collected by centrifugation and then resuspended in equal weight ratios to obtain the second-stage strictly anaerobic probiotic group B2.

[0051] Preparation Example 3: This preparation example provides a method for preparing multi-resistant probiotics, including the following steps: For Lactobacillus rhamnosus, Pediococcus lactis, Lactobacillus paracasei, Bifidobacterium adolescentis, and Bifidobacterium infantis, 12 parts by weight of each free bacterial suspension activated to the logarithmic growth phase were independently transferred to 110 parts by weight of fresh MRS liquid medium supplemented with 0.05 wt% L-cysteine ​​hydrochloride for parallel gradient acclimatization.

[0052] First, heat tolerance acclimatization was carried out, starting at 37℃, and then passaged three times each at 40℃, 42℃, 45℃ and 48℃. Subsequently, four batches were subjected to heat stress acclimatization at 50℃ and 55℃, and heat-resistant strains that remained viable after heat treatment at 55℃ for 30 minutes were screened out.

[0053] The obtained heat-resistant strains were acclimatized to gastric acid and sequentially inoculated into MRS liquid medium with decreasing pH gradients. The strains were passaged 3 times each at pH 5.0 and pH 4.0, and 4 times at pH 3.5. Acid-resistant strains that remained viable after 2.0 h of treatment at pH 3.5 were selected.

[0054] Next, the acid-resistant strains were acclimated to bile salts. They were inoculated into MRS liquid medium containing 0.05 wt% and 0.1 wt% ox bile salts, and passaged 3 times each. They were also passaged 4 times in MRS liquid medium containing 0.2 wt% ox bile salts. Bile salt-resistant strains that remained viable after 4 hours of treatment with 0.2 wt% ox bile salts were selected.

[0055] Bile salt-tolerant strains were acclimatized to osmotic pressure by being passaged three times in MRS liquid medium containing 2.0 wt% sodium chloride and four times in MRS liquid medium containing 4.0 wt% sodium chloride. Hyperosmotic-tolerant strains with an OD600 value greater than or equal to 1.8 after culturing in a hyperosmotic environment of 4.0 wt% sodium chloride for 24 h were selected.

[0056] Next, the osmotic-tolerant strains were subjected to oxidation acclimatization. In an environment with an oxygen volume concentration of 5.0% to 10.0%, the strains were passaged three times each in MRS liquid medium containing 0.1 mM and 0.2 mM hydrogen peroxide, and four times in MRS liquid medium containing 0.5 mM hydrogen peroxide. Oxidation-tolerant mutant strains that remained viable after 1 hour of treatment with 0.5 mM hydrogen peroxide were then screened.

[0057] Finally, the antioxidant mutant strains selected through sequential acclimatization were inoculated into a combined stress MRS liquid medium containing 50℃, pH 3.5, 0.1wt% ox bile salt, 2.0wt% sodium chloride, and 0.2mM hydrogen peroxide for 2.0 h to verify the combined stress effect. The strains that could be inoculated into fresh liquid medium after combined stress treatment and cultured for 48 h with a viable count restored to 5.0 × 10⁻⁶ cells / year were screened. 8 For each mutant strain with a CFU / mL or higher, Lactobacillus rhamnosus, Pediococcus lactis, and Lactobacillus paracasei mutant strains were collected by centrifugation and then resuspended in equal weight ratios to obtain the first-stage aerobic probiotic group C1; for Bifidobacterium adolescentis and Bifidobacterium infantis mutant strains, they were collected by centrifugation and then resuspended in equal weight ratios to obtain the second-stage strictly anaerobic probiotic group C2.

[0058] Example 1: This embodiment provides a method for preparing a fermented beverage containing edible fungi and probiotics, comprising the following steps: Wash and chop 10 parts by weight of freshly picked eryngii and oyster mushrooms. Add 150 parts by weight of deionized water at a material-to-liquid ratio of 1:15 and place the mixture in a pulping machine to grind and pulp, obtaining a single mushroom slurry. Then, adjust the pH of the mushroom slurry to 5.2 and raise the temperature to 40°C. Subsequently, add an outer layer enzyme combination consisting of 0.40 parts by weight of β-glucanase, 0.20 parts by weight of neutral protease, and 0.20 parts by weight of mannanase to the mushroom slurry. Stir and enzymatically hydrolyze the mixture at a constant temperature of 40°C for 2.0 hours, thereby destroying the outer layer structure of the edible fungus cell wall.

[0059] Subsequently, the pH of the enzymatic hydrolysis mixture was adjusted to 4.8 using hydrochloric acid, and the system was heated to 45°C. An inner layer enzyme combination consisting of 0.20 parts by weight of chitinase, 0.20 parts by weight of cellulase, and 0.10 parts by weight of lysozyme was added, and the enzymatic hydrolysis was carried out at a constant temperature of 45°C with stirring. At the 30-minute mark after the addition of the inner layer enzyme combination, an ultrasonic device with a power of 250W was started for ultrasonic-assisted treatment. After 15 minutes of treatment, the ultrasonic device was turned off, and the constant temperature stirring was continued under the continuous action of the inner layer enzyme, so that the total enzymatic hydrolysis time reached 2.0 hours, and a fully broken cell wall enzymatic hydrolysis mixture was obtained.

[0060] The enzymatic hydrolysis mixture was then rapidly heated to 90°C and kept at that temperature for 15 minutes to simultaneously inactivate the enzymes and sterilize before fermentation. After the mixture cooled to 25°C, it was filtered through a 200-mesh filter to obtain pure mushroom juice. Six parts by weight of sucrose were then added to the mushroom juice and stirred to dissolve, finally yielding the fermentation base liquid.

[0061] Next, 12 parts by weight of the first-stage aerobic probiotic group B1 were inoculated into the fermentation base liquid, and fermentation was carried out at a constant temperature of 37°C for 15 hours to rapidly consume the dissolved oxygen in the system. After the fermentation environment was in an anaerobic state, 12 parts by weight of the second-stage strictly anaerobic probiotic group B2 were inoculated into the system, and fermentation was carried out at a constant temperature and in a closed environment for 30 hours until the pH value of the system dropped to 4.0, at which point the fermentation ended.

[0062] Next, the fermented liquid is directly fed into a homogenizer and subjected to high-pressure homogenization at a temperature of 65℃ and a pressure of 18MPa. This process achieves physical anti-sedimentation and pasteurization to remove unwanted bacteria, while preserving the live core probiotics. After homogenization, the mixture is directly filled and sealed under aseptic conditions to obtain the edible fungi compound probiotic fermented beverage.

[0063] Example 2: This embodiment provides a method for preparing a fermented beverage containing edible fungi and probiotics, comprising the following steps: Wash and chop 10 parts by weight of freshly picked golden top oyster mushrooms, add 100 parts by weight of deionized water at a material-to-liquid ratio of 1:10, and place in a pulping machine to grind and pulp to obtain a single mushroom slurry. Then, adjust the pH of the mushroom slurry to 5.5 and raise the temperature to 38°C. Subsequently, add an outer layer enzyme combination consisting of 0.40 parts by weight of β-glucanase, 0.15 parts by weight of neutral protease, and 0.15 parts by weight of mannanase to the mushroom slurry. Stir and enzymatically hydrolyze at a constant temperature of 38°C for 1.5 hours to destroy the outer layer structure of the edible fungus cell wall.

[0064] Subsequently, the pH of the enzymatic hydrolysis mixture was adjusted to 5.0 using phosphoric acid, and the system was heated to 42°C. An inner layer enzyme combination consisting of 0.16 parts by weight of chitinase, 0.16 parts by weight of cellulase, and 0.08 parts by weight of lysozyme was added, and the enzymatic hydrolysis was carried out at a constant temperature of 42°C with stirring. At the 20-minute mark after the addition of the inner layer enzyme combination, an ultrasonic device with a power of 200W was started for ultrasonic-assisted treatment. After treatment for 18 minutes, the ultrasonic device was turned off, and the constant temperature stirring was continued under the continuous action of the inner layer enzyme, so that the total enzymatic hydrolysis time reached 1.5 hours, and a fully cell-wall-broken enzymatic hydrolysis mixture was obtained.

[0065] The enzymatic hydrolysis mixture was then rapidly heated to 90°C and kept at that temperature for 15 minutes to simultaneously inactivate the enzymes and sterilize before fermentation. After the mixture cooled to 25°C, it was filtered through a 150-mesh filter to obtain pure mushroom juice. Four parts by weight of sucrose were then added to the mushroom juice and stirred to dissolve, finally yielding the fermentation base liquid.

[0066] Next, 10 parts by weight of the first-stage aerobic probiotic group A1 were inoculated into the fermentation base liquid, and fermentation was carried out at a constant temperature of 35°C for 12 hours to rapidly consume the dissolved oxygen in the system. After the fermentation environment was in an anaerobic state, 10 parts by weight of the second-stage strictly anaerobic probiotic group A2 were inoculated into the system, and fermentation was carried out at a constant temperature and in a closed environment for 24 hours until the pH value of the system dropped to 4.2, at which point the fermentation ended.

[0067] Next, the fermented liquid is directly fed into a homogenizer and subjected to high-pressure homogenization at a temperature of 60℃ and a pressure of 15MPa. This process physically prevents sedimentation and also removes unwanted bacteria through pasteurization, while preserving the live core probiotics. After homogenization, the mixture is directly filled and sealed under aseptic conditions to obtain the edible fungi compound probiotic fermented beverage.

[0068] Example 3: This embodiment provides a method for preparing a fermented beverage containing edible fungi and probiotics, comprising the following steps: Ten parts by weight of freshly picked Boletus chinensis were washed, chopped, and added to 200 parts by weight of deionized water at a material-to-liquid ratio of 1:20. The mixture was then placed in a pulping machine to grind and pulp, obtaining a single mushroom slurry. The pH of the mushroom slurry was then adjusted to 5.0 and the temperature was raised to 42°C. Subsequently, an outer layer enzyme combination consisting of 0.50 parts by weight of β-glucanase, 0.25 parts by weight of neutral protease, and 0.25 parts by weight of mannanase was added to the mushroom slurry. The mixture was then kept at a constant temperature of 42°C and stirred for 2.0 hours to enzymatically hydrolyze the outer layer of the edible fungus cell wall.

[0069] Subsequently, the pH of the enzymatic hydrolysis mixture was adjusted to 4.5 using hydrochloric acid, and the system was heated to 48°C. An inner layer enzyme combination consisting of 0.30 parts by weight of chitinase, 0.30 parts by weight of cellulase, and 0.15 parts by weight of lysozyme was added, and the mixture was stirred at a constant temperature of 48°C. At the 40-minute mark after adding the inner layer enzyme combination, an ultrasonic device with a power of 300W was started for ultrasonic-assisted treatment. After 20 minutes of treatment, the ultrasonic device was turned off, and the mixture was stirred at a constant temperature under the continuous action of the inner layer enzymes, so that the total enzymatic hydrolysis time reached 2.0 hours, resulting in a fully disrupted enzymatic hydrolysis mixture.

[0070] The enzymatic hydrolysis mixture was then rapidly heated to 90°C and kept at that temperature for 15 minutes to simultaneously inactivate the enzymes and sterilize before fermentation. After the mixture cooled to 25°C, it was filtered through a 250-mesh filter to obtain pure mushroom juice. Eight parts by weight of sucrose were then added to the mushroom juice and stirred to dissolve, finally yielding the fermentation base liquid.

[0071] Next, 15 parts by weight of the first-stage aerobic probiotic group C1 were inoculated into the fermentation base liquid, and fermentation was carried out at a constant temperature of 40°C for 18 hours to rapidly consume the dissolved oxygen in the system. After the fermentation environment was in an anaerobic state, 15 parts by weight of the second-stage strictly anaerobic probiotic group C2 were inoculated into the system, and fermentation was carried out at a constant temperature and in a closed environment for 36 hours until the pH value of the system dropped to 3.8, at which point the fermentation ended.

[0072] Next, the fermented liquid is directly fed into a homogenizer and subjected to high-pressure homogenization at a temperature of 55℃ and a pressure of 20MPa. This process physically prevents sedimentation and also removes unwanted bacteria through pasteurization, while preserving the live core probiotics. After homogenization, the mixture is directly filled and sealed under aseptic conditions to obtain the edible fungi compound probiotic fermented beverage.

[0073] Example 4: This embodiment provides a method for preparing a fermented beverage containing edible fungi and probiotics, comprising the following steps: Mix 15 parts by weight of fresh mushrooms (including Pleurotus eryngii, Pleurotus ostreatus, and Boletus sinensis) in equal weight ratio. Wash and chop 15 parts by weight of the mixed fresh mushrooms. Add 180 parts by weight of deionized water at a material-to-liquid ratio of 1:12 and place the mixture in a pulping machine to grind and pulp, thus obtaining a compound mushroom slurry. Then, adjust the pH of the compound mushroom slurry to 5.3 and raise the temperature to 40°C. Subsequently, add an outer layer enzyme combination consisting of 0.40 parts by weight of β-glucanase, 0.20 parts by weight of neutral protease, and 0.20 parts by weight of mannanase to the compound mushroom slurry. Stir and enzymatically hydrolyze the mixture at a constant temperature of 40°C for 1.8 hours, thereby destroying the outer layer structure of the edible mushroom cell wall.

[0074] Subsequently, the pH of the enzymatic hydrolysis mixture was adjusted to 4.6 using phosphoric acid, and the system was heated to 46°C. An inner layer enzyme combination consisting of 0.20 parts by weight of chitinase, 0.20 parts by weight of cellulase, and 0.10 parts by weight of lysozyme was added, and the mixture was stirred at a constant temperature of 46°C. At 35 minutes after the addition of the inner layer enzyme combination, an ultrasonic device with a power of 280W was started for ultrasonic-assisted treatment. After 15 minutes of treatment, the ultrasonic device was turned off, and the mixture was stirred at a constant temperature under the continuous action of the inner layer enzymes, so that the total enzymatic hydrolysis time reached 1.8 hours, and a fully broken cell wall enzymatic hydrolysis mixture was obtained.

[0075] The enzymatic hydrolysis mixture was then rapidly heated to 90°C and kept at that temperature for 15 minutes to simultaneously inactivate the enzymes and sterilize before fermentation. After the mixture cooled to 25°C, it was filtered through a 200-mesh filter to obtain pure mushroom juice. Seven parts by weight of sucrose were then added to the mushroom juice and stirred to dissolve, finally yielding the fermentation base liquid.

[0076] Next, 13 parts by weight of the first-stage aerobic probiotic group B1 were inoculated into the fermentation base liquid, and fermentation was carried out at a constant temperature of 38°C for 16 hours to rapidly consume the dissolved oxygen in the system. After the fermentation environment was in an anaerobic state, 13 parts by weight of the second-stage strictly anaerobic probiotic group B2 were inoculated into the system, and fermentation was carried out at a constant temperature and in a sealed environment for 28 hours until the pH value of the system dropped to 3.9, at which point the fermentation ended.

[0077] Next, the fermented liquid is directly fed into a homogenizer and subjected to high-pressure homogenization at a temperature of 65℃ and a pressure of 16MPa. This process physically prevents sedimentation and also removes unwanted bacteria through pasteurization, while preserving the live core probiotics. After homogenization, the mixture is directly filled and sealed under aseptic conditions to obtain the edible fungi compound probiotic fermented beverage.

[0078] Comparative Example 1: Compared with Example 1, the difference is that the stepwise enzyme addition and cell wall breaking operation was not performed. Instead, the outer enzyme combination and the inner enzyme combination were added to the mushroom slurry at the beginning and the enzyme was stirred at a constant temperature of 45°C for 4.0 h. Then, on the 2.5 h after enzyme addition, an ultrasonic device with a power of 250W was started for 15 min of auxiliary treatment. All other aspects were the same.

[0079] Comparative Example 2: Compared with Example 1, the difference is that after adding the inner layer enzyme combination, the ultrasound was not started at 30 minutes and the treatment was controlled for only 15 minutes. Instead, the ultrasound device with a power of 250W was started immediately when the inner layer enzyme combination was added, and ultrasound-assisted treatment was continuously performed for 2.0 hours during the inner layer enzymatic hydrolysis. All other aspects are the same.

[0080] Comparative Example 3: Compared with Example 1, the difference is that the first-stage aerobic probiotic group B1 and the second-stage strictly anaerobic probiotic group B2 were not inoculated stepwise during the fermentation stage. Instead, 12 parts by weight of the first-stage aerobic probiotic group B1 and 12 parts by weight of the second-stage strictly anaerobic probiotic group B2 were combined and inoculated into the fermentation base liquid at the beginning of fermentation, and then directly carried out constant temperature closed fermentation for 45 hours. All other aspects are the same.

[0081] Comparative Example 4: Compared with Example 1, the difference is that after the fermentation environment was in an anaerobic state, the second stage of strictly anaerobic probiotic group B2 was not inoculated into the system. Instead, 24 parts by weight of the first stage of aerobic probiotic group B1 was directly inoculated at the beginning of fermentation, and the fermentation was carried out at a constant temperature and sealed at 37°C for 45 hours. All other aspects were the same.

[0082] Comparative Example 5: Compared with Example 1, the difference is that the first-stage aerobic probiotic group B1 and the second-stage strictly anaerobic probiotic group B2 were not domesticated by the gradient multiple stress system of the preparation example of the present invention. Instead, they were directly replaced by commercially available original pure live bacteria that had not undergone any domestication treatment, mixed in the same proportion. All other aspects are the same.

[0083] Test Example 1: The mushroom juice obtained after heating to 90°C and maintaining the temperature for 15 minutes, and cooling to 25°C and then filtering through a 200-mesh filter in Examples 1, 1, and 2 was used as the test sample. Control group 1 and control group 2 were set up. In control group 1, an equal amount of fresh Erycibe oyster mushroom was taken, without the addition of enzyme preparation, only deionized water was added, and the mixture was stirred at 45°C for 2.0 hours before filtration and extraction. Control group 2 adopted the two-step gradient enzyme addition process described in Example 1. The ultrasonic equipment was not activated throughout the entire treatment cycle. Twenty parallel samples were taken from each of Examples 1, 1, 2, control group 1, and control group 2 for endpoint sampling. At the same time, dynamic monitoring samples were taken every 30 minutes during the treatment cycle from 0 to 180 minutes.

[0084] The supernatant of different groups of material solutions was extracted, and phenol solution and concentrated sulfuric acid were added to trigger the colorimetric reaction. After the reaction solution was allowed to stand and cool to room temperature, the absorbance of the reaction system was measured at a wavelength of 490 nm using a UV-Vis spectrophotometer. A standard working curve was plotted using a glucose standard solution. Based on the theoretical total mass of polysaccharides from fresh eryngii and oyster mushrooms in the feed, the free polysaccharide dissolution rate of each parallel sample was calculated.

[0085] The intracellular nucleic acid leakage ultraviolet absorption method was used to characterize the cell wall structure. Equal volumes of the test group solution were taken and centrifuged at 4000 r / min for 10 min to remove large particles of residue. The supernatant was collected, and the absorbance of the supernatant at 260 nm was measured using a spectrophotometer. The sample that was continuously processed by an ultrasonic disruptor for 40 min was used as the total leakage reference. The relative cell wall disruption rate of each parallel sample was calculated. The control group 1 was not included in the comparison of relative cell wall disruption rate data because no enzyme preparation was added for cell wall disruption treatment.

[0086] The test results are shown in Table 1.

[0087] Table 1. Results of polysaccharide dissolution and cell wall disruption under different process conditions:

[0088] According to Table 1 and appendix Figure 1 and attached Figure 2The data shows that the average value of the free polysaccharide dissolution rate in Example 1 reached 81.3%. In Comparative Example 1, the outer and inner degrading enzymes were added to the reaction system simultaneously in the initial treatment stage, resulting in the inner degrading enzyme directly contacting the undecomposed outer polymer layer. Due to the steric hindrance effect, the direct contact area between the reaction substrate and the active center of the inner degrading enzyme was limited. The free polysaccharide dissolution rate of Comparative Example 1 increased in the early stage of treatment and then quickly entered a stagnant period, with an average value of only 54.2% at the end. Example 1 used a step-by-step intervention strategy, adding the inner targeting enzyme after pre-degrading the outer matrix, and the free polysaccharide dissolution rate maintained a continuous increase throughout the entire treatment cycle.

[0089] Both Example 1 and Comparative Example 2 were designed with a stepwise enzyme addition process. In Comparative Example 2, the ultrasonic device was started and continuously run at the same time as the inner layer targeted enzyme was added. The cell wall disruption rate of Comparative Example 2 stopped increasing after 90 minutes, and the average value at the endpoint remained at 73.8%. The long-term continuous output of ultrasonic energy directly changed the internal spatial conformation of proteins such as chitinase and cellulase, resulting in denaturation and inactivation of biological enzymes. In Example 1, the ultrasonic treatment was limited to a specific time interval. After the ultrasonic intervention ended, the cell wall disruption rate continued to increase, and the average value at the endpoint of the cell wall disruption rate reached 96.2%, maintaining the biocatalytic stability inside the reaction system.

[0090] Control group 1 was extracted with deionized water only, without the addition of enzyme preparations for destruction, and therefore could not induce cell wall structure disintegration and nucleic acid leakage. Thus, the cell wall disruption rate of control group 1 is marked as "—" in Table 1. Control group 2 relied solely on a two-step gradient enzyme addition strategy to eliminate steric hindrance. The reaction rate of the inner framework under the action of single biological enzyme degradation was limited, and the average value of the final free polysaccharide dissolution rate was 72.5%. Example 1 introduced time-limited ultrasound-assisted intervention, using the micro-jet mechanical shear force generated by acoustic cavitation to accelerate the peeling of metabolic attachments on the surface of deep substrates. The time-limited intervention operation promoted the renewal and exposure of the reaction substrate surface, avoiding damage to the enzymatic environment, and presenting the final result of the synergistic effect of physical mechanical intervention and biological enzyme degradation.

[0091] Test Example 2: Using the mushroom substrate liquid from Examples 1 and 2, which underwent cell wall disruption and sterilization, as the fermentation substrate, Examples 1 and 2 followed a stepwise fermentation process. In the first stage, the aerobic probiotic group B1 was inoculated and fermented at a constant temperature for 15 hours. In the second stage, the strictly anaerobic probiotic group B2 was inoculated and fermented at a constant temperature in a sealed environment for 30 hours. Fermentation samples from Comparative Example 3 were extracted. In Comparative Example 3, the aerobic probiotic group B1 and the strictly anaerobic probiotic group B2 were combined and introduced into the fermentation substrate liquid at the beginning of fermentation and fermented at a constant temperature in a sealed environment for 45 hours. Fermentation samples from Comparative Example 4 were extracted. In Comparative Example 4, the aerobic probiotic group B1 was directly inoculated at the beginning of fermentation and fermented at a constant temperature in a sealed environment for 45 hours. The strictly anaerobic probiotic group B2 was not inoculated during the entire fermentation stage. After fermentation, liquid samples were extracted from each fermentation tank as endpoint test samples.

[0092] Take equal volumes of endpoint test samples and perform serial dilutions using sterile physiological saline. Spread the appropriately diluted sample solution onto the surface of MRS solid medium and incubate at 37°C for 48 hours. Count the total number of colonies and convert it to the total viable count concentration. Take another equal volume of diluted sample solution and spread it onto the surface of BBL agar selective medium supplemented with mupirocin lithium salt. Incubate in an anaerobic incubator and count the number of colonies and convert it to the viable count concentration of Bifidobacterium.

[0093] A supernatant from the fermentation endpoint was added to a metaphosphate solution to precipitate the protein components in the reaction system. The treated solution was centrifuged at 10,000 r / min for 15 min. The clear supernatant was extracted and filtered through a 0.22 μm microporous membrane. The filtrate was transferred to a sample vial for a gas chromatograph. The concentrations of acetic acid, propionic acid, and butyric acid were determined by a gas chromatograph equipped with a flame ionization detector. The total amount of short-chain fatty acids was obtained by summing the concentrations.

[0094] The test results are shown in Table 2.

[0095] Table 2. Data on niche shift and metabolic complementarity at fermentation endpoints in different groups:

[0096] According to Table 2 and appendix Figure 3 and attached Figure 4 The data shows that in Comparative Example 3, when both aerobic probiotic group B1 and strictly anaerobic probiotic group B2 were introduced at the initial stage of fermentation, the free oxygen present in the initial environment caused oxidative toxicity damage to Bifidobacteria. Before a completely anaerobic environment was established, a large number of Bifidobacteria were inactivated, and the average number of viable Bifidobacteria remained at 0.8 × 10⁻⁶. 8CFU / mL, Example 1 employed a stepwise inoculation strategy. In the initial stage, the aerobic probiotic group B1 consumed dissolved oxygen and produced secondary metabolites. In the second stage, when the strictly anaerobic probiotic group B2 was inoculated, the fermentation system was switched to a strictly anaerobic state, eliminating the influence of toxicity. The metabolites from the initial stage were directly converted into carbon source substrates for use by Bifidobacteria, and the average viable count of Bifidobacteria reached 3.1 × 10⁻⁶ CFU / mL. 8 CFU / mL confirmed cross-generic niche replacement and cross-feeding mechanisms.

[0097] Comparative Example 4 was not inoculated with the strictly anaerobic probiotic group B2, and no live Bifidobacteria were present in the reaction system; the viable Bifidobacteria count was recorded as "—". The strictly anaerobic probiotic group B2 contains a specific fructose-6-phosphate phosphoketone hydrolase, possessing a specific biochemical pathway for metabolizing complex macromolecular oligosaccharides from fungi. Due to the lack of a specific degradative enzyme system, the complex carbon sources within the fermentation substrate in Comparative Example 4 could not be fully degraded, and the core synthesis pathway of short-chain fatty acids was blocked, resulting in an average decrease in the total short-chain fatty acid content to 12.42 mmol / L. Example 1, combined with specific time-point microbial replacement, constructed a dissolved oxygen scavenging mechanism and a complementary pathway for multi-microbial metabolic substrates within the same reaction system, ultimately increasing the average total viable count in the fermentation system to 6.8 × 10⁻⁶. 8 CFU / mL, the average production of short-chain fatty acids increased to 34.2 mmol / L.

[0098] Test Example 3: Mixed bacterial strain samples from Example 1, which were subjected to multiple stress system acclimatization at a maximum temperature of 65°C, and from Example 3, which were subjected to multiple stress system acclimatization at a maximum temperature of 55°C, were extracted respectively. The commercially available original mixed bacterial strain samples from Comparative Example 5, which were not subjected to pressure acclimatization, were also extracted. The mixed bacterial strain samples from different groups were resuspended in sterile physiological saline and the initial viable bacterial concentration was adjusted to the same baseline level to prepare the bacterial suspension to be tested.

[0099] Equal volumes of different groups of bacterial suspensions were measured and subjected to five single extreme environmental treatments: incubation at 65℃ for 30 min; adjustment of pH to 2.5 with hydrochloric acid and incubation at 2 h; addition of sodium deoxycholate to a final concentration of 0.3% and incubation at 4 h; addition of sodium chloride to a final concentration of 6% and incubation at 4 h; and addition of hydrogen peroxide to a final concentration of 1.0 mmol / L and incubation at 1 h.

[0100] Take equal volumes of different groups of bacterial suspensions and place them into a simulated extreme combined stress environment. The combined stress environment conditions are set as follows: temperature 55℃, pH 3.0, sodium deoxycholate concentration 0.2%, sodium chloride concentration 4%, and hydrogen peroxide concentration 0.5 mmol / L. The combined stress environment is continuously treated for 2 hours.

[0101] After each stress treatment reached its predetermined time point, the treatment solution was immediately extracted and serially diluted with sterile physiological saline. The treatment solution with an appropriate dilution was then spread onto the surface of MRS solid medium and incubated in a 37°C incubator for 48 hours. The total number of colonies generated on the surface of the solid medium was counted and divided by the initial number of viable bacteria in the bacterial suspension before the stress treatment to calculate the survival rate of the strains under different treatment conditions.

[0102] The test results are shown in Table 3.

[0103] Table 3. Survival rate test data of cross-protection in multiple stress systems:

[0104] According to Table 3 and appendix Figure 5 The data shows that Comparative Example 5 did not undergo stress system acclimatization. The survival rate of the commercially available original mixed strain sample in extreme environments was low, with an average survival rate of 8.5% after heat treatment at 65°C for 30 minutes. Example 3 underwent multiple stress acclimatization at a maximum of 55°C. The average survival rate after heat treatment at 65°C for 30 minutes, which exceeded the acclimatization temperature threshold, was 38.14%. Example 1 underwent multiple stress system acclimatization at a maximum of 65°C, with an average survival rate of 62.3%. Example 1 showed the largest area coverage in the polygonal region and the highest values ​​in multiple test indicators. Temperature and pressure gradient acclimatization showed the characteristic of stimulating cross-protective responses within microorganisms.

[0105] For example, Example 3, without targeted treatment at extremely high concentrations such as pH 2.5 and 6% sodium chloride, still exhibited cross-protection advantages, achieving average survival rates of 61.45% and 59.73% in these two tests, respectively. Example 1, which underwent a higher level of targeted combined extreme acclimatization, showed the strongest resistance advantage, achieving higher average survival rates of 75.6% and 72.8% in 2 hours of pH 2.5 treatment and 4 hours of 6% sodium chloride treatment, respectively. In 2 hours of combined stress treatment with multiple extreme conditions, Example 1 maintained an average survival rate of 62.8%. The test data reflect that gradient combined acclimatization operation promotes the construction of a multi-dimensional stress resistance structure in the fermentation community, providing the microbial community with a physiological barrier basis to resist the effects of industrial pasteurization terminal processing technology and the extreme environment of the human gastrointestinal tract.

[0106] Test Example 4: Seventy 8-week-old male C57BL / 6 mice of specific pathogen-free grade were selected and acclimatized for one week in a standard animal room with constant temperature and humidity. The 70 male C57BL / 6 mice were randomly assigned to 7 experimental groups, each containing 10 mice. The 7 experimental groups were defined as control group, HFD model group, comparative example 1 intervention group, comparative example 3 intervention group, comparative example 5 intervention group, example 1 intervention group, and example 4 intervention group.

[0107] The control group mice were fed a normal maintenance diet throughout the entire period, while the HFD model group and each intervention group mice were fed a high-fat model diet with 45% fat as the energy source. At fixed times each day, mice in each intervention group were given fluid perfusion using a sterile gavage needle. The intervention group of Comparative Example 1 was given the fermented beverage prepared in Comparative Example 1; the intervention group of Comparative Example 3 was given the fermented beverage prepared in Comparative Example 3; the intervention group of Comparative Example 5 was given the fermented beverage prepared in Comparative Example 5; the intervention group of Example 1 was given a single eryngii and oyster mushroom fermented beverage prepared in Example 1; and the intervention group of Example 4 was given a compound mushroom fermented beverage prepared in Example 4. The fluid perfusion dose for all intervention groups was uniformly set at 10 mL / kg mouse body weight. The control group and the HFD model group mice were given the same volume of sterile physiological saline per day. The feeding and fluid intervention were performed continuously for 12 weeks.

[0108] After the 12-week intervention period, all participating mice were fasted for 12 hours, with no restriction on water intake during the fasting period. Whole blood samples were collected by removing the mice's eyeballs. After standing, the samples were centrifuged at 3000 r / min for 15 min to separate and extract the supernatant serum. The mice were then sacrificed and the intact liver tissue was dissected. The surface residual blood was washed off with ice-cold physiological saline, the moisture was absorbed with filter paper, and the samples were weighed. Subsequently, the samples were flash-frozen in liquid nitrogen and transferred to a -80℃ constant temperature device for storage.

[0109] For biochemical index determination, an appropriate amount of serum sample was taken, and the concentrations of serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum total cholesterol (TC), and serum triglycerides (TG) were measured using a fully automated biochemical analyzer and matching kit. A fixed mass of liver tissue sample was taken, and the tissue was homogenized and mechanically pulverized. The supernatant was extracted, and the concentrations of total cholesterol (TC) and triglycerides (TG) in the liver were measured using an enzyme-linked immunosorbent assay (ELISA) reader and a tissue lipid assay kit.

[0110] The test results are shown in Table 4.

[0111] Table 4. Animal Experiment Data on the Comprehensive Verification of the Auxiliary Lipid-Lowering Efficacy:

[0112] According to Table 4 and appendix Figure 6The data showed that the average serum total cholesterol concentration in the control group was 2.12 mmol / L, while the average serum total cholesterol concentration in the HFD model group increased to 4.56 mmol / L, reflecting the lipid accumulation state induced by a high-fat diet.

[0113] The average liver triglyceride concentrations in the intervention groups of Comparative Example 1, Comparative Example 3, and Comparative Example 5 were 26.55 μmol / g, 25.42 μmol / g, and 27.68 μmol / g, respectively, which were close to those in the HFD model group. Comparative Example 1 did not introduce a gradient cell disruption step, resulting in a lower degree of free active ingredients. Comparative Example 3 used simultaneous fermentation, which led to free oxygen interfering with anaerobic bacterial metabolism. Comparative Example 5 used undomesticated original strains, resulting in a lower retention rate of live bacteria in the mouse digestive tract. None of the intervention groups of Comparative Example 1, Comparative Example 3, and Comparative Example 5 established a stable foundation for microecological regulation.

[0114] Example 1: The intervention group used a multi-stage cell wall disruption, stepwise fermentation and stress acclimatization process. The average serum alanine aminotransferase level decreased to 46.2 U / L and the average liver total cholesterol level decreased to 7.54 μmol / g. The macromolecular polysaccharides enriched in the finished product and the highly resistant probiotic population jointly intervened in the lipid metabolism network.

[0115] Example 4: The intervention group used a compound mushroom formula, and the average serum total cholesterol decreased to 2.65 mmol / L, and the average liver triglyceride decreased to 12.86 μmol / g. The biochemical test values ​​were closest to those of the control group. The diversified carbon sources promoted the formation of a wider range of metabolic channels in the fermentation system, proving that the overall process of this invention, combined with compound mushroom raw materials, has an intervention and regulatory effect on lipid deposition in organisms.

[0116] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to the equipment structure, process flow, step sequence, operating parameters, or working medium type in these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the core technical solutions disclosed above and their equivalent substitutions.

Claims

1. A fermented beverage containing edible fungi and probiotics, characterized in that, The fermented beverage is made from ingredients comprising the following parts by weight: Edible fungi: 10 to 15 servings; Deionized water: 100 to 200 parts; Outer enzyme assembly: 0.7 to 1.0 parts; Inner layer enzyme composition: 0.4 to 0.75 parts; Sucrose: 4 to 8 parts; Phase 1: 10 to 15 servings of oxygen-tolerant probiotics; Second stage: strictly anaerobic probiotics: 10 to 15 servings.

2. The edible fungi compound probiotic fermented beverage according to claim 1, characterized in that, The edible fungus is one or a combination of several of the following: Pleurotus eryngii, Pleurotus ostreatus, and Boletus sinensis.

3. The edible fungi compound probiotic fermented beverage according to claim 1, characterized in that, The outer enzyme assembly consists of β-glucanase, neutral protease and mannanase; The inner enzyme assembly consists of chitinase, cellulase and lysozyme.

4. The edible fungi compound probiotic fermented beverage according to claim 1, characterized in that, The first-stage aerobic probiotic group is composed of Lactobacillus rhamnosus mutant strain, Pediococcus lactis mutant strain and Lactobacillus paracasei mutant strain in equal weight ratios. The second stage of strictly anaerobic probiotics consists of a mixture of Bifidobacterium adolescentis mutant strain and Bifidobacterium infantis mutant strain in equal weight ratio; All mutant strains were obtained by screening after undergoing gradient acclimatization treatments in sequence to improve heat resistance, stomach acid resistance, bile salt resistance, osmotic pressure resistance, and oxidation resistance.

5. A method for preparing a fermented beverage containing compound probiotics from edible fungi according to any one of claims 1 to 4, characterized in that, The method includes the following steps: Edible fungi are mixed with deionized water and then crushed and pulped to obtain fungal slurry. The pH and temperature of the mushroom slurry were adjusted, and an outer layer enzyme combination was added for constant temperature stirring and enzymatic hydrolysis. Subsequently, the pH and temperature of the system were adjusted again, and an inner layer enzyme combination was added for constant temperature stirring and enzymatic hydrolysis. During the inner layer enzymatic hydrolysis, time-limited ultrasonic treatment was applied to obtain an enzymatic hydrolysis mixture. The enzymatic hydrolysate was heated to inactivate enzymes and sterilize before fermentation. After cooling, it was filtered to obtain the mushroom juice. Sucrose was added and stirred to dissolve, thus obtaining the fermentation base liquid. The first stage of aerobic probiotics was inoculated into the fermentation base liquid for constant temperature fermentation to consume dissolved oxygen. After the system became oxygen-deficient, the second stage of strictly anaerobic probiotics was inoculated and constant temperature closed fermentation continued until the fermentation was completed, and the fermented liquid was obtained. The fermented liquid is subjected to high-pressure homogenization, followed by aseptic filling and sealing to obtain a fermented beverage of edible fungi compound probiotics.

6. The preparation method of the edible fungus compound probiotic fermented beverage according to claim 5, characterized in that, The step of adding the outer layer enzyme combination for constant temperature stirring enzymatic hydrolysis includes: adjusting the pH of the mushroom slurry to 5.0 to 5.5, raising the temperature to 38 to 42°C, adding the outer layer enzyme combination, and stirring and hydrolyzing under constant temperature conditions for 1.5 to 2.0 hours. The step of adding the inner layer enzyme combination for isothermal stirring enzymatic hydrolysis includes: adjusting the pH of the system to 4.5 to 5.0 with inorganic acid, simultaneously raising the temperature to 42 to 48°C, adding the inner layer enzyme combination for isothermal stirring enzymatic hydrolysis, so that the total enzymatic hydrolysis time of the inner layer reaches 1.5 to 2.0 hours.

7. The preparation method of the edible fungus compound probiotic fermented beverage according to claim 5, characterized in that, The timed ultrasonic treatment step includes: starting an ultrasonic device with a power of 200 to 300W for ultrasonic-assisted treatment at the 20th to 40th minute after adding the inner layer enzyme combination, and turning off the ultrasonic device after 15 to 20 minutes of continuous treatment.

8. The preparation method of the edible fungus compound probiotic fermented beverage according to claim 5, characterized in that, The step of inoculating the first-stage aerobic probiotic group for constant temperature fermentation includes: inoculating the first-stage aerobic probiotic group and fermenting at a constant temperature of 35 to 40°C for 12 to 18 hours. The steps of inoculating the second-stage strictly anaerobic probiotic group and continuing constant-temperature closed fermentation include: after the fermentation environment is in an oxygen-deficient state, inoculating the second-stage strictly anaerobic probiotic group and continuing constant-temperature closed fermentation for 24 to 36 hours until the pH value of the system drops to 3.8 to 4.2, at which point the fermentation ends.

9. The preparation method of the edible fungus compound probiotic fermented beverage according to claim 5, characterized in that, The step of high-pressure homogenization of the fermented liquid includes: directly feeding the fermented liquid into a homogenizer and performing high-pressure homogenization at a temperature of 55 to 65°C and a pressure of 15 to 20 MPa.

10. The use of a fermented beverage containing edible fungi and probiotics as described in any one of claims 1 to 4 in the preparation of products that assist in lowering blood lipids or regulating lipid deposition in organisms.

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