Edible aerobic strain preservation method

Abstract

The invention belongs to the technical field of edible fungus preservation and cultivation, and particularly relates to an edible aerobic strain preservation method which comprises the following steps: S1, placing target hyphae in a solid culture medium for cultivation, then selecting the target hyphae with good growth vigor, and transferring the selected target hyphae to a culture medium slope of a sterile and nutrient-poor culture container for cultivation, stopping culturing when hyphae grow to 4 / 5 of the length of the inclined plane; s2, a covering solution is prepared, the covering solution comprises glycerin and paraffin oil, and the sterile preservation covering solution is applied to the culture container and completely submerges the slant of the culture medium; wherein the mass percentage of the glycerol is 60%-70%, and the mass percentage of the paraffin oil is 30%-40%; and S3, sealing the cultivation container by using a sealing element with controllable air permeability, and preserving in a dark place at normal temperature. Compared with the prior art, the technical problem that aerobic edible mushrooms cannot be well preserved by a paraffin oil method in the prior art is solved by setting the proportion of the glycerol to the paraffin oil.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of edible fungus preservation and cultivation, and particularly relates to a preservation method for edible aerobic fungus spores. BACKGROUND

[0002] Edible fungus spores are the cornerstone of the development of the edible fungus industry, and the stable maintenance of their excellent properties is directly related to the yield, quality and economic benefits of cultivation. The purpose of spore preservation is to maximize the reduction of metabolic activity of the spores by creating a specific environment, so that the spores are in a dormant state, thereby maintaining their activity and genetic stability for a long time, and avoiding the degradation of the spores caused by frequent transfer or improper preservation, such as reduced yield and weakened stress resistance.

[0003] At present, there are various preservation methods for edible fungus spores, each with its own scope of application and inherent drawbacks. The conventional low-temperature preservation method usually places high-temperature type spores in a 16℃ environment and low-temperature type spores in a 4℃ environment, which can effectively slow down the metabolism of the mycelium, but it is highly dependent on the continuous and stable operation of refrigeration equipment. Once there is a power interruption or equipment failure, the entire batch of spores is likely to be inactivated, which is high in application cost and risk for small and medium-sized enterprises with relatively weak infrastructure. Although the liquid nitrogen ultra-low temperature preservation method can achieve long-term (several decades) stable preservation of spores and is considered a standard long-term preservation method, it requires expensive equipment such as liquid nitrogen tanks and programmable cooling instruments, and the daily maintenance cost is high. The technical operation requirements are strict, and it is difficult to popularize in the majority of production enterprises. In addition, the periodic subculture preservation method used by some enterprises is not only labor-intensive, but more importantly, the risk of genetic mutation accumulation increases significantly in the repeated cycle of nutritional activation and starvation, which is one of the important reasons for the degradation of spores.

[0004] In comparison, mineral oil covering preservation method (such as paraffin oil method) as a classic preservation technology, shows unique application potential due to its no need of special equipment, low cost and preservation at room temperature. This method effectively isolates air and prevents evaporation of water in the culture medium by covering sterile paraffin oil on the spore slope, thereby delaying the aging of mycelium. However, the traditional paraffin oil preservation method also has obvious defects in practice: on the one hand, the paraffin oil may have potential toxicity to mycelium cells during long-term preservation, affecting the later recovery activity; on the other hand, and more importantly, its nearly complete sealed environment can easily lead to "asphyxia" of mycelium cells during preservation due to long-term lack of oxygen or extremely low oxygen, and excessive water loss leads to abnormal cell metabolic pathways, accumulation of reactive oxygen species, causing irreversible damage, ultimately resulting in difficulty in activating spores after preservation, slow recovery, weak growth, and even direct death, which seriously affects the preservation effect and application value. SUMMARY

[0005] The present invention aims to provide a method for preserving edible aerobic bacteria, mainly to solve the technical problem that the paraffin oil method in the prior art cannot preserve aerobic edible fungi well.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for preserving edible aerobic bacteria includes the following steps: S1: Place the target hyphae in a solid culture medium for cultivation, and then select the target hyphae with good growth and transfer them to a culture slant in a sterile, nutrient-poor culture container for cultivation. Stop cultivation when the hyphae grow to 4 / 5 of the length of the slant. S2: Prepare a covering solution containing glycerol and paraffin oil. Apply the sterile preservation covering solution to the culture vessel and completely submerge the culture medium slant. The glycerol comprises 60%–70% by mass, and the paraffin oil comprises 30%–40% by mass. S3: Seal the culture container with a sealant that has controllable air permeability and store it at room temperature away from light.

[0007] The core breakthrough of this invention lies in the specially formulated covering solution: the proportion of glycerol is increased to 60%-70%, and the paraffin oil is adjusted accordingly to 30%-40%. The higher proportion of glycerol not only enhances the system's moisture retention, but its molecular properties also significantly improve the permeability of the covering solution, allowing oxygen to penetrate at an extremely slow rate, maintaining the minimum necessary life activities for dormant mycelia. Simultaneously, this method abandons the traditional completely sealed approach, introducing sealing components with controllable permeability, such as silicone breathable plugs or hydrophobic breathable membranes. This design, in synergy with the improved covering solution, together constructs a stable "micro-aerobic" environment, creating a haven for the mycelia to safely hibernate.

[0008] Preferably, the sealing element is a silicone breathable plug or a test tube stopper with a built-in hydrophobic breathable membrane. This solution provides a specific, reliable, and easy-to-implement sealing method that ensures oxygen can permeate at a slow and controllable rate, while effectively preventing moisture evaporation and microbial contamination of the culture medium.

[0009] Preferably, the covering liquid also contains sterile porous material particles, which are microporous silica gel or molecular sieves. In this design, the porous material particles form microscopic oxygen channels in the covering liquid, enabling more precise control of oxygen supply. This is suitable for aerobic bacteria that are more sensitive to oxygen demand, further enhancing the oxygen exchange capacity of the covering liquid layer.

[0010] Preferably, the added mass of the porous material particles is 1.5% to 2% of the total mass of the covering liquid.

[0011] Preferably, before applying the preservation covering solution, the solid culture medium containing aerobic bacterial hyphae is subjected to stress-induction treatment, which includes adding 0.01% to 0.05% sodium chloride by weight to the pre-culture medium. In this method, stress-induction treatment can enhance the inherent ability of aerobic bacterial hyphae to resist stresses such as hypoxia and nutrient deficiency during storage. Through mild stress pretreatment, the stress protection mechanism of the hyphae is stimulated, thereby maintaining higher cell viability during storage.

[0012] Preferably, the covering solution also contains 0.001% to 0.005% vitamin E by mass. In this solution, vitamin E can alleviate the oxidative damage that aerobic bacteria may experience under low-oxygen conditions; the addition of trace amounts of safe antioxidants helps to remove reactive oxygen species accumulated in mycelial cells, protect the integrity of cell membranes and biomolecules, and extend the effective shelf life.

[0013] Preferably, the preparation method of the covering solution is as follows: glycerol, paraffin oil, and vitamin E are stirred and mixed at 80℃~90℃ for 20~30 minutes. After standing until the covering solution cools to room temperature, porous material particles are added and stirring is continued for 10~15 minutes. Before use, the solution is ultrasonically treated for 5~8 minutes and then immediately poured into a culture container and the culture container is sealed. In this scheme, mixing glycerol, paraffin oil, and vitamin E at high temperature can improve the mixing degree of each component. Ultrasonic treatment before use can reduce the air introduced by the porous material during the mixing process, thus playing a defoaming role and controlling (reducing) the oxygen content in the solution, avoiding excessive oxygen content in the covering solution from affecting the preservation effect.

[0014] A preservation device for preserving edible aerobic bacteria includes a tubular culture container with a cover device at the opening of the culture container that allows for freely adjustable air permeability. The air permeability device includes an air permeability window and a control device for controlling the air permeability of the air permeability window.

[0015] Preferably, the venting device includes a tube cap covering the opening of the culture container, and the venting window includes a perforated opening at the top of the tube cap, the perforated opening being covered with a hydrophobic and breathable membrane.

[0016] A covering solution for preserving edible aerobic bacteria comprises the following components by weight percentage: 60%–70% glycerol, 30%–40% paraffin oil, and 0.001%–0.005% vitamin E. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0018] Figure 1 This is a three-dimensional structural diagram of Scheme 1 of a preservation device for preserving edible aerobic bacteria according to the present invention patent; Figure 2 This is a three-dimensional structural diagram of Scheme 1 of a preservation device for preserving edible aerobic bacteria according to the present invention patent; Figure 3 This is an exploded view of the structure of Scheme 1 of the preservation device for preserving edible aerobic bacteria according to the present invention patent; Figure 4 This is a three-dimensional structural diagram of Scheme 2 of the preservation device for preserving edible aerobic bacteria according to the present invention patent; Figure 5 This is an exploded view of Scheme 2 of a preservation device for preserving edible aerobic bacteria according to the present invention patent; Figure 6 This invention patent provides a second embodiment of a preservation device for preserving edible aerobic bacteria strains. Figure 5 Enlarged view of point A; Figure 7 This is an exploded view of Scheme 2 of a preservation device for preserving edible aerobic bacteria according to the present invention patent; Figure 8 This is a top view of Scheme 2 of the preservation device for preserving edible aerobic bacteria according to the present invention patent; Figure 9 This invention patent provides a second embodiment of a preservation device for preserving edible aerobic bacteria strains. Figure 8 BB cross-section; Figure 10 This invention patent provides a second embodiment of a preservation device for preserving edible aerobic bacteria strains. Figure 9 Enlarged view of point C.

[0019] The reference numerals in the accompanying drawings include: culture container 1, tube cap 21, hydrophobic and breathable membrane 22, fixing ring 23, mounting port 24, shielding plate 25, slot 26, locking block 27, first baffle 31, second baffle 32, and rotating shaft 33. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0021] In the description of this invention, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. Where the terms "first," "second," and "third" are used for descriptive purposes and to distinguish technical features, they should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the sequential relationship of the indicated technical features.

[0022] The embodiments of the present invention will now be described.

[0023] Example 1: A method for preserving edible aerobic bacteria, including S1: Place the target hyphae in a solid culture medium for cultivation, and then select the target hyphae with good growth and transfer them to a culture slant in a sterile, nutrient-poor culture container for cultivation. Stop cultivation when the hyphae grow to 4 / 5 of the length of the slant. S2: Prepare a covering solution containing glycerol and paraffin oil. Apply the sterile preservation covering solution to the culture vessel and completely submerge the culture medium slant. The glycerol comprises 60% by mass and the paraffin oil comprises 40% by mass. S3: Seal the culture container with a sealant that has controllable air permeability and store it at room temperature away from light.

[0024] Preferably, the sealing element is a test tube stopper with a built-in hydrophobic and breathable membrane, which ensures that oxygen can permeate at a slow and controllable rate, while effectively preventing the evaporation of moisture from the culture medium and contamination by other microorganisms.

[0025] Preferably, the covering liquid further comprises sterile porous material particles dispersed therein, the added mass of which is 1.5% of the total mass of the covering liquid. The porous material particles are microporous silica gel or molecular sieves. The porous material particles form microscopic oxygen channels in the covering liquid, which can more precisely control the oxygen supply. This is suitable for aerobic bacteria that are more sensitive to oxygen demand, and further enhances the oxygen exchange capacity of the covering liquid layer.

[0026] Preferably, before applying the preservation covering solution, the solid culture medium containing aerobic bacterial hyphae is subjected to stress-induction treatment, which includes adding 0.01% sodium chloride by weight to the pre-culture medium. Stress-induction treatment enhances the inherent ability of aerobic bacterial hyphae to resist stresses such as hypoxia and nutrient deficiency during storage. Through mild stress pretreatment, the stress protection mechanism of the hyphae is activated, thereby maintaining higher cell viability during storage.

[0027] Preferably, the covering solution also contains 0.001% vitamin E by mass. Vitamin E can alleviate oxidative damage that may occur to aerobic bacteria under low-oxygen conditions; the addition of trace amounts of safe antioxidants helps to remove reactive oxygen species accumulated in mycelial cells, protect the integrity of cell membranes and biomolecules, and extend the effective shelf life.

[0028] Preferably, the preparation method of the covering solution is as follows: Glycerin, paraffin oil, and vitamin E are stirred and mixed at 80°C for 20 minutes. After standing until the covering solution cools to room temperature, porous material particles are added and stirring is continued for 10 minutes. Before use, the solution is ultrasonically treated for 5 minutes and then immediately poured into a culture container, which is then sealed. Mixing glycerin, paraffin oil, and vitamin E at high temperature can improve the mixing degree of each component. Ultrasonic treatment before use can reduce the air introduced by the porous material during the mixing process, thus playing a defoaming role and controlling (reducing) the oxygen content in the solution, avoiding excessive oxygen content in the covering solution from affecting the preservation effect.

[0029] Preferably, the overall structure of the culture container is as follows, with two structural examples: Option 1: This embodiment discloses a preservation device for preserving edible aerobic bacteria. Its core lies in the precise control of the oxygen content in the culture container 1 through an adjustable air permeability cover device, thereby providing a suitable growth and preservation environment for edible aerobic bacteria.

[0030] The preservation device includes a tubular culture container 1, which is used to contain edible aerobic bacteria and the corresponding culture medium, providing basic space for the growth and preservation of aerobic bacteria. A lid is installed at the opening of the culture container 1 to seal the opening of the culture container 1, while the air permeability can be freely adjusted. By controlling the air permeability, the amount of gas exchange between the inside and outside of the culture container 1 can be controlled, thereby regulating the oxygen content inside the container.

[0031] The covering device includes a tube cap 21, which is placed over the opening of the culture container 1 to achieve a basic seal of the opening. A rubber sealing layer is fixed to the inner top and inner side of the tube cap 21. This rubber sealing layer fits tightly against the end face of the opening of the culture container 1, enhancing the sealing performance between the tube cap 21 and the culture container 1. This prevents irregular gas leakage from the gap between the tube cap 21 and the culture container 1, ensuring that gas exchange between the inside and outside of the culture container 1 is precisely regulated only through subsequent ventilation windows.

[0032] The cap 21 is provided with a venting window, which includes a perforation at the top of the cap 21 that extends through the top end face of the cap 21, providing a channel for gas exchange. A hydrophobic and breathable membrane 22 covers the perforation, completely covering the opening area and tightly fixed to the edge of the perforation. This allows air and other gases to pass through, facilitating gas exchange between the inside and outside of the culture container 1, while effectively preventing moisture from the external environment from entering the culture container 1, preventing abnormal changes in the humidity of the culture medium, and ensuring the stability of the aerobic bacteria growth environment. A certain distance is maintained between the edge of the perforation and the inner surface of the cap 21. This distance is greater than the wall thickness at the opening of the culture container 1. When the cap 21 is placed over the opening of the culture container 1, the edge of the opening is located within the space formed by this distance, preventing direct contact with the surface of the hydrophobic and breathable membrane 22 and thus preventing deformation of the membrane at the contact point from reducing the sealing effect, ensuring the breathability stability and sealing reliability of the hydrophobic and breathable membrane 22.

[0033] The covering device also includes a control device for controlling the air permeability of the ventilation window. The control device is installed at the perforation at the top of the cover 21 and is used to adjust the effective ventilation area of ​​the perforation, thereby controlling the air permeability. The control device includes a fixing ring 23, which is fixedly installed on the outer periphery of the perforation at the top of the cover 21. Its inner wall is fixedly connected to the arc-shaped edge of the first baffle 31, providing fixed support for the first baffle 31. The upper and lower surfaces of the first baffle 31 are both semi-circular. A connecting plate is fixed at the center of the first baffle 31. The connecting plate is integrally formed with the center of the top surface of the first baffle 31, forming an extension of the first baffle 31. A rotating shaft 33 is vertically fixed at the center of the connecting plate. The rotating shaft 33 is perpendicular to the first baffle 31 and provides rotational support for the rotation of the second baffle 32.

[0034] The control device also includes a second baffle 32. The upper and lower surfaces of the second baffle 32 are also semi-circular, and their dimensions are exactly the same as those of the first baffle 31. A connecting plate is also fixed at the center of the second baffle 32. A through hole adapted to the outer diameter of the rotating shaft 33 is opened on the connecting plate. The second baffle 32 is rotatably sleeved on the rotating shaft 33 through the through hole, so that the second baffle 32 and the first baffle 31 are coaxially arranged and can rotate relative to the first baffle 31 around the rotating shaft 33. The end of the rotating shaft 33 is thickened to form a limiting structure to prevent the second baffle 32 from falling out. The first baffle 31 and the second baffle 32 are adjusted in relative position through the cooperation of the rotating shaft 33. When the two are completely offset, they can cover the entire opening. By rotating the second baffle 32 to change its overlapping area with the first baffle 31, the area of ​​the opening can be precisely adjusted, thereby realizing the free selection and precise control of the ventilation window's air permeability. This meets the differentiated oxygen content requirements of edible aerobic bacteria at different growth stages and ensures that the aerobic bacteria are always in a suitable gaseous environment for growth and preservation.

[0035] Option 2: Unlike Example 1, a tubular culture container 1 is first set up. This culture container 1 serves as a carrier for edible aerobic bacteria. Its material is preferably transparent glass or food-grade plastic. The transparent material makes it easy for staff to observe the growth and preservation status of the bacteria inside. The tubular structure can provide a stable growth space for the bacteria, and its open end is used for the subsequent installation of the covering device to achieve the control of the internal environment of the container.

[0036] A cap 21 is placed over the opening of the culture container 1. The cap 21, as the main structure of the covering device, is shaped to match the opening of the culture container 1, typically being a circular cap. The inner diameter of the cap 21 is slightly larger than the outer diameter of the opening of the culture container 1 to ensure that the cap 21 can be stably fitted over the opening of the culture container 1, achieving initial sealing. A rubber sealing layer is fixed to the inner wall and top of the cap 21. This rubber sealing layer is made of highly elastic food-grade rubber. When the cap 21 is placed over the opening of the culture container 1, the rubber sealing layer will fit tightly against the upper surface of the opening of the culture container 1. The elastic deformation of the rubber itself fills any tiny gaps that may exist between the cap 21 and the opening of the culture container 1, thereby significantly enhancing the sealing of the entire device. This prevents bacteria, dust, or moisture from the outside air from entering the interior of the culture container 1, and also avoids excessive gas leakage from the internal culture environment, creating a clean and stable preservation environment for aerobic bacteria.

[0037] A perforated opening is provided at the top of the cap 21, forming a ventilation window for the device. The shape of the perforation can be designed as circular, square, or other regular shapes according to actual needs. In this embodiment, a circular shape is preferred. The diameter of the perforation needs to be determined based on the volume of the culture container 1 and the oxygen requirement of the aerobic bacteria to ensure sufficient gas flow area. A hydrophobic and breathable membrane 22 is covered inside the perforation. The hydrophobic and breathable membrane 22 is made of polytetrafluoroethylene or other polymer materials with hydrophobic and breathable properties. It is fixed to the edge of the perforation by bonding or hot pressing to ensure a tight connection between the membrane and the perforation, with no gaps for gas leakage. The function of the hydrophobic and breathable membrane 22 is to allow oxygen from the outside air to enter the culture container 1 through the membrane, providing the oxygen required for the aerobic bacteria to breathe. At the same time, its hydrophobic properties can prevent moisture in the external environment (such as moisture in the air, accidentally spilled liquid, etc.) from entering the container through the membrane, avoiding abnormally high humidity inside the container and affecting the preservation quality of the bacteria.

[0038] It is important to note that a certain distance is maintained between the edge of the perforation and the inner surface of the cap 21, and this distance is greater than the wall thickness at the opening of the culture container 1. This design ensures that when the cap 21 is placed over the opening of the culture container 1, the edge of the opening is precisely within the distance between the inner surface of the cap 21 and the edge of the perforation, without directly contacting the surface of the hydrophobic and breathable membrane 22 fixed inside the perforation. Because the hydrophobic and breathable membrane 22 is relatively soft, if the edge of the opening of the culture container 1 directly touches the membrane, a flexible contact will occur. This contact method may not only reduce the sealing effect at the contact point (due to the potential for new gaps caused by membrane deformation), but may also cause compression damage to the hydrophobic and breathable membrane 22, affecting its air permeability and hydrophobic properties. By setting the aforementioned distance, this problem is effectively avoided, ensuring the normal function of the hydrophobic and breathable membrane 22 and the sealing reliability of the device.

[0039] A retaining ring 23 is also fixed to the top of the tube cap 21. The retaining ring 23 is coaxially arranged with the tube cap 21, that is, the central axis of the retaining ring 23 coincides with the central axis of the tube cap 21. This arrangement ensures that the movement trajectory of the subsequent control components corresponds to the center of the perforation and the hydrophobic and breathable membrane 22, thus ensuring the uniformity of air permeability control. The retaining ring 23 is made of a material with elastic deformation capability, such as elastic plastic or rubber. It is fixed to the top of the tube cap 21 by bonding or integral molding. The position of the retaining ring 23 corresponds to the top of the perforation, and its inner diameter is slightly larger than the diameter of the perforation, so as to completely cover the space above the perforation and the hydrophobic and breathable membrane 22, providing a range for the installation and movement of the subsequent shielding components.

[0040] The circumferential surface of the fixing ring 23 is provided with an installation opening 24. The installation opening 24 is opened along the circumference of the fixing ring 23, and its length covers 1 / 2 of the circumferential surface of the fixing ring 23. The width of the installation opening 24 is determined according to the thickness of the subsequently inserted shielding piece 25 to ensure that the shielding piece 25 can be inserted smoothly and will not cause excessive shaking after insertion.

[0041] The control device also includes a long strip-shaped shield 25. The length of the shield 25 is greater than the diameter of the fixing ring 23, and its width is adapted to the inner diameter of the fixing ring 23 to ensure that the shield 25 can fully or partially cover the space above the hydrophobic and breathable membrane 22 after being inserted into the fixing ring 23. The shield 25 can be made of rigid plastic or thin metal sheet to ensure that it has a certain structural strength and is not easily deformed during insertion and adjustment. Several slots 26 are arranged in an array along the length of the upper surface of the shield 25. The cross-sectional shape of the slots 26 is triangular or trapezoidal. The spacing between adjacent slots 26 is set according to the required air permeability adjustment level of the device. The smaller the spacing, the higher the adjustment accuracy of the air permeability.

[0042] A locking block 27 is fixed to the upper surface of the mounting port 24. The shape of the locking block 27 matches the shape of the slot 26 (either a shape consistent with the slot 26 or a cylindrical shape; the advantage of a cylindrical shape is that the smooth surface of the locking block 27 reduces the friction of the contact surface during the movement of the shielding plate 25, making it easier to push the shielding plate 25). The locking block 27 is fixed to the upper surface of the mounting port 24 by welding or bonding. When it is necessary to adjust the air permeability of the culture container 1, the shielding plate 25 is inserted into the mounting port 24 of the fixing ring 23. Since the fixing ring 23 has elastic deformation capability, the fixing ring 23 at the mounting port 24 will undergo slight elastic deformation during the insertion of the shielding plate 25, thereby providing sufficient space for the movement of the shielding plate 25. Push the shielding plate 25 along the mounting opening 24 into the fixing ring 23. At this time, the locking block 27 will slide on the upper surface of the shielding plate 25. When the shielding plate 25 moves to a suitable position (that is, the area of ​​the hydrophobic and breathable membrane 22 exposed to the air reaches the area corresponding to the required air permeability), stop pushing the shielding plate 25. The elastic deformation of the fixing ring 23 will be restored, and the locking block 27 will be embedded in the corresponding slot 26 on the shielding plate 25. Through the cooperation of the locking block 27 and the slot 26, the movement of the shielding plate 25 in the fixing ring 23 is restricted, so that the shielding plate 25 is kept in the current position, thereby fixing the exposed area of ​​the hydrophobic and breathable membrane 22. This achieves the purpose of controlling the airflow between the inside and outside of the culture container 1, providing a suitable concentration of oxygen for the aerobic bacteria in the tube, and meeting the oxygen requirements of different types of aerobic bacteria during the preservation process.

[0043] Meanwhile, since the shielding plate 25 is designed to be elongated and its length is greater than the diameter of the fixing ring 23, when the shielding plate 25 is inserted into the fixing ring 23, one end of it will fit against the inner surface of the fixing ring 23. This structural design can effectively prevent the shielding plate 25 from being completely pushed into the fixing ring 23 and then coming out from the top of the fixing ring 23 during the adjustment process, ensuring that the shielding plate 25 is always stably installed in the fixing ring 23, thus ensuring the reliability and practicality of the entire control device.

[0044] Example 2: Unlike Example 1, the glycerin content is 65% by mass and the paraffin oil content is 35% by mass. The mass of porous material particles added is 1.8% of the total mass of the covering liquid.

[0045] The covering solution contains 0.003% vitamin E by mass.

[0046] Example 3: Unlike Example 1, the mass percentage of glycerin is 70% and the mass percentage of paraffin oil is 30%. The mass of porous material particles added is 2% of the total mass of the covering liquid.

[0047] The covering solution contains 0.005% vitamin E by mass.

[0048] Comparative Example 1: Unlike Example 1, the mass percentage of glycerin is 50% and the mass percentage of paraffin oil is 50%. Comparative Example 2: Unlike Example 1, the mass percentage of glycerin is 40% and the mass percentage of paraffin oil is 60%. Comparative Example 3: Unlike Example 1, no porous material particles were added to the covering liquid.

[0049] Comparative Example 4: Unlike Example 1, the seal is not a seal with controllable permeability, but an airtight, corrosion-resistant rubber plug.

[0050] Effect Experiment: 1. Experimental Materials and Methods 1.1 Test strains To ensure the representativeness and comparability of the experiments, Lentinula edodes was selected as the test strain: all experiments used robust mycelia that had been activated and grown to the late logarithmic phase.

[0051] 1.2 Culture medium Activation and propagation medium: PDA (potato dextrose agar) medium.

[0052] Culture medium before preservation: To simulate the "nutrient-poor" conditions in the patent, diluted PDA culture medium (1 / 5 the composition of standard PDA) was used.

[0053] 1.3 Experimental Grouping The experiment consisted of 7 treatment groups, as shown in the table below. Each treatment group had 3 replicates.

[0054] 1.4 Preparation and Preservation Process of Preservation Covering Solution Preparation of the covering solution: Prepare strictly according to the method described in the patent. Taking Example 1 as an example: Accurately weigh 60g glycerin, 40g paraffin oil, and 0.001g vitamin E, place them in an Erlenmeyer flask, and magnetically stir (300 r / min) for 20 minutes in an 80℃ water bath. After cooling to room temperature, add 1.5g sterile microporous silica gel particles (particle size 100-200 mesh), and continue stirring for 10 minutes. Before use, ultrasonically treat with an ultrasonic cleaner (100W power) for 5 minutes, and use immediately.

[0055] Mycelial culture and treatment: Inoculate the mycelia into diluted PDA slant medium containing 0.01% sodium chloride and culture at 25°C until the mycelia grow to 4 / 5 of the slant.

[0056] Preservation procedure: Aseptically pour the prepared covering solution into the culture container, completely immersing the mycelial slant. Cover with the corresponding seal and store at room temperature (25±2℃) away from light.

[0057] 1.5 Detection Indicators and Methods The preservation period is 24 months. Samples will be taken and tested at 6, 12, 18, and 24 months after preservation.

[0058] 1.5.1 Mycelial survival rate Detection method: 1. Resuscitation: Under aseptic conditions, take out a small amount of preserved mycelium (about the size of a soybean) and blot dry the surface covering liquid with sterile filter paper.

[0059] 2. Transfer: Transfer the mycelial block to the center of a fresh PDA plate.

[0060] 3. Incubation: Incubate in a constant temperature incubator at 25℃.

[0061] 4. Observation and counting: Observe and record the time it takes for the mycelium to recover and grow (i.e., "revival time") every day. After culturing for 7 consecutive days, count the number of transfer blocks that can recover and grow (new mycelium grows around the mycelial block).

[0062] Survival rate calculation: Survival rate (%) = (Number of transfer blocks that resumed growth / Total number of transfer blocks) × 100%. Each treatment group tested 10 duplicate transfer blocks per test.

[0063] 1.5.2 Colony growth rate and morphology Detection method: For the colonies that were successfully revived, the diameter of the colonies was measured using the cross-sectional method on the 3rd and 5th days of incubation.

[0064] Calculate: Colony growth rate (mm / day) = (diameter on day 5 - diameter on day 3) / 2. Simultaneously observe and record whether the colony morphology is robust and dense, and whether there are any signs of degeneration such as sparse hyphae or abnormal pigmentation.

[0065] 1.5.3 Physiological state of hyphae (indicators of oxidative damage) Detection indicator: Malondialdehyde (MDA) content. MDA is the end product of cell membrane lipid peroxidation, and its content can reflect the degree of oxidative damage to the cell membrane.

[0066] Detection method: The thiobarbituric acid (TBA) method was used. 0.5g of vigorously growing mycelium after resuscitation was ground in liquid nitrogen and analyzed using a kit (such as those from Nanjing Jiancheng Bioengineering Institute). The specific steps were performed according to the instructions. MDA content is expressed as nmol / g fresh weight.

[0067] 2. Experimental Results and Data Analysis 2.1 Effect of different glycerol ratios on preservation effect The table below shows the test results of different glycerol / paraffin oil ratio treatment groups after 24 months of storage.

[0068] Note: Different letters after the data in the same column indicate that the differences between treatments are significant at the P<0.05 level.

[0069] The data clearly show that when the glycerol volume percentage exceeds 50% (Examples 1-3), the survival rate, reactivation rate, and growth viability of the hyphae are significantly better than those of Comparative Examples 1 and 2, where the glycerol percentage is ≤50%. A higher glycerol percentage provides a more suitable microenvironment, effectively maintaining hyphal activity. Simultaneously, the high glycerol percentage group exhibits significantly lower MDA content, indicating stronger antioxidant capacity and better preservation of cell membrane integrity.

[0070] 2.2 Influence of porous material microparticles on preservation effect Compare the data of Example 1 and Comparative Example 3 (the only difference being whether or not porous material particles were added) after 18 months of storage.

[0071] Adding porous material particles can significantly improve the long-term survival rate and greatly reduce the colony degradation rate. This proves that the "microscopic oxygen channels" formed by porous materials can more precisely regulate oxygen supply, meet the weak respiratory needs of aerobic bacteria, and prevent them from dying or degenerating due to being in a "hypoxia-suffocation" edge state for a long time.

[0072] 2.3 The Influence of Controllable Air Permeability Seals on Preservation Effect Compare the data of Example 1 and Comparative Example 4 (the only difference being the type of seal) after 12 months of storage.

[0073] The use of controllable breathable seals is crucial for the long-term survival of mycelia. Comparative Example 4 (impermeable) almost entirely died within a year because complete oxygen isolation suffocated aerobic bacteria, while the accumulation of metabolic waste and the inability to exchange even trace amounts of moisture led to a deterioration of the culture medium environment (drying and turbidity). Controllable breathable plugs, on the other hand, prevent contamination while ensuring slow gas exchange, providing trace amounts of oxygen while avoiding excessive moisture evaporation, thus creating a stable preservation microenvironment.

[0074] 3. Overall Conclusion This experiment confirmed through systematic detection methods that: 1. A glycerol content of more than 50% (preferably 60%-70%) is the basis for forming a covering solution with good mycelial protection and suitable viscosity, which is crucial for maintaining long-term mycelial activity.

[0075] 2. Adding porous material particles can significantly enhance the controllable aeration function of the covering liquid, making it particularly suitable for long-term preservation and effectively preventing mycelial degeneration.

[0076] 3. The use of controllable breathable seals is a prerequisite for the successful preservation of aerobic bacteria at room temperature, as it can create an aerobic and stable microenvironment.

[0077] This invention achieves safe preservation of edible aerobic bacteria strains at room temperature with long-term, high activity and low degradation through the synergistic effect of a high-glycerol-proportion covering liquid, porous material microparticles, and controllable breathable sealing components, with significantly better results than traditional methods.

[0078] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is obvious that many changes and variations can be made based on the above teachings. Although embodiments of the invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. The purpose of selecting and describing exemplary embodiments is to explain the specific principles of the invention and its practical application, so that those skilled in the art, after reading this specification, can make modifications, substitutions, variations, and various choices and changes to the embodiments as needed without departing from the principles and spirit of the invention, provided that such modifications, substitutions, variations, and choices and changes are within the scope of the claims of the invention and are protected by patent law.

Claims

1. A method for preserving edible aerobic bacteria strains, characterized in that, Includes the following steps: S1: Place the target hyphae in a solid culture medium for cultivation, and then select the target hyphae with good growth and transfer them to a culture slant in a sterile, nutrient-poor culture container for cultivation. Stop cultivation when the hyphae grow to 4 / 5 of the length of the slant. S2: Prepare a covering solution containing glycerol and paraffin oil. Apply the sterile preservation covering solution to the culture vessel and completely submerge the culture medium slant. The glycerol comprises 60%–70% by mass, and the paraffin oil comprises 30%–40% by mass. S3: Seal the culture container with a sealant that has controllable air permeability and store it at room temperature away from light.

2. The method for preserving edible aerobic bacteria according to claim 1, characterized in that, The sealing element is a silicone breathable plug or a test tube plug with a built-in hydrophobic breathable membrane.

3. The method for preserving edible aerobic bacteria according to claim 2, characterized in that, The covering liquid also contains sterile porous material particles, which are microporous silica gel or molecular sieves.

4. The method for preserving edible aerobic bacteria according to claim 3, characterized in that, The added mass of the porous material particles is 1.5% to 2% of the total mass of the covering liquid.

5. The method for preserving edible aerobic bacteria according to claim 1, characterized in that, Before applying the preservation covering solution, the solid culture medium on which aerobic mycelia are growing is subjected to stress induction treatment, which includes adding sodium chloride at a mass percentage of 0.01% to 0.05% to the pre-culture medium.

6. The method for preserving edible aerobic bacteria according to claim 1, characterized in that, The covering solution also contains 0.001% to 0.005% vitamin E by mass.

7. The method for preserving edible aerobic bacteria according to claim 6, characterized in that, The preparation method of the covering liquid is as follows: Glycerin, paraffin oil and vitamin E are stirred and mixed at 80℃~90℃ for 20~30min. After standing until the covering liquid cools to room temperature, porous material particles are added and stirring is continued for 10~15min. Before use, the liquid is ultrasonically treated for 5-8min and then immediately poured into the culture container and the culture container is sealed.

8. A preservation device for preserving edible aerobic bacteria strains, used in the method for preserving edible aerobic bacteria strains according to claim 1, characterized in that, The invention includes a tubular culture container, the opening of which is equipped with a cover device that allows for adjustable air permeability. The air permeability device includes an air permeability window and a control device for controlling the air permeability of the air permeability window.

9. A preservation device for preserving edible aerobic bacteria according to claim 8, characterized in that, The venting device includes a tube cap covering the opening of the culture container, and the venting window includes a perforated opening at the top of the tube cap, the perforated opening being covered with a hydrophobic and breathable membrane.

10. A covering solution for preserving edible aerobic bacteria strains, used in the method for preserving edible aerobic bacteria strains according to claim 1, characterized in that, The venting device includes a cap that covers the opening of the culture container, and the venting window includes a component comprising the following mass percentages: 60%–70% glycerol, 30%–40% paraffin oil, and 0.001%–0.005% vitamin E.

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