Method for separating and cultivating strains from tricholoma matsutake sporocarp

Abstract

The invention relates to the technical field of strain cultivation, in particular to a method for separating and cultivating strains from matsutake sporocarps, which comprises the following steps: collecting 70-80% of matured insect-free matsutake, sterilizing with alcohol, dividing into two halves along the longitudinal direction, removing mushroom flesh at pileus and stipe, and inoculating to a slant culture medium to obtain primary strains; inoculating the primary strain into a culture solution, shaking by a shaking table to obtain a primary shake-flask strain, inoculating the primary shake-flask strain into the culture solution, carrying out shaking culture by the shaking table, and collecting the metabolite concentration, the dissolved oxygen content in the culture solution and the pH value in real time to construct a strain growth time prediction model to predict the stage culture duration; determining a cell proliferation trend based on the number of tricholoma matsutake hypha cells so as to determine an actual culture duration corresponding to a strain culture stage; the shake-flask culture duration of the strain in the shake-flask culture is determined based on the stage culture duration and the actual culture duration, the adjustment mode of the shake-flask speed / culture solution pH value is determined based on the shake-flask culture duration and the predicted culture duration, the shake-flask culture conditions are effectively optimized, and the strain quality is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of strain cultivation, and particularly to a method for isolating and cultivating strains from the fruiting bodies of Tricholoma matsutake. Background Art

[0002] Tricholoma matsutake is a second-class endangered protected species in China. It has strict requirements for the growth environment, grows extremely slowly, generally takes 5 - 6 years, has extremely low yields, and its wild resources are facing threats such as over-picking. Moreover, Tricholoma matsutake is rich in various bioactive substances, and its anti-tumor activity is much higher than that of Ganoderma lucidum, but it is expensive. Countries around the world and major scientific research institutions are actively conducting research on the artificial cultivation of wild Tricholoma matsutake and the isolation and cultivation of strains, aiming to increase the yield of Tricholoma matsutake through artificial cultivation and reduce the market price.

[0003] Chinese Patent Publication No.: CN110199778A, discloses a production process for producing Tricholoma matsutake mycelium by fermenting corn flour, including: taking the fruiting body of Tricholoma matsutake with unbroken fungal membranes, repeatedly performing surface sterilization with 70% ethanol by mass fraction, then using sterile forceps and a knife to cut off a small piece, inoculating it into a slant medium, sealing it well, putting it into an incubator, and culturing it under the condition of constant temperature at 25°C. After the mycelium fills the slant, the slant strain is inoculated into a shake flask corn flour liquid seed medium and cultured on a shaker at 25°C and 190 r / min for 8 days. Then, it is aseptically filtered to obtain mycelium, which is rinsed clean with sterile water, diluted with four times the volume of physiological saline and a small amount of glass beads, the mycelial balls are broken and shaken well to obtain liquid seeds. The fermentation tank is sterilized under high pressure, and under aseptic conditions, the sterilized corn flour liquid fermentation medium is added to the fermentation tank. Then, the cultured liquid seeds are transferred into the fermentation tank, and air filtered through a 0.45 μm sterile filter membrane is introduced for culturing, and fermentation culture is carried out for one week. The culture solution is filtered through a 40-mesh filter screen to separate the mycelium and the fermentation broth. After the mycelium is rinsed several times with deionized water, it is vacuum filtered and dried to a constant weight in an oven at 60°C, and weighed with an analytical balance to obtain the dry matter of Tricholoma matsutake mycelium.

[0004] The highest yield of the dry matter of the mycelium produced by the above technical solution is above 24.82 g / L, the yield of the dry matter of the mycelium is stable and the output is high. However, it does not consider the influence of shake flask parameters on the growth rate of Tricholoma matsutake strains. And although the prior art can determine the optimal culture conditions for Tricholoma matsutake strains through orthogonal experiments, in the actual culture process, there are individual differences among different batches of Tricholoma matsutake strains, and it is difficult to keep the culture environment consistent. The optimal conditions obtained by orthogonal experiments cannot be completely suitable for the culture of each batch. Summary of the Invention

[0005] To this end, the present invention provides a method for isolating and cultivating strains from Tricholoma matsutake fruiting bodies, so as to overcome the problems in the prior art of not considering the influence of shaking bottle parameters on the proliferation rate of Tricholoma matsutake strains when isolating and cultivating strains from Tricholoma matsutake fruiting bodies, and not considering the dynamic changes of relevant parameters during the cultivation process when determining the cultivation conditions.

[0006] To achieve the above object, the present invention provides a method for separating and cultivating strains of Tricholoma matsutake fruiting bodies, comprising: Step S1, collecting 70-80% mature insect-free matsutake mushrooms, disinfecting the insect-free matsutake mushroom fruiting bodies with alcohol under sterile conditions, splitting the disinfected matsutake mushrooms into two halves longitudinally, cutting mushroom flesh of a preset size at the cap and stipe, and inoculating them into a test tube filled with a slant culture medium. After the inoculation is completed, culturing at a constant temperature of 24-26° C. in the absence of light until the mycelium fills the test tube, thereby obtaining a primary strain; Step S2, inoculating the primary strain into the culture solution at an inoculum rate of 0.5% to 1.2%, culturing at a constant temperature of 24 to 26° C., and shaking on a shaker to obtain a primary shake flask strain; Step S3, inoculating the primary shake flask strain into the culture solution at an inoculum rate of 9.5% to 11%, maintaining a constant temperature of 24-26° C., and shaking and culturing on a shaker, so that the separation and cultivation of the Tricholoma matsutake strains is completed; Wherein, the step S3 comprises: Step S31, collecting metabolite concentration, dissolved oxygen content in culture solution, and pH value in real time to build a strain growth time prediction model, and predicting the stage culture duration corresponding to the strain culture stage based on the strain growth time prediction model, wherein the strain culture stage includes an initial adjustment period, a logarithmic growth period, and a stable period; Step S32, collecting bacterial liquid in the culture solution in real time and determining the number of Tricholoma matsutake mycelial cells, determining the cell growth trend based on the number of Tricholoma matsutake mycelial cells, and determining the actual culture time corresponding to the strain culture stage based on the cell growth trend; Step S33, determining the predicted culture time and shake flask culture time of the strain in the shaking table culture based on the stage culture time and the actual culture time, and determining the adjustment method of the shaking table speed / culture solution pH value based on the predicted culture time and the shake flask culture time; Step S34, determining the completion time of separation and cultivation of Tricholoma matsutake strains based on the strain cultivation stage.

[0007] Furthermore, in parts by mass, the slant culture medium consists of 2 parts of glucose, 0.1 parts of magnesium sulfate, 2 parts of agar and 0.5 parts of yeast extract, and the culture solution consists of 2 parts of glucose, 2 parts of sucrose, 1 part of yeast powder, 1 part of milk powder, 0.12 parts of peptone, 0.15 parts of potassium dihydrogen phosphate, 0.01 parts of magnesium sulfate, 0.005 parts of vitamin B1 and 0.005 parts of vitamin B2.

[0008] Further, in the step S31, it includes: Performing noise removal and normalization processing on the metabolite concentration, dissolved oxygen content in the culture solution, and pH value; Respectively determining the characteristic correlation coefficients among the metabolite concentration, dissolved oxygen content, and pH value according to the metabolite concentration, dissolved oxygen content, and pH value; Constructing the prediction model for the strain growth time based on the characteristic correlation coefficients, metabolite concentration, dissolved oxygen content, and pH value; The characteristic correlation coefficients include a first correlation coefficient between the metabolite concentration and the dissolved oxygen content, a second correlation coefficient between the metabolite concentration and the pH value, and a third correlation coefficient between the dissolved oxygen content and the pH value.

[0009] Further, in the step S31, based on the first correlation coefficient, the second correlation coefficient, and the third correlation coefficient, determining the respective weights of the metabolite concentration, dissolved oxygen content, and pH value in the strain growth time prediction model.

[0010] Further, in the step S31, it further includes: determining the stage segmentation points in the strain culture stage based on the strain growth time prediction model, and determining the corresponding stage culture durations based on the stage segmentation points.

[0011] Further, in the step S32, constructing a cell growth curve of the Tricholoma matsutake hyphal cells according to the number of Tricholoma matsutake hyphal cells, determining the cell proliferation characteristics according to the cell growth curve, determining the corresponding strain culture stage according to the cell proliferation characteristics, and determining the actual culture duration according to the current strain culture stage.

[0012] Further, in the step S33, determining the predicted culture duration based on the stage culture duration, and determining the shake flask culture duration based on the actual culture duration.

[0013] Further, in the step S33, the adjustment method includes: if the shake flask culture duration is greater than the predicted culture duration, increasing the shaker speed or adjusting the pH value of the culture solution.

[0014] Further, in the step S33, determining the speed increase amount of the shaker speed according to the culture solution volume, the culture container volume, the shake flask culture duration, the predicted culture duration, and the initial shaker speed.

[0015] Further, in the step S33, adjusting the pH value of the culture solution includes: If the pH value of the culture solution is greater than the preset pH value, lowering the pH value of the culture solution; If the pH value of the culture solution is less than the preset pH value, raising the pH value of the culture solution.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: By combining constant temperature and shaker oscillation cultivation at different cultivation stages, it provides a suitable temperature environment and sufficient oxygen supply for the growth of Tricholoma matsutake strains. At the same time, by collecting the concentration of metabolites, dissolved oxygen content, and pH value in the culture solution in real time to construct a prediction model for the growth time of the strains, it can more accurately predict the corresponding stage cultivation duration in the strain cultivation stage; meanwhile, by determining the number of Tricholoma matsutake hyphal cells in the culture solution in real time, and then determining the cell proliferation trend and actual cultivation duration, it is possible to more precisely grasp the growth status of the strains at different cultivation stages, determine the shake flask cultivation duration of the strains in the shaker cultivation according to the stage cultivation duration and actual cultivation duration, and adjust the shaker speed or frequency, flexibly optimize the cultivation conditions, so as to conduct personalized cultivation for the specific growth situation of each batch of strains, make up for the influence brought by individual differences and environmental changes, and make the cultivation results more stable and reliable, thereby further promoting the growth and metabolism of Tricholoma matsutake hyphae, and improving the growth rate and quality of the strains.

[0017] Furthermore, the present invention preprocesses the concentration of metabolites, dissolved oxygen content, and pH value in the culture solution, effectively removes the noise and outliers in the original data, and constructs dynamic change curves for each parameter respectively, intuitively grasping the dynamic evolution of environmental factors during the growth process of the strains, so as to determine the characteristic correlation coefficients between the concentration of metabolites, dissolved oxygen content, and pH value, clarify the linear correlation degree and direction between variables, effectively construct a prediction model for the growth time of the strains, provide guidance for optimizing cultivation conditions, and further improve the cultivation efficiency and quality of the strains.

[0018] Furthermore, the present invention determines the shake flask cultivation duration of the strains in the shaker cultivation based on the stage cultivation duration, actual cultivation duration, and actual cultivation duration, comprehensively considering the theoretical and actual cultivation situations, which helps to adjust the shaker parameters subsequently, improve the accuracy and controllability of the strain cultivation process, stably and efficiently cultivate strains that meet the requirements, and improve the cultivation success rate and product quality.

[0019] Furthermore, the present invention determines the shake flask cultivation duration of the strains in the shaker cultivation based on the stage cultivation duration and the actual cultivation duration, and determines the adjustment method of the shaker speed / culture solution pH value according to the shake flask cultivation duration and the predicted cultivation duration, significantly enhancing the mixing degree of the culture solution, greatly promoting the dissolved oxygen efficiency, enabling nutrients to be distributed more quickly and evenly around the Tricholoma matsutake hyphal cells, providing more sufficient oxygen and nutrients for their growth, thereby effectively accelerating the growth rate of the hyphae, while ensuring that the quality of the strains is not affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a step diagram for separating and cultivating Tricholoma matsutake fruiting bodies in an embodiment of the present invention; Figure 2A diagram showing the steps of shaking culture on a shaking table according to an embodiment of the present invention; Figure 3 A diagram showing the steps of constructing a strain growth time prediction model according to an embodiment of the present invention; Figure 4 A decision diagram for determining an adjustment method according to an embodiment of the present invention. DETAILED DESCRIPTION

[0021] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0023] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings. This is merely for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0024] See also Figure 1 , Figure 2 As shown, Figure 1 This is a diagram of the steps for separating and cultivating strains of Tricholoma matsutake fruiting bodies according to an embodiment of the present invention. Figure 2 This is a step diagram of shaking culture on a shaking table according to an embodiment of the present invention; it includes: Step S1, collecting 70-80% mature insect-free matsutake mushrooms, disinfecting the insect-free matsutake mushroom fruiting bodies with alcohol under sterile conditions, splitting the disinfected matsutake mushrooms into two halves longitudinally, cutting mushroom flesh of a preset size at the cap and stipe, and inoculating them into a test tube filled with a slant culture medium. After the inoculation is completed, culturing at a constant temperature of 24-26° C. in the absence of light until the mycelium fills the test tube, thereby obtaining a primary strain; Step S2, inoculating the primary strain into the culture solution at an inoculum rate of 0.5% to 1.2%, culturing at a constant temperature of 24 to 26° C., and shaking on a shaker to obtain a primary shake flask strain; Step S3, inoculating the primary shake flask strain into the culture solution at an inoculum rate of 9.5% to 11%, maintaining a constant temperature of 24-26° C., and shaking and culturing on a shaker, so that the separation and cultivation of the Tricholoma matsutake strains is completed; Wherein, the step S3 comprises: Step S31, collect the metabolite concentration, dissolved oxygen content in the culture medium, and pH value in real time to construct a prediction model for the growth time of the strain. Based on the prediction model for the growth time of the strain, predict the stage culture duration corresponding to the strain culture stage, where the strain culture stage includes the initial adjustment period, logarithmic growth period, and stationary period; Step S32, collect the bacterial liquid in the culture medium in real time and determine the number of Tricholoma matsutake hyphal cells. Based on the number of Tricholoma matsutake hyphal cells, determine the cell proliferation trend, and based on the cell proliferation trend, determine the actual culture duration corresponding to the strain culture stage; Step S33, determine the predicted culture duration and shake flask culture duration of the strain in the shaker culture based on the stage culture duration and the actual culture duration, and determine the adjustment method for the shaker speed / culture medium pH value based on the predicted culture duration and shake flask culture duration; Step S34, determine the completion time of the isolation and cultivation of Tricholoma matsutake strain based on the strain culture stage.

[0025] It can be understood that the physiological state of the Tricholoma matsutake fruiting body is suitable for strain isolation when the maturity is 70% - 80% and there are no insects. Taking the mycelium at the cap and stipe is beneficial to the growth and germination of the mycelium. The primary strain obtained through Step S1 is already active. Steps S2 and S3 adopt a hierarchical inoculation and culture method to gradually expand the culture area, which is beneficial for the strain to adapt to different culture environments and also beneficial for cultivating a large number of strains.

[0026] It can be understood that the strain will go through the initial adjustment period (adaptation period), logarithmic growth period, stationary period, and decline period during cultivation. During the cultivation process, the metabolite concentration, dissolved oxygen content in the culture medium, and pH value usually change as follows: In the initial adjustment period, the strain adapts to the environment and metabolizes slowly, the metabolite concentration increases relatively slowly, the strain consumes relatively less oxygen, the dissolved oxygen content does not decrease significantly, and the pH value is also relatively stable at this time. Entering the logarithmic growth period, the strain multiplies in large numbers and the metabolic activity is vigorous, the metabolite concentration will rise rapidly, the change range of the pH value increases, at the same time the number of strains increases rapidly, the respiration increases, and the oxygen demand increases significantly, so the dissolved oxygen content will drop rapidly. In the stationary period, due to the consumption of nutrients and the limitation of environmental conditions, the strain still needs a certain amount of oxygen for metabolic activities, the production rate of metabolites gradually stabilizes, the concentration growth slows down, and the change of the pH value gradually becomes gentle. It is best to stop the culture at the time point when transitioning from the logarithmic growth period to the stationary period. At this time, a large number of strains with strong vitality and activity can be obtained.

[0027] It is understandable that the stage culture duration is based on the prediction model of strain growth time, which mainly indirectly considers the influence of factors such as metabolite concentration, dissolved oxygen content in culture medium and pH value on the growth stage of strains. The actual culture duration is obtained by collecting the number of matsutake mycelium cells in the culture medium and then analyzing the cell proliferation characteristics, which provides an intuitive basis for obtaining the growth time of strains. The two durations are determined from different angles. The predicted culture duration and the shake flask culture duration are determined by the stage culture duration and the actual culture duration, which can more comprehensively reflect the actual growth of strains under the current culture conditions. By comparing the predicted culture duration with the shake flask culture duration, it is possible to promptly discover whether the current culture process deviates from expectations, and then determine whether the current culture situation is normal, so as to adjust the culture conditions (such as shaker speed / culture solution pH value) to achieve effective monitoring and optimization of the culture process, so as to ensure that the culture process achieves the expected results.

[0028] In a specific embodiment, mature 70-80% insect-free matsutake mushrooms are collected, and the insect-free matsutake mushroom fruiting bodies are disinfected with 75% alcohol under sterile conditions in a sterile room, and then the matsutake mushrooms are cut into two halves longitudinally with a scalpel, and the mushroom flesh with a preset size of 0.5 cm³ to 0.6 cm³ is cut at the cap and stipe, and transferred to a test tube with a height of 20 cm and a diameter of 3 cm containing a slant culture medium. After inoculation, the mixture is cultured at a constant temperature of 24-26°C without light for 8-10 days until the mycelium fills the test tube to form a primary strain; the culture solution is prepared, and the primary strain is inoculated at 0.5% to 1.2% into a 250 ml triangular flask containing 100 ml of culture solution, and the mixture is cultured on a shaker at a constant temperature of 24-26°C for 6-9 days to become a primary shake flask strain; the primary shake flask strain is inoculated at 9.5% to 11% into a 500 ml triangular flask containing 200 ml of culture solution, and the mixture is cultured on a shaker at a constant temperature of 24-26°C. At this point, the separation and cultivation of matsutake strains is completed. Preferably, the primary strain is inoculated at 1% into a 250ml triangular flask containing 100ml of culture solution, and the primary shake flask strain is inoculated at 10% into a 500ml triangular flask containing 200ml of culture solution. In implementation, the value range and preferred value of the preset size can be determined according to actual conditions, and are not specifically limited here and will not be repeated.

[0029] The present invention combines constant temperature and shaker oscillation culture at different culture stages to provide a suitable temperature environment and sufficient oxygen supply for the growth of Tricholoma matsutake strains. At the same time, by collecting the concentration of metabolites, dissolved oxygen content, and pH value in the culture medium in real time to construct a strain growth time prediction model, it can more accurately predict the corresponding stage culture duration at the strain culture stage. At the same time, by determining the number of Tricholoma matsutake hyphal cells in the culture medium in real time, the cell proliferation trend and actual culture duration are determined, and then the growth status of the strain at different culture stages can be grasped more accurately. According to the stage culture duration and actual culture duration, the shake flask culture duration of the strain in the shaker culture is determined, and the shaker speed or frequency is adjusted to flexibly optimize the culture conditions, so as to carry out personalized culture for the specific growth situation of each batch of strains, make up for the influence brought by individual differences and environmental changes, make the culture results more stable and reliable, and further promote the growth and metabolism of Tricholoma matsutake hyphae, and improve the growth speed and quality of the strain.

[0030] Specifically, by mass, the slant medium is composed of 2 parts of glucose, 0.1 part of magnesium sulfate, 2 parts of agar, and 0.5 part of yeast extract, and the culture medium is composed of 2 parts of glucose, 2 parts of sucrose, 1 part of yeast powder, 1 part of milk powder, 0.12 part of peptone, 0.15 part of potassium dihydrogen phosphate, 0.01 part of magnesium sulfate, 0.005 part of vitamin B1, and 0.005 part of vitamin B2.

[0031] It can be understood that the components contained in the culture medium can provide various nutrients required for the growth of Tricholoma matsutake. Glucose can be directly absorbed and utilized by Tricholoma matsutake cells, providing energy and material basis for its metabolic activities and the construction of cell structure. Sucrose can be decomposed into glucose and fructose by extracellular enzymes and then absorbed. The rich carbon source helps the growth and reproduction of Tricholoma matsutake mycelium. Yeast powder contains rich nutrients such as amino acids, vitamins, and minerals. Milk powder is rich in proteins, fats, vitamins, etc. Peptone is the hydrolysis product of protein and contains various amino acids. These nitrogen sources can provide nitrogen elements for Tricholoma matsutake to synthesize biological macromolecules such as proteins and nucleic acids, meeting its growth and metabolic needs. Potassium dihydrogen phosphate provides phosphorus and potassium elements, and magnesium sulfate provides magnesium elements. These mineral elements are indispensable in the growth process of Tricholoma matsutake and participate in various physiological and biochemical reactions of cells. At the same time, the addition of vitamin B1 and vitamin B2 also helps the growth of Tricholoma matsutake. As components of coenzymes, they participate in various metabolic processes in cells.

[0032] Please refer to Figure 3 as shown, which is a step diagram for constructing a strain growth time prediction model in an embodiment of the present invention. Specifically, in step S31, it includes: Step S311, performing noise removal and normalization processing on the metabolite concentration, dissolved oxygen content, and pH value in the culture medium; Step S312: Determine the characteristic correlation coefficients among the metabolite concentration, dissolved oxygen content, and pH value respectively based on the metabolite concentration, dissolved oxygen content, and pH value. Step S313: Construct the prediction model for the strain growth time based on the characteristic correlation coefficients, metabolite concentration, dissolved oxygen content, and pH value. The characteristic correlation coefficients include a first correlation coefficient between the metabolite concentration and the dissolved oxygen content, a second correlation coefficient between the metabolite concentration and the pH value, and a third correlation coefficient between the dissolved oxygen content and the pH value.

[0033] It can be understood that through preprocessing, abnormal data in the data can be removed, and the metabolite concentration, dissolved oxygen content in the culture solution, and pH value can be standardized, which is convenient for constructing the prediction model for the strain growth time. By constructing the dynamic change curves corresponding to the metabolite concentration, dissolved oxygen content, and pH value respectively, the change trends of the corresponding indicators of the strain at different culture stages can be observed, so as to determine the characteristic correlation coefficients between each other, and then construct the prediction model for the strain growth time.

[0034] In a specific embodiment, , the , the , and then construct the prediction model for the strain growth time according to the first correlation coefficient, the second correlation coefficient, the third correlation coefficient, the metabolite concentration, the dissolved oxygen content, and the pH value.

[0035] The present invention preprocesses the metabolite concentration, dissolved oxygen content in the culture solution, and pH value, effectively removes the noise and outliers in the original data, and constructs the dynamic change curves of each parameter respectively, intuitively grasps the dynamic evolution of environmental factors during the strain growth process, determines the characteristic correlation coefficients among the metabolite concentration, dissolved oxygen content, and pH value, clarifies the linear association degree and direction between each variable, effectively constructs the prediction model for the strain growth time, provides guidance for optimizing the culture conditions, and further improves the strain culture efficiency and quality.

[0036] Specifically, in the step S31, determine the respective weights corresponding to the metabolite concentration, dissolved oxygen content, and pH value in the prediction model for the strain growth time based on the first correlation coefficient, the second correlation coefficient, and the third correlation coefficient.

[0037] It can be understood that the positive and negative of the first correlation coefficient, the second correlation coefficient, and the third correlation coefficient can reflect the action degree between two different variables. When constructing the prediction model for the strain growth time, this action degree can help judge the influence degree of each variable on the strain growth time, and then allocate appropriate weights to each variable. After determining these weights, the prediction model for the strain growth time can be constructed more accurately, and the fitting degree and prediction accuracy of the model to the actual situation can be improved.

[0038] In a specific embodiment, the first correlation coefficient is the coefficient between the metabolite concentration and the dissolved oxygen content, the second correlation coefficient is the coefficient between the metabolite concentration and the pH value, and the third correlation coefficient is the coefficient between the dissolved oxygen content and the pH value. , , , after determining their respective corresponding weights, the input features of the strain growth time prediction model are the metabolite concentration and the corresponding weight, the dissolved oxygen content and the corresponding weight, and the pH value and the corresponding weight, and the output feature is the culture duration. Algorithms such as machine learning and neural networks can be used to construct this model, which is not specifically limited here.

[0039] Specifically, in the step S31, it includes: determining the stage segmentation points of the strain culture stage based on the strain growth time prediction model, and determining the corresponding stage culture duration based on the stage segmentation points.

[0040] It can be understood that the stage segmentation points are the time nodes of the strain at different growth stages, which are the segmentation points for the strain to enter the logarithmic growth phase from inoculation and to transition from the logarithmic growth phase to the stationary phase. The time interval between two adjacent stage segmentation points is the stage culture duration of the corresponding stage. By determining the stage segmentation points and the stage culture duration, the culture conditions can be precisely controlled according to the characteristics of the strain at each stage, and the culture time can be shortened.

[0041] In a specific embodiment, algorithms such as decision tree, random forest, and support vector machine can be used to train the strain growth time prediction model. During the training process, the model will learn the characteristic patterns of the corresponding data in different strain culture stages. For example, in the logarithmic growth phase, characteristic patterns such as the rapid increase in metabolite concentration, the rapid decrease in dissolved oxygen content, and the possible change in pH value due to metabolite accumulation. The time points corresponding to the data with changed characteristics according to the characteristic patterns are used as the stage segmentation points. In practice, the stage segmentation points can also be determined according to the actual situation, which is not specifically limited here and will not be elaborated further.

[0042] Specifically, in the step S32, a cell growth curve of Tricholoma matsutake hyphal cells is constructed according to the number of Tricholoma matsutake hyphal cells, the cell proliferation characteristics are determined according to the cell growth curve, the corresponding strain culture stage is determined according to the cell proliferation characteristics, and the actual culture duration is determined according to the current strain culture stage.

[0043] It can be understood that a cell growth curve is plotted with the culture time as the abscissa and the number of Tricholoma matsutake hyphal cells as the ordinate. This curve can intuitively reflect the cell proliferation characteristics of Tricholoma matsutake hyphal cells during the culture process, such as rapid proliferation, slow proliferation, or cessation of proliferation. According to the cell proliferation characteristics, it is possible to determine which stage of strain culture the Tricholoma matsutake hyphae are in to determine the corresponding actual culture duration, facilitating precise control and management of the entire culture process.

[0044] In a specific embodiment, during the culture of Tricholoma matsutake hyphae, a suitable method (such as counting with a hemocytometer, plate colony counting method, turbidimetry, etc.) is used to measure the number of hyphal cells. A cell growth curve is plotted based on the number of Tricholoma matsutake hyphal cells and the corresponding culture time, and the cell proliferation characteristics are determined according to the slope of the cell growth curve. When the cell proliferation characteristic of the cell growth curve is slow growth, the Tricholoma matsutake hyphae are in the initial adjustment period, manifested as a slow upward slope, and the slope of the corresponding cell growth curve is less than 0.75. When the cell proliferation characteristic of the cell growth curve is exponential growth, the Tricholoma matsutake hyphae are in the logarithmic growth phase, manifested as a large and steep curve slope greater than 0.75. When the cell proliferation characteristic corresponding to the cell growth curve is stable growth, the Tricholoma matsutake hyphae are in the stable period. Then, the actual culture duration can be determined according to the strain culture stage.

[0045] Specifically, in step S33, the predicted culture duration is determined based on the stage culture duration, and the shake flask culture duration is determined based on the actual culture duration.

[0046] It can be understood that the stage culture duration is the corresponding culture duration for each culture stage (initial adjustment period, logarithmic growth phase, and stable period) under the prediction model, and the actual culture duration is the corresponding culture duration for each culture stage during the actual culture process.

[0047] In a specific embodiment, the predicted culture duration is the sum of the stage culture durations corresponding to the initial adjustment period and the logarithmic growth phase of the prediction model, and the shake flask culture duration is the sum of the actual culture durations corresponding to the initial adjustment period and the logarithmic growth phase during the actual culture process.

[0048] The present invention determines the culture duration of the strain in the shaker culture based on the stage culture duration, the actual culture duration, the stage culture duration, and the actual culture duration, comprehensively considering the theoretical and actual culture situations, which helps to adjust the shaker parameters subsequently, improve the accuracy and controllability of the strain culture process, stably and efficiently cultivate strains that meet the requirements, and improve the culture success rate and product quality.

[0049] Please refer to Figure 4As shown, it is a decision diagram for determining the adjustment method in an embodiment of the present invention. Specifically, in step S33, the adjustment method includes: if the shaking flask culture duration is greater than the predicted culture duration, increase the shaking table speed or adjust the pH value of the culture medium.

[0050] It can be understood that if the shaking flask culture duration is greater than the predicted culture duration, it means that the growth rate of the strain is slow or the culture conditions are not ideal enough, and the shaking table speed or the pH value of the culture medium can be adjusted. Increasing the shaking table speed can increase the turbulence degree of the culture medium, make oxygen dissolve better in the culture medium, improve the dissolved oxygen amount, provide more sufficient oxygen for the strain, promote its metabolism, and accelerate the growth rate. At the same time, it makes the nutrients evenly distributed in the culture medium, facilitating the absorption by the Tricholoma matsutake hypha cells, shortening the culture time. Adjusting the pH value can also promote the growth of the strain and balance the acidity and alkalinity of the culture environment.

[0051] The present invention determines the shaking flask culture duration of the strain in the shaking table culture based on the stage culture duration and the actual culture duration, and determines the adjustment method for the shaking table speed / culture medium pH value according to the shaking flask culture duration and the predicted culture duration, significantly enhancing the mixing degree of the culture medium, greatly promoting the dissolved oxygen efficiency, enabling the nutrients to be more quickly and evenly distributed around the Tricholoma matsutake hypha cells, providing more sufficient oxygen and nutrients for its growth, thus effectively accelerating the growth rate of the hypha while ensuring that the quality of the strain is not affected.

[0052] Specifically, in step S33, the speed increase amount of the shaking table speed is determined according to the volume of the culture medium, the volume of the culture container, the shaking flask culture duration, the predicted culture duration, and the initial shaking table speed.

[0053] It can be understood that if the culture container is larger or the volume of the culture medium is more, in order to ensure sufficient oxygen supply and uniform distribution of nutrients, it is necessary to appropriately increase the shaking table speed; on the contrary, if the culture container is smaller or the culture medium is less, a lower shaking table speed can meet the requirements. Therefore, the volume of the culture medium and the volume of the culture container are considered in the process of determining the speed increase amount, and the initial shaking table speed is the corresponding initial shaking table speed at different strain culture stages.

[0054] In a specific embodiment, the value range of the initial shaking table speed is 120 - 140 revolutions / min, preferably, the value of the initial shaking table speed is 130 revolutions / min. The speed increase amount of the shaking table speed = In practice, the value range and the preferred value of the initial shaking table speed can be determined according to the actual situation, and no specific limitation is made here and will not be elaborated further.

[0055] Specifically, in step S33, adjusting the pH value of the culture medium includes: If the pH value of the culture medium is greater than the preset pH value, lower the pH value of the culture medium; If the pH value of the culture medium is less than the preset pH value, raise the pH value of the culture medium.

[0056] It can be understood that when the culture time is greater than the preset time, the adjustment direction of pH needs to be determined according to the preset pH value. If the strain produces alkaline substances or consumes a large amount of acidic substances during metabolism, it will cause the pH of the culture medium to rise. At this time, acidic substances need to be added to lower the pH to the preset pH value. If the strain ferments to produce acidic metabolites such as lactic acid, acetic acid, citric acid, etc., it will cause the pH of the culture medium to drop. At this time, alkaline substances need to be added to raise the pH.

[0057] In a specific embodiment, the value range of the preset pH value is 5 to 6. Preferably, the value of the preset pH value is 5.5. Preferably, the value range and the preferred value of the preset pH value can be determined according to the actual situation, and no specific limitation is made here and will not be elaborated further.

[0058] Example 1: Primary strain cultivation: Collect 75% mature and insect-free Tricholoma matsutake. Under sterile conditions in a sterile room, disinfect the insect-free Tricholoma matsutake fruiting body with 75% alcohol. Then, use a scalpel to longitudinally cut the Tricholoma matsutake in half, and cut out a 0.5 cm³ piece of mycelium at the cap and stipe. Transfer it to a test tube with a height of 20 cm and a diameter of 3 cm containing a slant medium. After inoculation, culture it at a constant temperature of 25 °C in the dark for 9 days until the mycelium fills the test tube to form a primary strain. The slant medium consists of 2 parts of glucose, 0.1 part of magnesium sulfate, 2 parts of agar, and 0.5 part of yeast extract.

[0059] First-stage shake flask: Prepare a culture medium, which consists of 2 parts of glucose, 2 parts of sucrose, 1 part of yeast powder, 1 part of milk powder, 0.12 part of peptone, 0.15 part of potassium dihydrogen phosphate, 0.01 part of magnesium sulfate, 0.005 part of vitamin B1, and 0.005 part of vitamin B2. Inoculate the primary strain into a 250 ml Erlenmeyer flask containing 100 ml of the culture medium at an inoculation amount of 1%, and culture it on a shaker at a constant temperature of 25 °C for 8 days to obtain a first-stage shake flask strain; Second-stage shake flask: Inoculate the first-stage shake flask strain into a 500 ml Erlenmeyer flask containing 200 ml of the culture medium at an inoculation amount of 10%, and culture it on a shaker at a constant temperature of 25 °C until the separation and cultivation of the Tricholoma matsutake strain are completed; Among them, during the secondary shake flask culture, starting from after the culture, the metabolite concentration, dissolved oxygen content in the culture solution, and pH value are collected in real time to construct a strain growth time prediction model, and the cell proliferation trend is determined by the number of Tricholoma matsutake hyphal cells. During the initial adjustment period, based on the strain growth time prediction model, the predicted stage culture duration of the initial adjustment period is 2 days, the logarithmic growth period is 6 days, the stationary phase is 5 days, and then it begins to decline. The actually measured actual culture duration of the initial adjustment period is 2.5 days, the logarithmic growth period is 7 days, and then it enters the stationary phase. After the Tricholoma matsutake strain enters the stationary phase from the logarithmic growth period, the isolation and cultivation of the Tricholoma matsutake strain are completed. The predicted culture duration corresponding to the strain culture stage is 8 days, and the shake flask culture duration is 9.5 days, lagging behind the predicted culture duration by 1.5 days.

[0060] Example 2 The difference from Example 1 is that the actually measured duration of the initial adjustment period is 0.5 days longer than the predicted initial adjustment period. Then, starting from 12 h after the initial adjustment period culture, the method of the present invention is used to adjust the shaker speed. The initial shaker speed before adjustment is 130 r / min, the pH value of the culture solution is 6.5, and the adjusted shaker speed is 140 r / min. The culture duration is shown in Table 1 below.

[0061] Example 3 The difference from Example 1 is that the actually measured actual stage duration of the logarithmic growth period is 1 day longer than the predicted stage culture duration. Then, starting from the 4th day, the method of the present invention is used to adjust the shaker speed. The shaker speed before adjustment is 130 r / min, the pH value of the culture solution is 4.8, and the adjusted shaker speed is 140 r / min. The culture duration is shown in Table 1 below.

[0062] Example 4 The difference from Example 2 is that the actually measured duration of the initial adjustment period is 0.5 days longer than the predicted initial adjustment period. Then, starting from 12 h after the initial adjustment period culture, the method of the present invention is used to adjust the pH value of the culture solution. The pH value of the culture solution before adjustment is 4.8, and the adjusted value is 5.5. The culture duration is shown in Table 1 below.

[0063] Example 5 The difference from Example 3 is that the actually measured actual stage duration of the logarithmic growth period is 1 day longer than the predicted stage culture duration. Then, starting from the 4th day, the method of the present invention is used to adjust the pH value of the culture solution. The pH value of the culture solution before adjustment is 5.6, and the adjusted value is 5.8. The culture duration is shown in Table 1 below.

[0064] Table 1 Comparison table of the corresponding culture durations under the predicted and actual conditions after the culture parameters are adjusted

[0065] As can be seen from Table 1 above, in Example 1, the shaker speed and the pH value of the culture medium were not adjusted, and the difference between the predicted culture duration and the actual culture duration was 1.5 days. In Example 2, starting from 12 hours after the start of cultivation, the shaker speed was adjusted to 140 rpm, and the pH value of the culture medium was adjusted to 5.5, that is, the adjustment was made during the initial adjustment period. The predicted culture duration was 7.8 days, and the actual culture duration was 8.5 days, with a difference of 0.7 days. In Example 3, starting from the fourth day of cultivation, the shaker speed was adjusted to 140 rpm, and the pH value of the culture medium was adjusted to 5.5, with a difference of 0.25 days. It is proved that appropriately increasing the shaker speed can make air dissolve better in the culture medium, providing more oxygen for the microorganisms to carry out respiration. Sufficient oxygen supply is beneficial to the metabolism of the microorganisms, thus accelerating the growth speed and shortening the culture duration.

[0066] Moreover, as can be seen from Example 4 and Example 5, adjusting the pH value of the culture medium to 5.5 is closer to the optimal pH range for the growth of Tricholoma matsutake. When the pH value is within the appropriate range, the enzyme activity in the microbial cells is relatively high, which helps the cells carry out various biochemical reactions, such as the absorption of nutrients and the synthesis of metabolites, thus promoting the growth and reproduction of the microorganisms and shortening the culture duration.

[0067] The present invention combines constant temperature + shaker oscillation cultivation at different cultivation stages to provide a suitable temperature environment and sufficient oxygen supply for the growth of Tricholoma matsutake strains. At the same time, by collecting the concentration of metabolites, the dissolved oxygen content, and the pH value in the culture medium in real time to construct a prediction model for the growth time of the strains, it is possible to more accurately predict the stage culture duration corresponding to the strain cultivation stage; at the same time, by determining the number of Tricholoma matsutake hyphal cells in the culture medium in real time, the cell proliferation trend and the actual culture duration can be determined, and then the growth status of the strains at different cultivation stages can be grasped more accurately. According to the stage culture duration and the actual culture duration, the shake flask culture duration of the strains in the shaker culture can be determined and the shaker speed or frequency can be adjusted to flexibly optimize the culture conditions, further promoting the growth and metabolism of Tricholoma matsutake hyphae and improving the growth speed and quality of the strains.

[0068] So far, the technical solution of the present invention has been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the protection scope of the present invention.

Claims

1. A method for separating and culturing strains from the fruiting body of Tricholoma matsutake, characterized in that, include: Step S1, collecting 70-80% mature insect-free matsutake mushrooms, disinfecting the insect-free matsutake mushroom fruiting bodies with alcohol under sterile conditions, splitting the disinfected matsutake mushrooms into two halves longitudinally, cutting mushroom flesh of a preset size at the cap and stipe, and inoculating them into a test tube filled with a slant culture medium. After the inoculation is completed, culturing at a constant temperature of 24-26° C. in the absence of light until the mycelium fills the test tube, thereby obtaining a primary strain; Step S2, inoculating the primary strain into the culture solution at an inoculum rate of 0.5% to 1.2%, culturing at a constant temperature of 24 to 26° C., and shaking on a shaker to obtain a primary shake flask strain; Step S3, inoculating the primary shake flask strain into the culture solution at an inoculum rate of 9.5% to 11%, maintaining a constant temperature of 24-26° C., and shaking and culturing on a shaker, so that the separation and cultivation of the Tricholoma matsutake strains is completed; Wherein, the step S3 comprises: Step S31, collecting metabolite concentration, dissolved oxygen content in culture solution, and pH value in real time to build a strain growth time prediction model, and predicting the stage culture duration corresponding to the strain culture stage based on the strain growth time prediction model, wherein the strain culture stage includes an initial adjustment period, a logarithmic growth period, and a stable period; Step S32, collecting bacterial liquid in the culture solution in real time and determining the number of Tricholoma matsutake mycelial cells, determining the cell growth trend based on the number of Tricholoma matsutake mycelial cells, and determining the actual culture time corresponding to the strain culture stage based on the cell growth trend; Step S33, determining the predicted culture time and shake flask culture time of the strain in the shaking table culture based on the stage culture time and the actual culture time, and determining the adjustment method of the shaking table speed / culture solution pH value based on the predicted culture time and the shake flask culture time; Step S34, determining the completion time of separation and cultivation of Tricholoma matsutake strains based on the strain cultivation stage.

2. The method for separating and cultivating strains from Tricholoma matsutake fruit bodies according to claim 1, characterized in that, In parts by mass, the slant culture medium consists of 2 parts of glucose, 0.1 parts of magnesium sulfate, 2 parts of agar and 0.5 parts of yeast extract, and the culture solution consists of 2 parts of glucose, 2 parts of sucrose, 1 part of yeast powder, 1 part of milk powder, 0.12 parts of peptone, 0.15 parts of potassium dihydrogen phosphate, 0.01 parts of magnesium sulfate, 0.005 parts of vitamin B1 and 0.005 parts of vitamin B2.

3. The method for separating and culturing strains from the fruiting bodies of Tricholoma matsutake according to claim 1, characterized in that, In the step S31, it also includes: Performing noise removal and standardization processing on the concentration of the metabolites, the amount of dissolved oxygen in the culture solution, and the pH value; According to the concentration of metabolites, the amount of dissolved oxygen and the pH value, the characteristic correlation coefficients between the concentration of metabolites, the amount of dissolved oxygen and the pH value are determined respectively; Constructing the bacterial strain growth time prediction model based on the characteristic correlation coefficient, metabolite concentration, dissolved oxygen content and pH value; The characteristic correlation coefficients include a first correlation coefficient between the metabolite concentration and the dissolved oxygen content, a second correlation coefficient between the metabolite concentration and the pH value, and a third correlation coefficient between the dissolved oxygen content and the pH value.

4. The method for separating and culturing strains from the fruiting bodies of Tricholoma matsutake according to claim 3, characterized in that, In the step S31, the corresponding weights of the metabolite concentration, dissolved oxygen content and pH value in the bacterial growth time prediction model are determined based on the first correlation coefficient, the second correlation coefficient and the third correlation coefficient.

5. The method for separating and culturing strains from the fruiting body of Tricholoma matsutake according to claim 4, characterized in that, In the step S31, it further includes: determining a stage segmentation point of the strain culture stage based on the strain growth time prediction model, and determining the corresponding stage culture duration based on the stage segmentation point.

6. The method for separating and cultivating strains from the fruiting bodies of Tricholoma matsutake according to claim 1, characterized in that, In the step S32, constructing a cell growth curve of the Tricholoma matsutake hyphae cells according to the number of Tricholoma matsutake hyphae cells, determining the cell proliferation characteristics according to the cell growth curve, determining the corresponding strain culture stage according to the cell proliferation characteristics, and determining the actual culture duration according to the current strain culture stage.

7. The method for separating and cultivating strains from the fruiting bodies of Tricholoma matsutake according to claim 1, characterized in that, In the step S33, determining the predicted culture duration based on the stage culture duration, and determining the shaking flask culture duration based on the actual culture duration.

8. The method for separating and cultivating strains from the fruiting bodies of Tricholoma matsutake according to claim 7, wherein, In the step S33, the adjustment method includes: if the shaking flask culture duration is greater than the predicted culture duration, increasing the shaker speed or adjusting the pH value of the culture solution.

9. The method for separating and cultivating strains from Tricholoma matsutake fruit bodies according to claim 8, characterized in that, In the step S33, determining the speed increase amount of the shaker speed according to the volume of the culture solution, the volume of the culture container, the shaking flask culture duration, the predicted culture duration, and the initial shaker speed.

10. The method for separating and cultivating strains from Tricholoma matsutake fruit bodies according to claim 9, characterized in that, In the step S33, adjusting the pH value of the culture solution includes: if the pH value of the culture solution is greater than the preset pH value, lowering the pH value of the culture solution; if the pH value of the culture solution is less than the preset pH value, increasing the pH value of the culture solution.

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