A method for isolating and cultivating tricholoma matsutake fruiting body strain

Abstract

This invention relates to the field of fungal strain cultivation technology, and particularly to a method for isolating and cultivating fungal strains from matsutake fruiting bodies. The method involves collecting matsutake mushrooms that are 70%–80% mature and free from insects, disinfecting them with alcohol, longitudinally splitting them in half, removing the flesh from the cap and stipe, and inoculating them onto a slant culture medium to obtain a primary strain. This primary strain is then inoculated into a culture medium and shaken to obtain a primary shake flask strain, which is then inoculated into a culture medium. The strain is then shaken and cultured, and the concentration of metabolites, dissolved oxygen, and pH in the culture medium are collected in real time to construct a strain growth time prediction model to predict the stage culture duration. The cell proliferation trend is determined based on the number of matsutake mycelial cells. Based on a comparison of the actual culture time during the initial adjustment period and the stage culture time, the adjustment method for the logarithmic growth phase of the current batch is determined. Based on a comparison of the shake flask culture time and the predicted culture time, the adjustment method for the initial culture conditions of the next batch is determined. This invention effectively optimizes shake flask culture conditions and improves strain quality.

Description

Technical Field

[0001] This invention relates to the field of fungal strain cultivation technology, and in particular to a method for isolating and cultivating fungal strains from matsutake fruiting bodies. Background Technology

[0002] Matsutake mushrooms are a Class II endangered protected species in my country. They have demanding growth requirements, grow extremely slowly (generally 5-6 years), and have very low yields. Wild resources face threats such as over-harvesting. Furthermore, matsutake mushrooms are rich in various bioactive substances, with anti-tumor activity far exceeding that of Ganoderma lucidum (reishi mushroom), but they are expensive. Countries and major research institutions worldwide are actively conducting research on the artificial cultivation and strain isolation of wild matsutake mushrooms to increase yields and lower market prices.

[0003] Chinese Patent Publication No. CN110199778A discloses a production process for matsutake mushroom mycelium using corn flour fermentation. The process includes: taking unruptured matsutake mushroom fruiting bodies, repeatedly surface-sterilizing them with 70% ethanol, removing a small piece with sterile forceps and a knife, inoculating it into a slant culture medium, sealing it, and placing it in an incubator for constant temperature cultivation at 25℃. After the mycelium has fully grown on the slant, the slant culture is inoculated into a shake flask of corn flour liquid seed culture medium and cultured on a shaker at 25℃ and 190 rpm for 8 days. The mycelium is then sterilized by filtration, rinsed with sterile water, diluted with four times its volume of physiological saline and a small amount of glass beads, the mycelial balls are broken up, and shaken well to obtain liquid seed. The fermenter is then autoclaved. Under sterile conditions, the sterilized corn flour liquid fermentation medium is added to the fermenter, followed by the transfer of the cultured liquid seed to the fermenter. Air filtered through a 0.45 μm sterile filter membrane is introduced for fermentation for one week. The culture medium was filtered through a 40-mesh filter to separate the mycelium and fermentation broth. The mycelium was washed several times with deionized water, then vacuum filtered and dried in a 60°C oven to constant weight. The dry matter of matsutake mycelium was obtained by weighing with an analytical balance.

[0004] The above-mentioned technical solutions produce mycelial dry matter fermentation with a maximum yield of over 24.82 g / L. The mycelial dry matter yield is stable and high. However, the influence of shake flask parameters on the growth rate of matsutake mushroom strains is not considered. Although existing technologies can determine the optimal culture conditions for matsutake mushroom strains through orthogonal experiments, in actual culture, there are individual differences between different batches of matsutake mushroom strains, and it is difficult to keep the culture environment consistent. Therefore, the optimal conditions obtained by orthogonal experiments cannot be completely suitable for each batch of culture. Summary of the Invention

[0005] Therefore, the present invention provides a method for isolating and cultivating Matsutake mushroom spawn from fruiting bodies, which overcomes the problems in the prior art that the influence of shaking flask parameters on the Matsutake spawn multiplication rate is not considered when isolating and cultivating Matsutake mushroom spawn from fruiting bodies, and that the dynamic changes of relevant parameters during the cultivation process are not considered when determining the cultivation conditions.

[0006] To achieve the above objectives, the present invention provides a method for isolating and cultivating fungal strains from matsutake fruiting bodies, comprising: Step S1: Collect 70%–80% mature insect-free matsutake mushrooms, disinfect the fruiting bodies of insect-free matsutake mushrooms with alcohol under sterile conditions, cut the disinfected matsutake mushrooms in half longitudinally, cut off the pre-sized flesh at the cap and stipe, and inoculate it into a test tube containing slant culture medium. After inoculation, culture at a constant temperature of 24℃–26℃ in the dark until the mycelium fills the test tube to obtain the primary strain. Step S2: Inoculate the primary bacterial strain into the culture medium at an inoculation rate of 0.5% to 1.2%, and culture at a constant temperature of 24℃ to 26℃ on a shaker to obtain the primary shake flask bacterial strain; Step S3: Inoculate the primary shake flask strain into the culture medium at an inoculation rate of 9.5% to 11%, and culture at a constant temperature of 24℃ to 26℃ on a shaker. At this point, the isolation and cultivation of matsutake mushroom strain is completed. Step S3 includes: Step S31: Collect the concentration of metabolites, dissolved oxygen in the culture medium and pH value in real time to construct a bacterial growth time prediction model. Based on the bacterial growth time prediction model, predict the stage culture time corresponding to the bacterial culture stage. The bacterial culture stage includes the initial adjustment period, the logarithmic growth period and the stationary period. Step S32: Collect bacterial culture in the culture medium in real time and determine the number of matsutake mycelial cells. Determine the cell proliferation trend based on the number of matsutake mycelial cells. Determine the actual culture time corresponding to the culture stage based on the cell proliferation trend. Step S33: Based on the stage culture time and the actual culture time, determine the predicted culture time and shake flask culture time of the strain in shaker culture, and based on the predicted culture time and shake flask culture time, determine the adjustment method of shaker speed / culture medium pH value; Step S34: Determine the timing of completion of matsutake mushroom strain isolation and cultivation based on the strain culture stage.

[0007] Furthermore, step S33 also includes determining the adjustment method for the current batch's logarithmic growth phase based on the comparison results between the actual culture duration of the initial adjustment period and the stage culture duration of the initial adjustment period.

[0008] Further, step S31 includes: The concentrations of the metabolites, dissolved oxygen in the culture medium, and pH values ​​were subjected to noise removal and standardization. The characteristic correlation coefficients between metabolite concentration, dissolved oxygen, and pH value were determined based on the concentration of metabolites, dissolved oxygen, and pH value, respectively. A growth time prediction model for the bacterial strain is constructed based on the aforementioned feature correlation coefficient, metabolite concentration, dissolved oxygen content, and pH value. The characteristic correlation coefficients include a first correlation coefficient between metabolite concentration and dissolved oxygen, a second correlation coefficient between metabolite concentration and pH value, and a third correlation coefficient between dissolved oxygen value and pH value.

[0009] Further, in step S31, the weights of metabolite concentration, dissolved oxygen 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.

[0010] Furthermore, in step S31, the method further includes: determining the 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.

[0011] Further, in step S32, a cell growth curve of matsutake mycelial cells is constructed based on the number of matsutake mycelial cells, cell proliferation characteristics are determined based on the cell growth curve, the corresponding culture stage of the strain is determined based on the cell proliferation characteristics, and the actual culture time is determined based on the current culture stage of the strain.

[0012] Furthermore, in step S33, the predicted culture time is determined based on the stage culture time, and the shake flask culture time is determined based on the actual culture time.

[0013] Further, in step S33, the adjustment method includes: if the shake flask culture time is longer than the predicted culture time, then increase the shaker speed or adjust the pH value of the culture medium for the initial culture conditions of the next batch. If the actual culture time of the initial adjustment period is longer than the corresponding stage culture time, then increase the shaking speed of the current batch during the logarithmic growth phase or adjust the pH of the culture medium.

[0014] Further, in step S33, the increase in the shaking speed of the initial culture conditions for the next batch is determined based on the current batch's culture medium volume and culture container volume, the shake flask culture time, the predicted culture time, and the initial shaking speed. The rate increase of the shaker speed during the current batch's logarithmic growth phase is determined based on the deviation between the actual culture time during the initial adjustment period and the stage culture time.

[0015] Further, 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, then the pH value of the culture medium is lowered. If the pH value of the culture medium is lower than the preset pH value, then the pH value of the culture medium is increased.

[0016] Compared with existing technologies, the beneficial effects of this invention lie in the combination of constant temperature and shaking culture at different cultivation stages, providing a suitable temperature environment and sufficient oxygen supply for the growth of matsutake mushroom strains. Simultaneously, by collecting real-time data on metabolic product concentration, dissolved oxygen content in the culture medium, and pH value, a growth time prediction model for the strain can be constructed, enabling relatively accurate prediction of the corresponding cultivation stage duration. Furthermore, by collecting real-time data on the mycelial cell count in the culture medium, the number of matsutake mycelial cells can be determined, thereby identifying cell proliferation trends and actual cultivation time. This allows for more precise control of the strain's growth status at different cultivation stages. Based on the stage cultivation time and actual cultivation time, the shake-flask cultivation time in the shaking culture can be determined, and the shaking speed or frequency can be adjusted to flexibly optimize cultivation conditions. This allows for personalized cultivation tailored to the specific growth conditions of each batch of strains, compensating for the influence of individual differences and environmental changes, making the cultivation results more stable and reliable. This further promotes the growth and metabolism of matsutake mycelium, improving the growth rate and quality of the strain.

[0017] Furthermore, this invention preprocesses the concentration of metabolites, dissolved oxygen in the culture medium, and pH value to effectively remove noise and outliers from the original data, and constructs dynamic change curves for each parameter to intuitively grasp the dynamic evolution of environmental factors during the growth of the strain. This allows for the determination of the characteristic correlation coefficients between the concentration of metabolites, dissolved oxygen, and pH value, clarifying the degree and direction of linear correlation between each variable, and effectively constructing a strain growth time prediction model. This provides guidance for optimizing culture conditions and further improves the efficiency and quality of strain cultivation.

[0018] Furthermore, this invention determines the shake flask culture time of the strain in shaker culture based on the stage culture time, the actual culture time, and the actual culture time. By comprehensively considering the theoretical and actual culture conditions, it helps to adjust the shaker parameters in the future, 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.

[0019] Furthermore, this invention determines the shake-flask culture time of the strain in shaker culture based on the stage culture time and the actual culture time, and determines the adjustment method of shaker speed / culture medium pH value according to the shake-flask culture time and the predicted culture time, which significantly enhances the mixing degree of culture medium, promotes a significant increase in dissolved oxygen efficiency, and allows nutrients to be distributed more quickly and evenly around the matsutake mycelial cells, providing them with more sufficient oxygen and nutrients for growth, thereby effectively accelerating the growth rate of mycelium, while ensuring that the quality of the strain is not affected. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the steps of isolating and cultivating fungal strains from matsutake fruiting bodies according to an embodiment of the present invention; Figure 2 This is a flowchart illustrating the steps of shaker culture according to an embodiment of the present invention; Figure 3 A flowchart illustrating the steps involved in constructing a microbial growth time prediction model according to an embodiment of the present invention; Figure 4 This is a determination diagram for identifying the adjustment method in an embodiment of the present invention. Detailed Implementation

[0021] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0022] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0023] It should be noted that in the description of this invention, the terms "upper", "lower", "left", "right", "inner", "outer", etc., which indicate directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and is not intended to indicate or imply that the device or element must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of this invention.

[0024] Please see Figure 1 , Figure 2 As shown, Figure 1 This is a diagram illustrating the steps of isolating and cultivating fungal strains from matsutake fruiting bodies according to an embodiment of the present Figure 2 This is a flowchart illustrating the steps of a shaker culture according to an embodiment of the present invention; including: Step S1: Collect 70%–80% mature insect-free matsutake mushrooms, disinfect the fruiting bodies of insect-free matsutake mushrooms with alcohol under sterile conditions, cut the disinfected matsutake mushrooms in half longitudinally, cut off the pre-sized flesh at the cap and stipe, and inoculate it into a test tube containing slant culture medium. After inoculation, culture at a constant temperature of 24℃–26℃ in the dark until the mycelium fills the test tube to obtain the primary strain. Step S2: Inoculate the primary bacterial strain into the culture medium at an inoculation rate of 0.5% to 1.2%, and culture at a constant temperature of 24℃ to 26℃ on a shaker to obtain the primary shake flask bacterial strain; Step S3: Inoculate the primary shake flask strain into the culture medium at an inoculation rate of 9.5% to 11%, and culture at a constant temperature of 24℃ to 26℃ on a shaker. At this point, the isolation and cultivation of matsutake mushroom strain is completed. Step S3 includes: Step S31: Collect the concentration of metabolites, dissolved oxygen in the culture medium and pH value in real time to construct a bacterial growth time prediction model. Based on the bacterial growth time prediction model, predict the stage culture time corresponding to the bacterial culture stage. The bacterial culture stage includes the initial adjustment period, the logarithmic growth period and the stationary period. Step S32: Collect bacterial culture in the culture medium in real time and determine the number of matsutake mycelial cells. Determine the cell proliferation trend based on the number of matsutake mycelial cells. Determine the actual culture time corresponding to the culture stage based on the cell proliferation trend. Step S33: Based on the stage culture time and the actual culture time, determine the predicted culture time and shake flask culture time of the strain in shaker culture; based on the predicted culture time and shake flask culture time, determine the adjustment method for the shaker speed / culture medium pH value, wherein the adjustment method includes... Adjustments to the shaking speed / culture medium pH for the current batch. And, adjustments to the shaker speed / culture medium pH for the next batch; Step S34: Determine the timing of completion of matsutake mushroom strain isolation and cultivation based on the strain culture stage.

[0025] It is understandable that matsutake mushrooms with a maturity of 70% to 80% and free from insects are in a physiological state suitable for strain isolation. Taking the flesh from the cap and stipe is beneficial for the growth and germination of mycelium. The primary strain obtained through step S1 is already active. Steps S2 and S3 adopt a graded inoculation culture method to gradually expand the culture area, which is conducive to the strain adapting to different culture environments and also conducive to the cultivation of a large number of strains.

[0026] Understandably, bacterial strains undergo an initial adjustment phase (adaptation phase), a logarithmic growth phase, a stationary phase, and a decline phase during cultivation. During this process, the concentration of metabolites, dissolved oxygen in the culture medium, and pH typically change as follows: During the initial adjustment phase, the strain's metabolism adapts slowly, the concentration of metabolites increases slowly, oxygen consumption is relatively low, dissolved oxygen levels do not decrease significantly, and the pH is relatively stable. Entering the logarithmic growth phase, the strain proliferates rapidly, metabolic activity is vigorous, the concentration of metabolites rises rapidly, pH fluctuations increase, the strain population increases rapidly, respiration intensifies, oxygen demand increases significantly, and dissolved oxygen levels drop rapidly. During the stationary phase, due to nutrient consumption and environmental limitations, the strain still requires some oxygen for metabolic activity. The rate of metabolite production gradually stabilizes, the concentration increase slows, and pH changes gradually level off. Stopping cultivation at the point when the strain transitions from the logarithmic growth phase to the stationary phase is optimal, as this yields a large number of highly viable and active strains.

[0027] It is understandable that the staged culture duration is derived from a strain growth time prediction model, primarily indirectly considering the influence of factors such as metabolite concentration, dissolved oxygen content in the culture medium, and pH value on the strain growth stages. The actual culture duration, on the other hand, is obtained by collecting data on the number of Matsutake mycelial cells in the culture medium and analyzing their cell proliferation characteristics, providing a direct basis for determining the strain growth time. The two durations are determined from different perspectives; using the staged culture duration and the actual culture duration to determine the predicted culture duration and shake-flask culture duration respectively provides a more comprehensive reflection of the strain's actual growth under current culture conditions. Comparing the predicted culture duration with the shake-flask culture duration allows for timely detection of deviations from expectations, thus determining whether the current culture situation is normal. Adjustments to culture conditions (such as shaker speed / culture medium pH value) can then be made to effectively monitor and optimize the culture process, ensuring that the culture achieves the expected results.

[0028] In one specific embodiment, 70%–80% mature, insect-free matsutake mushrooms were collected. The fruiting bodies were disinfected with 75% alcohol under sterile conditions in a sterile room. Then, the matsutake mushrooms were longitudinally cut in half with a scalpel, and a pre-determined 0.5 cm section was cut from the cap and stem. ~0.6 The fungal flesh was transferred to test tubes (20cm high, 3cm in diameter) containing slant culture medium. After inoculation, the tubes were incubated at a constant temperature of 24℃–26℃ in the dark for 8–10 days until the mycelium completely covered the tubes, forming the primary strain. A culture medium was prepared by inoculating the primary strain at a rate of 0.5%–1.2% into a 250ml Erlenmeyer flask containing 100ml of culture medium and incubating at a constant temperature of 24℃–26℃ on a shaker for 6–9 days to obtain the primary shake flask strain. The primary shake flask strain was then inoculated at a rate of 9.5%–11% into a 500ml Erlenmeyer flask containing 200ml of culture medium and incubated at a constant temperature of 24℃–26℃ on a shaker. This completes the isolation and cultivation of the matsutake mushroom strain. Preferably, the primary strain is inoculated at a rate of 1% into a 250ml Erlenmeyer flask containing 100ml of culture medium, and the primary shake flask strain is inoculated at a rate of 10% into a 500ml Erlenmeyer flask containing 200ml of culture medium. In practice, the range and preferred values ​​of the preset size can be determined according to the actual situation. No specific limitations are made here, and they will not be elaborated further.

[0029] This invention combines constant temperature and shaking culture at different cultivation stages to provide a suitable temperature environment and sufficient oxygen supply for the growth of matsutake mushroom spores. Simultaneously, by collecting real-time data on metabolic product concentration, dissolved oxygen content in the culture medium, and pH value, a spore growth time prediction model is constructed, which can accurately predict the corresponding cultivation duration for each stage of the spore cultivation process. Furthermore, by collecting real-time data on the mycelial cell count in the culture medium, the number of matsutake mycelial cells is determined, thereby identifying cell proliferation trends and actual cultivation duration. This allows for more precise control of the spore growth at different cultivation stages. Based on the stage cultivation duration and the actual cultivation duration, the shake-flask cultivation time in the shaking culture is determined, and the shaking speed or frequency is adjusted to flexibly optimize cultivation conditions. This allows for personalized cultivation tailored to the specific growth conditions of each batch of spores, compensating for the influence of individual differences and environmental changes, making the cultivation results more stable and reliable. This further promotes the growth and metabolism of matsutake mycelium, improving the growth rate and quality of the spores.

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

[0031] Understandably, the components of the culture medium provide a variety of nutrients necessary for matsutake mushroom growth. Glucose can be directly absorbed and utilized by matsutake cells, providing energy and a material basis for their metabolic activities and cell structure construction. Sucrose can be broken down into glucose and fructose by extracellular enzymes and then absorbed. The abundant carbon source helps the growth and reproduction of matsutake mycelium. Yeast powder contains abundant amino acids, vitamins, and minerals, while milk powder is rich in protein, fat, and vitamins. Peptone, a hydrolysis product of protein, contains various amino acids. These nitrogen sources provide nitrogen for matsutake to synthesize proteins, nucleic acids, and other biomolecules, meeting its growth and metabolic needs. Potassium dihydrogen phosphate provides phosphorus and potassium, and magnesium sulfate provides magnesium. These minerals are indispensable for matsutake growth, participating in various physiological and biochemical reactions within the cells. Simultaneously, the addition of vitamins B1 and B2 also contributes to matsutake growth; as components of coenzymes, they participate in various intracellular metabolic processes.

[0032] Step S3 includes: Step S31: Collect the concentration of metabolites, dissolved oxygen in the culture medium and pH value in real time to construct a bacterial growth time prediction model. Based on the bacterial growth time prediction model, predict the stage culture time corresponding to the bacterial culture stage. The bacterial culture stage includes the initial adjustment period, the logarithmic growth period and the stationary period. Please seeFigure 3 The diagram illustrates the steps involved in constructing a microbial growth time prediction model according to an embodiment of the present invention. Specifically, step S31 includes: Step S311: Noise removal and standardization are performed on the concentration of the metabolites, dissolved oxygen in the culture medium, and pH value. Step S312: Determine the characteristic correlation coefficients between metabolite concentration, dissolved oxygen, and pH value based on the metabolite concentration, dissolved oxygen, and pH value, respectively. Step S313: Construct a growth time prediction model for the bacterial strain based on the feature correlation coefficient, metabolite concentration, dissolved oxygen, and pH value. The characteristic correlation coefficients include a first correlation coefficient between metabolite concentration and dissolved oxygen, a second correlation coefficient between metabolite concentration and pH value, and a third correlation coefficient between dissolved oxygen value and pH value.

[0033] Understandably, preprocessing removes outliers and standardizes metabolite concentrations, dissolved oxygen levels, and pH values ​​in the culture medium, facilitating the construction of a microbial growth time prediction model. By constructing dynamic change curves for each of the metabolite concentrations, dissolved oxygen levels, and pH values, the changing trends of these indicators at different culture stages can be observed, thereby determining the characteristic correlation coefficients between them and ultimately constructing a microbial growth time prediction model.

[0034] In one specific embodiment, at least 30 historical batches of matsutake mushroom cultivation data were collected. The following information was recorded for each batch: Metabolic product concentration (g / L), dissolved oxygen (%), and pH value were collected every 2 hours within the first 4 hours after inoculation, for a total of 3 sets of data (hour 0, hour 2, and hour 4).

[0035] The stage breakpoint for this batch: T1: The end date (in days) of the initial adjustment period, defined as the first time the slope of the cell growth curve exceeds 0.75. T2: The time point (days) at which the logarithmic growth phase ends, with the criterion being that the slope of the cell growth curve drops to near 0; For each historical batch, the average of the three sets of data collected within the first four hours is used as the input feature: , , , Where C(t) is the concentration of metabolites at time t (g / L), D(t) is the dissolved oxygen at time t (%), P(t) is the pH value at time t, and t=0,2,4 represent the 0th hour, the 2nd hour, and the 4th hour, respectively. Output target: the initial adjustment period stage split point T1 and the logarithmic growth period stage split point T2 for this batch (unit: days); The input features C of all training samples in all historical batches a D a P a The data are aggregated, and the first, second, and third correlation coefficients are calculated respectively.

[0036] Suppose there are n historical batches (n≥30), then: , , , Where: C ai D ai P ai C represents the i-th batch. a D a P a value, μ C μ D μ P C respectively a D a P a The mean of the historical batches, where n is the number of historical batches (n≥30); First correlation coefficient , Second correlation coefficient , The third correlation coefficient , Then, a model for predicting bacterial growth time was constructed based on the first correlation coefficient, the second correlation coefficient, the third correlation coefficient, as well as the concentration of metabolites, dissolved oxygen, and pH value.

[0037] This invention preprocesses the concentration of metabolites, dissolved oxygen in the culture medium, and pH value to effectively remove noise and outliers from the original data. It also constructs dynamic change curves for each parameter to intuitively grasp the dynamic evolution of environmental factors during the growth of the strain. This allows for the determination of the characteristic correlation coefficients between the concentration of metabolites, dissolved oxygen, and pH value, clarifying the degree and direction of linear correlation between each variable, and effectively constructing a strain growth time prediction model. This provides guidance for optimizing culture conditions and further improves the efficiency and quality of strain cultivation.

[0038] Specifically, in step S31, the weights of metabolite concentration, dissolved oxygen 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.

[0039] Understandably, the signs of the first, second, and third correlation coefficients reflect the degree of influence between two different variables. When constructing a model to predict the growth time of a microbial strain, this degree of influence helps determine the impact of each variable on the growth time, thus allowing for the allocation of appropriate weights to each variable. Determining these weights enables a more accurate construction of the microbial growth time prediction model, improving its fit to the actual situation and its predictive accuracy.

[0040] In one specific embodiment, the first correlation coefficient is the coefficient between metabolite concentration and dissolved oxygen, the second correlation coefficient is the coefficient between metabolite concentration and pH value, and the third correlation coefficient is the coefficient between dissolved oxygen and pH value. Dissolved oxygen content in culture medium weighting pH weighting The original input features are weighted and transformed to obtain the model input variables: , , , Using a multiple linear regression algorithm, two regression equations are established respectively: Where: T3 is the predicted end time of the initial adjustment period (unit: days), T4 is the predicted end time of the logarithmic growth period (unit: days), and a1, b1, c1, d1 and a2, b2, c2, d2 are the regression coefficients and intercept terms; The training samples were fitted using the least squares method, and the optimal values ​​of the regression coefficients and intercept terms for the two sets were solved respectively.

[0041] Specifically: Let the number of training samples be n. Construct the input matrix X (n×4) and output vectors Y1 (n×1) and Y2 (n×1): X=[1,X11,X21,X31;1,X12,X22,X32;...;1,X1n,X2n,X3n] Y1=[T11,T12,...,T1n] ᵀ Y2=[T21,T22,...,T2n] ᵀ The least squares solution for the regression coefficient vectors β1 and β2 is: β1 = (X ᵀ X) -1 X ᵀ Y1, B2 = (X ᵀ X) -1 X ᵀ Y2 Where β1=[d1,a1,b1,c1] ᵀ β2=[d2,a2,b2,c2] ᵀ .

[0042] The above solution process is well known to those skilled in the art, and the specific regression coefficient values ​​can be calculated based on historical batch data.

[0043] The model accuracy was validated using five batches of data not used in training (a reserved validation set). For each sample in the validation set, the absolute error between the predicted values ​​T3 and T4 and the actual values ​​T1 and T2 was calculated. When the mean absolute error of all validation samples was less than 0.2 days, the model accuracy met the requirements and could be used for real-time prediction of the current batch. If the mean absolute error was greater than or equal to 0.2 days, the amount of historical batch data was increased, and the model was retrained until the accuracy met the standard. This validation process was completed before the model was put into use and did not involve the training process of the current batch.

[0044] Specifically, in step S31, after completing the construction of the microbial growth time prediction model, the method further includes: Step S314: Determine the stage segmentation points of the strain culture stage based on the strain growth time prediction model, and determine the corresponding stage culture duration based on the stage segmentation points.

[0045] Understandably, the stage division point is a time point in the growth of the microbial strain, specifically the point where the strain transitions from inoculation to the logarithmic growth phase and then to the stationary phase. The time interval between two adjacent stage division points is the corresponding stage incubation duration. By determining the stage division points and stage incubation durations, culture conditions can be precisely controlled according to the characteristics of the microbial strain at each stage, thus shortening the incubation time.

[0046] Specifically, after the current batch is inoculated, data on metabolite concentration, dissolved oxygen, and pH value are collected for the first 4 hours (collected every 2 hours, for a total of 3 sets of data). The average value is then weighted and transformed according to the steps above. Substituting these values ​​into the two regression equations, we obtain: The initial adjustment period's training duration is T3. The duration of the logarithmic growth phase = T4 - T3. Predicted culture duration = T4 This prediction can be made in the early stages of cultivation (after the first 4 hours), without waiting for the cultivation process to end.

[0047] Step S32: Collect bacterial culture in the culture medium in real time and determine the number of matsutake mycelial cells. Determine the cell proliferation trend based on the number of matsutake mycelial cells. Determine the actual culture time corresponding to the culture stage based on the cell proliferation trend. Specifically, in step S32, a cell growth curve of matsutake mycelium cells is constructed based on the number of matsutake mycelium cells, cell proliferation characteristics are determined based on the cell growth curve, the corresponding culture stage of the strain is determined based on the cell proliferation characteristics, and the actual culture time is determined based on the current culture stage of the strain.

[0048] Understandably, plotting a cell growth curve with culture time on the x-axis and the number of matsutake mycelial cells on the y-axis provides a clear visual representation of the cell proliferation characteristics of matsutake mycelial cells during culture, such as rapid proliferation, slow proliferation, or cessation of proliferation. Based on these cell proliferation characteristics, the stage of matsutake mycelium culture can be determined, thus specifying the corresponding actual culture time and facilitating precise control and management of the entire culture process.

[0049] In one specific embodiment, during the matsutake mycelium culture process, the number of mycelial cells is determined using appropriate methods (such as hemocytometer counting, plate colony counting, turbidimetry, etc.). A cell growth curve is plotted based on the number of matsutake mycelial cells and the corresponding culture time, and the cell proliferation characteristics are determined based on the slope of the cell growth curve. When the cell proliferation characteristic of the cell growth curve is slow growth, the matsutake mycelium is in the initial adjustment phase, characterized by a slowly increasing slope, with the slope of the corresponding cell growth curve being less than 0.75. When the cell proliferation characteristic of the cell growth curve is exponential growth, the matsutake mycelium is in the logarithmic growth phase, characterized by a large and steep slope greater than 0.75. When the cell proliferation characteristic corresponding to the cell growth curve is stable growth, the matsutake mycelium is in the stationary phase. The actual culture time can then be determined based on the stage of mycelium culture.

[0050] Step S33: Based on the stage culture time and the actual culture time, determine the predicted culture time and shake flask culture time of the strain in shaker culture, and based on the predicted culture time and shake flask culture time, determine the adjustment method of shaker speed / culture medium pH value; Specifically, in step S33, the predicted culture time is determined based on the stage culture time, and the shake flask culture time is determined based on the actual culture time.

[0051] It is understood that the stage cultivation duration refers to the cultivation duration corresponding to each cultivation stage (initial adjustment period, logarithmic growth period, and stationary period) under the prediction model, while the actual cultivation duration refers to the cultivation duration corresponding to each cultivation stage in the actual cultivation process.

[0052] In one specific embodiment, the predicted culture duration is the sum of the initial adjustment period and the logarithmic growth period corresponding to the prediction model, and the shake flask culture duration is the sum of the actual culture duration corresponding to the initial adjustment period and the logarithmic growth period in the actual culture process.

[0053] This invention determines the culture time of the strain in the shaker culture by considering both the stage culture time and the actual culture time. This comprehensive consideration of theoretical and actual culture conditions helps to adjust the shaker parameters in the future, improves the accuracy and controllability of the strain culture process, and stably and efficiently cultivates strains that meet the requirements, thereby improving the success rate of culture and product quality.

[0054] Please see Figure 4 As shown, this is a determination diagram for the adjustment method in an embodiment of the present invention. Specifically, in step S33, the adjustment method includes: If the shake flask culture time is longer than the predicted culture time, increase the shaker speed or adjust the pH of the culture medium for the initial culture conditions of the next batch. If the actual culture time of the initial adjustment period is longer than the corresponding stage culture time, then increase the shaking speed of the current batch during the logarithmic growth phase or adjust the pH of the culture medium.

[0055] Understandably, if the actual culture time of the initial adjustment period is longer than the stage culture time of the initial adjustment period for the current batch, it means that the strain is growing slowly during the initial adjustment period. In this case, the shaking speed can be increased or the pH of the culture medium can be adjusted during the logarithmic growth phase of the current batch to promote the growth of the strain.

[0056] For the next batch of adjustments, if the shake-flask culture time exceeds the predicted time, it indicates that the fungal growth rate is slow or the culture conditions are not ideal. In this case, the shaker speed or the pH of the culture medium can be adjusted in the next batch. Increasing the shaker speed increases the turbulence of the culture medium, allowing oxygen to dissolve better, increasing dissolved oxygen levels, providing more oxygen for the fungal strain, promoting its metabolism, and accelerating its growth. Simultaneously, it ensures that nutrients are evenly distributed in the culture medium, facilitating absorption by the matsutake mycelium cells and shortening the culture time. Adjusting the pH can also promote fungal growth and balance the acidity / alkalinity of the culture environment.

[0057] This invention determines the shake-flask culture time of the strain in shaker culture based on the stage culture time and the actual culture time. It also determines the adjustment method for the shaker speed / culture medium pH value of the initial culture conditions for the next batch based on the shake-flask culture time and the predicted culture time. Furthermore, it determines the adjustment method for the shaker speed / culture medium pH value of the current batch during the logarithmic growth phase based on the stage culture time of the initial adjustment period and the actual culture time of the initial adjustment period. This significantly enhances the mixing degree of the culture medium, promotes a substantial increase in dissolved oxygen efficiency, and allows nutrients to be distributed more rapidly and evenly around the Matsutake mycelial cells, providing them with more sufficient oxygen and nutrients for growth. This effectively accelerates the mycelial growth rate while ensuring that the quality of the strain remains unaffected.

[0058] Specifically, in step S33, for the next batch adjustment, the increase in the shaking speed of the initial culture conditions for the next batch is determined based on the culture medium volume and culture container volume of the current batch, the shake flask culture time, the predicted culture time, and the initial shaking speed. For the current batch adjustment, the increase in shaker speed during the logarithmic growth phase of the current batch is determined based on the deviation between the actual culture time of the initial adjustment period and the stage culture time of the initial adjustment period.

[0059] Understandably, if the culture container is large or the culture medium volume is large, a higher shaking speed is needed to ensure sufficient oxygen supply and uniform nutrient distribution; conversely, if the culture container is small or the culture medium volume is small, a lower shaking speed is sufficient. Therefore, the culture medium volume and culture container volume are taken into account in determining the increase in speed, and the initial shaking speed refers to the initial shaking speed corresponding to different stages of microbial culture.

[0060] In one specific embodiment, the initial shaking speed ranges from 120 rpm to 140 rpm, preferably 130 rpm. For the next batch adjustment, the increase in shaking speed is = ; The initial shaking speed of the next batch = the initial shaking speed of the current batch + the speed increase.

[0061] For the current batch adjustment, the increase in shaker speed is determined based on the deviation between the actual culture time during the initial adjustment period and the stage culture time: for every 0.5 days increase in deviation, the shaker speed increases by 10 r / min. In practice, the range and preferred value of the initial shaker speed can be determined according to the actual situation, and are not specifically limited here, nor will they be elaborated further.

[0062] Specifically, in step S33, adjusting the pH of the culture medium includes: If the pH value of the culture medium is greater than the preset pH value, then the pH value of the culture medium is lowered. If the pH value of the culture medium is lower than the preset pH value, then the pH value of the culture medium is increased.

[0063] Understandably, when the incubation time exceeds the preset time, the direction of pH adjustment needs to be determined based on the preset pH value. If the strain produces alkaline substances during metabolism or consumes a large amount of acidic substances, the pH of the culture medium will rise. In this case, acidic substances need to be added to lower the pH to the preset pH value. If the strain produces acidic metabolic products, such as lactic acid, acetic acid, or citric acid, the pH of the culture medium will drop. In this case, alkaline substances need to be added to raise the pH.

[0064] In one specific embodiment, the preset pH value is in the range of 5 to 6, preferably 5.5. Preferably, the range and preferred value of the preset pH value can be determined according to the actual situation, and are not specifically limited here, nor will they be elaborated further.

[0065] Step S34: Determine the timing of completion of matsutake mushroom strain isolation and cultivation based on the strain culture stage.

[0066] Specifically, when the slope of the cell growth curve first drops to near 0, the logarithmic growth phase is considered to have ended and the strain has entered the stationary phase, at which point the isolation and cultivation of the matsutake mushroom strain is complete. Stopping cultivation at the point when the logarithmic growth phase transitions to the stationary phase is optimal, as this allows for the acquisition of a large quantity of strains with strong viability and activity.

[0067] Example 1: Primary strain cultivation: Collect 75% mature, insect-free matsutake mushrooms. Disinfect the fruiting bodies with 75% alcohol in a sterile room under sterile conditions. Then, use a scalpel to longitudinally halve the matsutake mushrooms, and cut off a predetermined size of 0.5 cm from the cap and stem. The bacterial culture medium was transferred into a test tube 20 cm high and 3 cm in diameter containing slant culture medium. After inoculation, it was cultured at a constant temperature of 25°C in the dark for 9 days until the mycelium filled the test tube and formed the primary strain. The slant culture medium consisted of 2 parts glucose, 0.1 parts magnesium sulfate, 2 parts agar and 0.5 parts yeast extract.

[0068] Primary shake flask: Prepare the culture medium, which consists of 2 parts glucose, 2 parts sucrose, 1 part yeast powder, 1 part milk powder, 0.12 parts peptone, 0.15 parts potassium dihydrogen phosphate, 0.01 parts magnesium sulfate, 0.005 parts vitamin B1, and 0.005 parts vitamin B2. Inoculate the primary strain at a rate of 1% into a 250ml Erlenmeyer flask containing 100ml of culture medium and incubate at a constant temperature of 25℃ on a shaker for 8 days to obtain the primary shake flask strain. Secondary shake flask: Inoculate the primary shake flask strain at a rate of 10% into a 500ml Erlenmeyer flask containing 200ml of culture medium, and incubate at a constant temperature of 25℃ on a shaker until the Matsutake mushroom strain isolation and cultivation are completed; During the secondary shake-flask culture, metabolite concentrations, dissolved oxygen levels, and pH values ​​were collected in real-time after culture to construct a predictive model for the fungal growth time. The cell proliferation trend was determined by the number of Matsutake mycelial cells. In the initial adjustment period, the predicted culture duration based on the fungal growth time prediction model was 2 days, with a logarithmic growth phase of 6 days and a stationary phase of 5 days, after which the growth began to decline. The actual measured culture duration for the initial adjustment period was 2.5 days, with a logarithmic growth phase of 7 days, followed by a stationary phase. Once the fungus entered the stationary phase from the logarithmic growth phase, the Matsutake fungal strain isolation and cultivation were completed. The predicted culture duration for the fungal culture phase was 8 days, while the shake-flask culture duration was 9.5 days, lagging behind the predicted culture duration by 1.5 days.

[0069] Example 2 The difference from Example 1 is that the actual measured initial adjustment period was 0.5 days longer than the predicted initial adjustment period. Therefore, starting from the 12th hour of the initial adjustment period, the shaking speed was adjusted using the method described in this invention. The initial shaking speed before adjustment was 130 rpm, the pH value of the culture medium was 6.5, and the shaking speed after adjustment was 140 rpm. The culture time is shown in Table 1 below.

[0070] Example 3 The difference from Example 1 is that the actual measured duration of the logarithmic growth phase was one day longer than the predicted duration. Therefore, starting from the 4th day, the shaking speed was adjusted using the method described in this invention. The shaking speed before adjustment was 130 rpm, the pH of the culture medium was 4.8, and the shaking speed after adjustment was 140 rpm. The culture duration is shown in Table 1 below.

[0071] Example 4 The difference from Example 2 is that the actual measured initial adjustment period was 0.5 days longer than the predicted initial adjustment period. Therefore, starting from the 12th hour of the initial adjustment period, the pH value of the culture medium was adjusted using the method described in this invention. The pH value of the culture medium before adjustment was 4.8, and the pH value after adjustment was 5.5. The culture time is shown in Table 1 below.

[0072] Example 5 The difference from Example 3 is that the actual measured duration of the logarithmic growth phase was one day longer than the predicted duration. Therefore, starting from the fourth day, the pH of the culture medium was adjusted using the method described in this invention. The pH of the culture medium before adjustment was 5.6, and the pH after adjustment was 5.8. The culture duration is shown in Table 1 below.

[0073] Table 1. Comparison of predicted and actual culture durations after adjusting culture parameters.

[0074] As shown in Table 1 above, in Example 1, the shaking speed and pH of the culture medium were not adjusted, and the predicted and actual culture times differed by 1.5 days. In Example 2, the shaking speed was adjusted to 140 rpm and the pH of the culture medium was adjusted to 5.5 starting after 12 hours of culture, i.e., adjustments were made during the initial adjustment period. The predicted culture time was 7.8 days, and the actual culture time was 8.5 days, a difference of 0.7 days. In Example 3, the shaking speed was adjusted to 140 rpm and the pH of the culture medium was adjusted to 5.5 starting after the fourth day of culture, with a time difference of 0.25 days. This demonstrates that appropriately increasing the shaking speed allows air to dissolve better in the culture medium, providing more oxygen for microorganisms to use for respiration. Sufficient oxygen supply is beneficial to the metabolism of microorganisms, thereby accelerating growth and shortening the culture time.

[0075] Furthermore, as can be seen from Examples 4 and 5, adjusting the pH of the culture medium to 5.5 is closer to the optimal pH range for matsutake growth. When the pH is within a suitable range, the enzyme activity within the microbial cells is higher, which helps the cells carry out various biochemical reactions, such as the absorption of nutrients and the synthesis of metabolites, thereby promoting the growth and reproduction of microorganisms and shortening the culture time.

[0076] It should be noted that Examples 2-5 show experimental data with parameter adjustments within the current batch. The differences in the predicted culture time are due to individual differences between different batches. The different concentrations of metabolites, dissolved oxygen, and pH values ​​in the first 4 hours after inoculation lead to different outputs of the prediction model.

[0077] This application further proposes a next-batch adjustment scheme based on the complete cycle deviation (Examples 6-9). This scheme uses a unified prediction model to predict the culture duration to remain at 8 days, in order to more clearly verify the adjustment effect. The two adjustment methods can be used separately or in combination, both of which can effectively optimize the culture conditions. The verification data are shown in Table 2 and Examples 6-9.

[0078] Example 6: The difference from Example 1 is that, after completing the entire culture cycle, the shake flask culture time was 9.5 days, while the predicted culture time was 8 days, meaning the shake flask culture time was 1.5 days longer than the predicted culture time. This indicates that the overall growth of this batch was slow.

[0079] The comparison results were used to preset the initial culture conditions for the next batch: when cultivating the next batch, the initial shaking speed was increased from 130 r / min to 140 r / min.

[0080] The results of the next batch of culture: the predicted culture time was 8 days, and the actual culture time in shake flasks was 8.5 days, reducing the deviation to 0.5 days. Compared with the unadjusted Example 1, the deviation was reduced from 1.5 days to 0.5 days, verifying the effectiveness of the adjustment for the next batch. The results are shown in Table 2.

[0081] Example 7: The difference from Example 1 is that after completing the entire culture cycle, the shake flask culture time was 9.5 days, the predicted culture time was 8 days, the shake flask culture time was 1.5 days longer than the predicted culture time, and the pH value was lower during the culture process.

[0082] The comparison results were used to preset the initial culture conditions for the next batch: when conducting the next batch of cultivation, the pH of the initial culture medium was adjusted from 5.0 to 5.5.

[0083] The results of the next batch of culture: the predicted culture time was 8 days, the actual culture time in shake flasks was 8.4 days, the deviation was reduced to 0.4 days, the quality of the strain was good, and the results are shown in Table 2.

[0084] Example 8: The difference from Example 6 is that when using the comparison results to preset the initial culture conditions for the next batch, the initial shaking speed and initial pH value are adjusted simultaneously: the initial shaking speed is increased from 130 r / min to 140 r / min, and the initial pH value is adjusted from 5.0 to 5.5.

[0085] The results of the next batch of culture: the predicted culture time was 8 days, and the actual culture time in shake flasks was 8.3 days, with the deviation reduced to 0.3 days, which verified the effectiveness of the combination adjustment. The results are shown in Table 2.

[0086] Example 9: Three batches of matsutake mushroom spawn were cultivated consecutively, with each batch using the same adjustment method as the next batch: Batch 1: Initial shaker speed 130 r / min, actual shake flask culture time 9.5 days, predicted culture time 8 days, deviation +1.5 days.

[0087] Batch 2: Based on the deviation of Batch 1, the initial shaking speed was increased to 140 r / min. The actual shake flask culture time was 8.5 days, the predicted culture time was 8 days, and the deviation was +0.5 days.

[0088] Batch 3: Based on the deviation of batch 2, the initial shaking speed was increased to 145 r / min. The actual shake flask culture time was 8.2 days, the predicted culture time was 8 days, and the deviation was +0.2 days.

[0089] Through multiple batches of iterative optimization, the cultivation conditions gradually approached the optimal state, and the deviation in cultivation time was reduced batch by batch. The results are shown in Table 2.

[0090] Table 2 Experimental results of the next batch adjustment and multi-batch iterative optimization

[0091] As can be seen from Table 2: By adjusting the conditions in the next batch (Examples 6 and 7), the overall deviation of the current batch was used as the initial culture conditions for the next batch, and the deviation was reduced from 1.5 days to 0.5 days and 0.4 days, respectively, verifying the effectiveness of the adjustment in the next batch. By simultaneously adjusting the speed and pH value (Example 8), the deviation was further reduced to 0.3 days. Through multi-batch iterative optimization (Example 9), the deviation was reduced batch by batch to 0.2 days, verifying the continuous optimization effect of the technical solution of this application.

[0092] This invention combines constant temperature and shaking culture at different cultivation stages to provide a suitable temperature environment and sufficient oxygen supply for the growth of matsutake mushroom spores. Simultaneously, by collecting real-time data on metabolic product concentration, dissolved oxygen content in the culture medium, and pH value, a spore growth time prediction model is constructed, which can accurately predict the corresponding cultivation duration for each stage of the spore cultivation process. Furthermore, by collecting real-time data on the mycelial cell count in the culture medium, the number of matsutake mycelial cells is determined, thereby identifying cell proliferation trends and actual cultivation duration. This allows for more precise monitoring of the spore's growth status at different cultivation stages. Based on the stage cultivation duration and the actual cultivation duration, the shake-flask cultivation time in the shaking culture is determined, and the shaking speed or frequency is adjusted to flexibly optimize cultivation conditions, further promoting the growth and metabolism of matsutake mycelium and improving the growth rate and quality of the spore.

[0093] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles 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 scope of protection of the present invention.

Claims

1. A method for isolating and cultivating fungal strains from matsutake fruiting bodies, characterized in that, include: Step S1: Collect 70%–80% mature insect-free matsutake mushrooms, disinfect the fruiting bodies of insect-free matsutake mushrooms with alcohol under sterile conditions, cut the disinfected matsutake mushrooms in half longitudinally, cut off the pre-sized flesh at the cap and stipe, and inoculate it into a test tube containing slant culture medium. After inoculation, culture at a constant temperature of 24℃–26℃ in the dark until the mycelium fills the test tube to obtain the primary strain. Step S2: Inoculate the primary bacterial strain into the culture medium at an inoculation rate of 0.5% to 1.2%, and culture at a constant temperature of 24℃ to 26℃ on a shaker to obtain the primary shake flask bacterial strain; Step S3: Inoculate the primary shake flask strain into the culture medium at an inoculation rate of 9.5% to 11%, and culture at a constant temperature of 24℃ to 26℃ on a shaker. At this point, the isolation and cultivation of matsutake mushroom strain is completed. Step S3 includes: Step S31: Collect the concentration of metabolites, dissolved oxygen in the culture medium and pH value in real time to construct a bacterial growth time prediction model. Based on the bacterial growth time prediction model, predict the stage culture time corresponding to the bacterial culture stage. The bacterial culture stage includes the initial adjustment period, the logarithmic growth period and the stationary period. Step S32: Collect bacterial culture in the culture medium in real time and determine the number of matsutake mycelial cells. Determine the cell proliferation trend based on the number of matsutake mycelial cells. Determine the actual culture time corresponding to the culture stage based on the cell proliferation trend. Step S33: Based on the stage culture time and the actual culture time, determine the predicted culture time and shake flask culture time of the strain in shaker culture, and based on the predicted culture time and shake flask culture time, determine the adjustment method of shaker speed / culture medium pH value; Step S34: Determine the timing of completion of matsutake mushroom strain isolation and cultivation based on the strain culture stage.

2. The method for isolating and cultivating Matsutake mushroom spawn according to claim 1, characterized in that, Step S33 also includes determining the adjustment method for the current batch's logarithmic growth phase based on the comparison between the actual culture duration of the initial adjustment period and the stage culture duration of the initial adjustment period.

3. The method for isolating and cultivating Matsutake fruiting bodies according to claim 1, characterized in that, Step S31 further includes: The concentrations of the metabolites, dissolved oxygen in the culture medium, and pH values ​​were subjected to noise removal and standardization. The characteristic correlation coefficients between metabolite concentration, dissolved oxygen, and pH value were determined based on the concentration of metabolites, dissolved oxygen, and pH value, respectively. A growth time prediction model for the bacterial strain is constructed based on the aforementioned feature correlation coefficient, metabolite concentration, dissolved oxygen content, and pH value. The characteristic correlation coefficients include a first correlation coefficient between metabolite concentration and dissolved oxygen, a second correlation coefficient between metabolite concentration and pH value, and a third correlation coefficient between dissolved oxygen value and pH value.

4. The method for isolating and cultivating Matsutake fruiting bodies according to claim 3, characterized in that, In step S31, the weights of metabolite concentration, dissolved oxygen 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 isolating and cultivating Matsutake mushroom spawn according to claim 4, characterized in that, Step S31 further includes: determining the 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 isolating and cultivating Matsutake fruiting bodies according to claim 1, characterized in that, In step S32, a cell growth curve of matsutake mycelial cells is constructed based on the number of matsutake mycelial cells, cell proliferation characteristics are determined based on the cell growth curve, the corresponding culture stage of the strain is determined based on the cell proliferation characteristics, and the actual culture time is determined based on the current culture stage of the strain.

7. The method for isolating and cultivating Matsutake fruiting bodies according to claim 2, characterized in that, In step S33, the predicted culture time is determined based on the stage culture time, and the shake flask culture time is determined based on the actual culture time.

8. The method for isolating and cultivating Matsutake mushroom spawn according to claim 7, characterized in that, In step S33, the adjustment method includes: If the shake flask culture time is longer than the predicted culture time, increase the shaker speed or adjust the pH of the culture medium for the initial culture conditions of the next batch. If the actual culture time of the initial adjustment period is longer than the corresponding stage culture time, then increase the shaking speed of the current batch during the logarithmic growth phase or adjust the pH of the culture medium.

9. The method for isolating and cultivating Matsutake mushroom spawn according to claim 8, characterized in that, In step S33, the increase in the shaking speed of the initial culture conditions for the next batch is determined based on the current batch's culture medium volume and culture container volume, the shake flask culture time, the predicted culture time, and the initial shaking speed. The rate increase of the shaker speed during the current batch's logarithmic growth phase is determined based on the deviation between the actual culture time during the initial adjustment period and the stage culture time.

10. The method for isolating and cultivating Matsutake fruiting bodies according to claim 9, characterized in that, In step S33, adjusting the pH of the culture medium includes: If the pH value of the culture medium is greater than the preset pH value, then the pH value of the culture medium is lowered. If the pH value of the culture medium is lower than the preset pH value, then the pH value of the culture medium is increased.

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