Saccharomyces cerevisiae-fungal mycelium composite membrane based on co-culture as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a saccharomyces cerevisiae-fungal mycelium composite membrane based on co-culture as well as a preparation method and application of the saccharomyces cerevisiae-fungal mycelium composite membrane. The composite membrane is formed by mutually interweaving mucor mycelia to form a three-dimensional network structure, and saccharomyces cerevisiae cells are uniformly attached to the surfaces of the mucor mycelia and the interior of the three-dimensional network structure. The composite membrane has a large specific surface area and good mechanical properties, and is a potential excellent cell immobilization carrier. The invention also optimizes the co-culture conditions of mucor mycelium and saccharomyces cerevisiae, and solves the problem that saccharomyces cerevisiae cells cannot be efficiently loaded on active mucor mycelium membranes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing a co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane. Background Technology

[0002] In traditional fermentation industries, yeast cell immobilization technology is crucial for improving fermentation efficiency, stabilizing the fermentation process, and enabling the reuse of yeast cells. Common yeast immobilization carriers include synthetic or natural polymers such as sodium alginate and carrageenan; however, these carriers suffer from insufficient mechanical strength, affecting product quality. New yeast immobilization solutions are needed. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing a co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane, in order to solve the problem of insufficient mechanical strength of yeast immobilization carriers in the prior art.

[0004] Furthermore, during the co-culture of *Saccharomyces cerevisiae* cells and *Mucor* mycelium, the *Saccharomyces cerevisiae* cells inhibit the normal growth of *Mucor* mycelium, making it difficult to form a structurally intact and high-performance mycelial membrane loaded with yeast cells. This invention also establishes specific co-culture conditions for *Mucor* mycelium and *Saccharomyces cerevisiae*, solving the problem of *Saccharomyces cerevisiae* cells not being efficiently loaded onto active *Mucor* mycelial membranes.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane is characterized in that the composite membrane is composed of a three-dimensional network structure formed by interwoven Mucor mycelia, and Saccharomyces cerevisiae cells are uniformly attached to the surface of the Mucor mycelia and the interior of the three-dimensional network structure.

[0006] Preferably, the *Mucor* species includes at least one of *Mucor radiata* CICC40252, *Mucor radiata* CICC3118, and *Mucor radiata* CICC40701.

[0007] Preferably, the brewing yeast includes at least one of Saccharomyces cerevisiae W303A, Saccharomyces cerevisiae S288C, and Saccharomyces cerevisiae PJ16.

[0008] The preparation method of the co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane includes the following steps: Mucor was cultured until the spore concentration reached 1×10⁻⁶. 6 Cells / mL ~ 1×108 The concentration of Mucor spores was increased to 1 / mL to obtain a Mucor spore solution. The inoculated Mucor spore solution was transferred to a solid culture medium and incubated upside down at 28-30℃ for 48 hours to obtain mycelial membranes. Yeast cells were cultured to a cell concentration of 1×10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL was used to obtain a high concentration of yeast culture; Mycelial membranes were cultured in a high concentration of yeast culture to obtain composite membranes.

[0009] Preferably, the volume ratio of the Mucor spore liquid to the solid culture medium is 1:125.

[0010] Preferably, the solid culture medium has a diameter of 9-10 mm; the ratio between the mycelial membrane and the high-concentration yeast solution is 9-10 mm in diameter: 2 mL of yeast solution.

[0011] Preferably, the culture medium used to prepare Mucor spore liquid includes at least one of potato dextrose liquid medium, malt extract liquid medium, and Czapek's liquid medium.

[0012] Preferably, the solid culture medium includes at least one of potato dextrose agar solid medium, malt extract agar medium, and Czapek's medium; the culture medium used for yeast cell culture is yeast extract peptone glucose liquid medium.

[0013] Preferably, the mycelial membrane is cultured in a high concentration of yeast solution at 30-32°C for 24-28 hours.

[0014] Active Mucor mycelium membranes loaded with Saccharomyces cerevisiae were used to prepare biosensors.

[0015] The principle of this invention is as follows: (1) Natural formation of mucor mycelial membrane The fungus utilized in this invention, under suitable culture conditions such as specific temperature and nutrient substrate, exhibits continuous growth, extension, and intertwining of hyphae. Utilizing its natural hyphal entanglement ability, it spontaneously forms a mycelial membrane with a three-dimensional network structure. The formation of this structure depends on the fungus's own growth and metabolic characteristics. The mycelia are interconnected through cell wall components such as chitin and glucan, constructing a stable spatial network with a certain mechanical strength; the tensile strength can reach 9.020 ± 0.523 N / mm². 2 Nutrients such as glucose and peptone in the liquid culture medium provide energy and material basis for fungal growth, ensuring the continuous proliferation of mycelium and the formation of a complete membrane structure.

[0016] (2) Solving the problem of growth inhibition In this invention, when brewing yeast and Mucor are co-cultured, there is nutrient competition or metabolite inhibition. For example, yeast may secrete certain substances that inhibit the growth of Mucor. However, by first preparing a fungal mycelium membrane and then co-culturing it with the inoculated yeast solution, the inhibition of Mucor by yeast can be reduced, while ensuring that yeast cells fully attach to the mycelium membrane, thus achieving a balance between "normal growth of Mucor to form a membrane structure" and "highly efficient yeast loading".

[0017] (3) Loading of brewer's yeast In this invention, yeast cells attach to the mycelial membrane through bioaffinity and physical adsorption during co-culture. The three-dimensional network structure of the Mucor mycelial membrane has a large specific surface area and abundant surface sites such as polysaccharides and proteins on the mycelial surface, which have bioaffinity with the surface of Saccharomyces cerevisiae cells, such as charge attraction and hydrogen bonding. During co-culture, while the yeast cells grow and reproduce, they come into contact with the mycelial membrane through Brownian motion or active migration, and are fixed on its surface and inside the network structure through physical adsorption, forming a stable loading system.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a co-culture-based brewer's yeast-fungus mycelium composite membrane, wherein the composite membrane is composed of a three-dimensional network structure formed by interwoven mucor mycelia, and brewer's yeast cells are uniformly attached to the surface of the mucor mycelia and the interior of the three-dimensional network structure.

[0019] (2) The present invention optimizes the co-culture conditions of Mucor mycelium and Saccharomyces cerevisiae, so that Saccharomyces cerevisiae cells can be efficiently loaded onto the active Mucor mycelium membrane; the interaction between them can produce or transform products that are difficult to obtain by single pure culture, or improve production efficiency.

[0020] (3) The active mucor mycelium membrane carrier loaded with brewing yeast prepared by the present invention has excellent performance. It utilizes the natural three-dimensional network structure of mucor mycelium as a fixed platform, which has a large specific surface area and can provide sufficient attachment space for brewing yeast cells, which is conducive to increasing the loading capacity of yeast cells. At the same time, the good mechanical properties of the mycelium membrane make it less prone to breakage during fermentation, which can maintain the stability of the structure and ensure the smooth progress of the fermentation process.

[0021] (4) The preparation method of the present invention is simple to operate, requires no complicated equipment or special reagents, and can be completed through conventional microbial culture and simple separation operations, making it easy to achieve industrial production.

[0022] (5) The mycelium membrane prepared by this invention, as an emerging bio-based material, has shown great application potential in food packaging, biomedicine, environmental protection and other fields due to its excellent biodegradability, abundant raw material sources and unique physicochemical properties. Attached Figure Description

[0023] Figure 1 This is a diagram showing the growth of the mycelial membrane of *Rhizopus radiata* in Example 1 of the present invention.

[0024] Figure 2 This is a microscopic observation of the mycelial membrane of *Mucor radiata* in Embodiment 1 of the present invention.

[0025] Figure 3 The Saccharomyces cerevisiae-Rhizopus dauricum mycelium composite membrane is obtained in Example 1 of the present invention.

[0026] Figure 4 The image shown is a microscopic observation of the Saccharomyces cerevisiae-Rhizopus radiata mycelium composite membrane of Embodiment 1 of the present invention. The arrow points to the yeast cells seen on the membrane surface. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials, reagents, etc. used are all commercially available.

[0028] The strain used in this invention is *Mucor radiata* CICC40252, purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0029] PDB liquid medium represents potato dextrose liquid medium; PDA solid medium is potato dextrose agar medium; YPD liquid medium is yeast extract peptone dextrose medium.

[0030] Example 1 The preparation steps of a co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane are as follows: S1: Prepare PDB liquid culture medium, PDA solid culture medium and YPD liquid culture medium, and sterilize them.

[0031] S2: Strain activation to obtain a pure strain of *Rhizopus yazhiense* CICC40252. *Rhizopus yazhiense* CICC40252 was activated and cultured in PDB liquid medium at 28℃ on a shaker at 180 rpm until the spore concentration reached 1×10⁻⁶. 6 The number of spores per mL was used to harvest the Mucor spore liquid.

[0032] S3: Inoculate 200 μL of Mucor spore solution into PDA solid medium; add 25 mL of PDA solid medium to a disposable plate with a diameter of 10 cm. After the medium solidifies, take 200 μL of the spore solution harvested in step S2 and inoculate it into PDA solid medium. Incubate upside down in an incubator at a temperature of 28℃ for 48 h.

[0033] S4: During the cultivation process, regularly observe the growth status of *Mucor radiata* CICC 40252 mycelium and the formation of the mycelial membrane. Once the mycelial membrane is fully formed, take photos and record the observations. Harvest the mycelial membrane. Figure 1 As shown, its structure under the microscope is as follows Figure 2 As shown.

[0034] S5: Activation of the yeast strain to obtain yeast culture. Saccharomyces cerevisiae W303A was activated and cultured in YPD liquid medium at 30℃ on a shaker at 180 rpm until the cell concentration reached 1×10⁻⁶ cells / mL. 7 CFU / mL.

[0035] S6: Transfer 20 mL of the high-concentration yeast culture harvested in S5 to a sterile plate. Place the mycelial membrane harvested in S4 into the high-concentration yeast culture and incubate in a 30℃ oven for 24 hours. Harvest the composite membrane, as shown below. Figure 3 As shown.

[0036] The composite membrane was washed twice with sterile distilled water to remove yeast cells from its surface. The membrane was then stained with methylene blue; viable yeast cells were stained colorless and transparent. Their morphology was observed under an electron microscope, and the results are as follows: Figure 4 As shown, yeast cells are active. Colorless and transparent yeast cells can be observed under a 40x electron microscope objective; the arrow points to the yeast cells visible on the membrane surface.

[0037] Example 2 The preparation steps of a co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane are as follows: S1: Prepare PDB liquid culture medium, PDA solid culture medium and YPD liquid culture medium, and sterilize them.

[0038] S2: Strain activation to obtain a pure strain of *Rhizopus yazhiense* CICC40252. *Rhizopus yazhiense* CICC40252 was activated and cultured in PDB liquid medium at 28℃ on a shaker at 180 rpm until the spore concentration reached 1×10⁻⁶. 8 The number of spores per mL was used to harvest the Mucor spore liquid.

[0039] S3: Inoculate 200 μL of Mucor spore solution into PDA solid medium; add 25 mL of PDA solid medium to a disposable plate with a diameter of 10 cm. After the medium solidifies, take 200 μL of the spore solution harvested in step S2 and inoculate it into PDA solid medium. Incubate upside down in an incubator at a temperature of 30℃ for 48 h.

[0040] S4: During the cultivation process, regularly observe the growth status of the mycelium of *Rhizopus yazhiensis* CICC40252 and the formation of the mycelial membrane. Harvest the mycelial membrane when it is fully formed and has a diameter of 100 mm.

[0041] S5: Activation of the yeast strain to obtain yeast culture; Saccharomyces cerevisiae W303A was activated and cultured in YPD liquid medium at 30℃ on a shaker at 180 rpm until the cell concentration reached 1×10⁻⁶. 9 CFU / mL.

[0042] S6: Transfer 20 mL of the high-concentration yeast culture harvested in S5 to a sterile plate. Place the mycelial membrane harvested in S4 into the high-concentration yeast culture and incubate in an oven at 30℃ for 28 h to harvest the composite membrane.

[0043] Example 3 The preparation steps of a co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane are as follows: S1: Prepare PDB liquid culture medium, PDA solid culture medium and YPD liquid culture medium, and sterilize them.

[0044] S2: Strain activation to obtain a pure strain of *Rhizopus yazhiense* CICC40252. *Rhizopus yazhiense* CICC40252 was activated and cultured in PDB liquid medium at 28℃ on a shaker at 180 rpm until the spore concentration reached 1×10⁻⁶. 6 The number of spores per mL was used to harvest the Mucor spore liquid.

[0045] S3: Inoculate 200 μL of Mucor spore solution into PDA solid medium; add 25 mL of PDA solid medium to a disposable plate with a diameter of 10 cm. After the medium solidifies, take 200 μL of the spore solution harvested in step S2 and inoculate it into PDA solid medium. Incubate upside down in an incubator at a temperature of 30℃ for 48 h.

[0046] S4: During the cultivation process, regularly observe the growth status of the mycelium of *Rhizopus yazhiensis* CICC40252 and the formation of the mycelial membrane. Harvest the mycelial membrane when it is fully formed and has a diameter of 90 mm.

[0047] S5: Activation of the yeast strain to obtain yeast culture; Saccharomyces cerevisiae W303A was activated and cultured in YPD liquid medium at 30℃ on a shaker at 180 rpm until the cell concentration reached 1×10⁻⁶. 7 CFU / mL.

[0048] S6: Transfer 20 mL of the high-concentration yeast culture harvested in S5 to a sterile plate. Place the mycelial membrane harvested in S4 into the high-concentration yeast culture and incubate in an oven at 32℃ for 24 h to harvest the composite membrane.

[0049] To test the properties of the composite membrane, the following experiments were conducted: Experiment 1: Tensile strength test of composite membranes co-cultured with *Rhizopus radiata* CICC40252 and *Saccharomyces cerevisiae* W303A The tensile strength of the composite film prepared in Example 1 was measured by an instrument, and the results are shown in Table 1 below.

[0050] Table 1 Mechanical Properties of Composite Membranes Experiment 2: Self-repair test of composite membrane formed by co-culture of *Rhizopus radiata* CICC 40252 and *Saccharomyces cerevisiae* W303A In Example 1, the composite membrane prepared by S6 was taken out, a slit was made in the middle, and then it was placed in a high concentration of yeast solution. After 72 hours, it was taken out and observed, and the slit was found to have disappeared.

[0051] Application Example 1: A sensor based on a co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane.

[0052] Based on Example 1, through genetic modification, a plasmid containing an ERE estrogen-sensing element and the yEGFP gene was inserted into wild-type yeast, transforming the yeast into yeast that can emit green fluorescence after sensing external estrogen. The composite membrane was prepared according to the method of Example 1. The harvested composite membrane emitted green fluorescence under 488nm excitation light after sensing estrogen.

[0053] Comparative Example 1 The preparation steps of a co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane are as follows: S1: Prepare PDB liquid culture medium, PDA solid culture medium and YPD liquid culture medium, and sterilize them.

[0054] S2: Strain activation to obtain a pure strain of *Rhizopus yazhiense* CICC40252. *Rhizopus yazhiense* CICC40252 was activated and cultured in PDB liquid medium at 28℃ on a shaker at 180 rpm until the spore concentration reached 1×10⁻⁶. 6The number of spores per mL was used to harvest the Mucor spore liquid.

[0055] S3: Inoculate 200 μL of Mucor spore solution into PDA solid medium; add 25 mL of PDA solid medium to a disposable plate with a diameter of 10 cm. After the medium solidifies, take 200 μL of the spore solution harvested in step S2 and inoculate it into PDA solid medium. Incubate upside down in an incubator at a temperature of 30℃ for 48 h.

[0056] S4: During the cultivation process, regularly observe the growth status of the mycelium of *Rhizopus yazhiensis* CICC40252 and the formation of the mycelial membrane. Once the mycelial membrane is fully formed, take photos and record the data, and harvest the mycelial membrane.

[0057] S5: Activation of the yeast strain to obtain yeast culture. Saccharomyces cerevisiae W303A was activated and cultured in YPD liquid medium at 30℃ on a shaker at 180 rpm until the cell concentration reached 1×10⁻⁶ cells / mL. 7 CFU / mL.

[0058] S6: Inoculate 2 mL of the high-concentration yeast culture harvested in S5 into 100 mL of PDB liquid medium. Place the mycelial membrane harvested in S4 into a medium inoculated with Saccharomyces cerevisiae and culture it in a shaker at 30℃ and 180 rpm for 24 h. Harvest the composite membrane.

[0059] The membrane obtained by this method is relatively thick, and the yeast loading rate of the composite membrane is low. The yeast is embedded inside the mycelium membrane and cannot be observed by electron microscopy. The mycelium of *Rhizopus yazhiensis* CICC40252 inhibits the growth of yeast.

[0060] Comparative Example 2 S1: Prepare PDB liquid culture medium, PDA solid culture medium and YPD liquid culture medium, and sterilize them.

[0061] S2: Strain activation to obtain a pure strain of *Rhizopus yazhiense* CICC40252. *Rhizopus yazhiense* CICC40252 was activated and cultured in PDB liquid medium at 28℃ on a shaker at 180 rpm until the spore concentration reached 1×10⁻⁶. 6 The number of spores per mL was used to harvest the Mucor spore liquid.

[0062] S3: Activation of the yeast strain to obtain yeast culture. Saccharomyces cerevisiae W303A was activated and cultured in YPD liquid medium at 30℃ in a shaker at 180 rpm until the cell concentration reached 1×10⁻⁶ cells / mL. 7 CFU / mL.

[0063] S4: The Rhizopus spore liquid harvested in S2 and the high-concentration yeast liquid harvested in S3 were inoculated into 100 mL of PDB liquid medium at different ratios, with the yeast liquid accounting for 10%, 20%, 40%, and 80% of the total inoculation ratio, and cultured for 96 h in a shaker at 30 °C and 180 rpm.

[0064] S5: Filter the mixed mycelium cultured in S4 using a vacuum filtration flask to remove the culture medium, harvest the yeast-loaded mycelium, wash it three times with sterile distilled water to remove unloaded yeast cells.

[0065] S6: Place the S5 mycelium into the mold and adjust its shape to form a composite film.

[0066] The composite film harvested by this method suffers from yeast aging due to the long cultivation time, and the yeast inhibits the growth of Mucor mycelium. Even when the yeast concentration accounts for 10% of the inoculum concentration, the growth of mycelium is severely inhibited, making it impossible to harvest the composite film.

[0067] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane, characterized in that, The composite membrane is formed by interwoven Mucor mycelia to create a three-dimensional network structure, with Saccharomyces cerevisiae cells uniformly attached to the surface of the Mucor mycelia and inside the three-dimensional network structure.

2. The composite membrane according to claim 1, characterized in that, The *Mucor* species include at least one of *Mucor radiata* CICC40252, *Mucor radiata* CICC3118, and *Mucor radiata* CICC40701.

3. The composite membrane according to claim 1, characterized in that, The brewing yeast includes brewing yeast W303A ( Saccharomyces cerevisiae ), Saccharomyces cerevisiae S288C ( Saccharomyces cerevisiae ), brewer's yeast PJ16 ( Saccharomyces cerevisiae At least one of the following.

4. A method for preparing a co-cultured Saccharomyces cerevisiae-fungus mycelium composite membrane as described in any one of claims 1 to 3, characterized in that, Includes the following steps: Mucor was cultured until the spore concentration reached 1×10⁻⁶. 6 Cells / mL ~ 1×10 8 The concentration of Mucor spores was increased to 1 / mL to obtain a Mucor spore solution. The inoculated Mucor spore solution was transferred to a solid culture medium and incubated upside down at 28-30℃ for 48 hours to obtain mycelial membranes. Yeast cells were cultured to a cell concentration of 1×10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL was used to obtain a high concentration of yeast culture; Mycelial membranes were cultured in a high concentration of yeast culture to obtain composite membranes.

5. The method according to claim 4, characterized in that, The volume ratio of the Mucor spore liquid to the solid culture medium is 1:

125.

6. The method according to claim 4, characterized in that, The solid culture medium has a diameter of 9-10 mm; the ratio between the mycelial membrane and the high-concentration yeast solution is 9-10 mm in diameter: 2 mL of yeast solution.

7. The method according to claim 4, characterized in that, The culture medium used to prepare Mucor spore liquid includes at least one of potato dextrose liquid medium, malt extract liquid medium, and Czapek's liquid medium.

8. The method according to claim 4, characterized in that, The solid culture medium includes at least one of potato dextrose agar solid medium, malt extract agar medium, and Czapek's medium; the culture medium used for yeast cell culture is yeast extract peptone glucose liquid medium.

9. The method according to claim 4, characterized in that, The mycelial membrane was cultured in a high-concentration yeast solution at 30-32°C for 24-28 hours.

10. The application of the active *Mucor* mycelium membrane loaded with *Saccharomyces cerevisiae* as described in any one of claims 1 to 3, characterized in that, Active Mucor mycelium membranes loaded with Saccharomyces cerevisiae were used to prepare biosensors.

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