Method for accurately analyzing matrix utilization rate of phlebopus portentosus

Abstract

The invention relates to the technical field of edible fungus cultivation, in particular to a method for accurately analyzing the matrix utilization rate of phlebopus portentosus, which comprises the following steps: performing conventional cultivation on phlebopus portentosus by using a cultivation matrix with grains as nutrient substances; after matrix samples at different cultivation stages are rubbed, grains are separated and dried, and shriveled grains are removed; randomly selecting 100 grains from the grain sample, and weighing to obtain the dry weight of 100 grains; 100 grain samples selected at different cultivation stages are hulled, the dry weight of 100 grains is calculated, and then the weight loss percentage of the rice grains and the accumulated weight loss percentage are calculated; and analyzing to obtain the utilization conditions of the phlebopus portentosus on the substrate in different cultivation stages. According to the method, the utilization condition of the matrix can be accurately analyzed in the culture process of the phlebopus portentosus, and timely and accurate adjustment reference data is provided for subsequent artificial cultivation of the phlebopus portentosus.

Description

Technical Field

[0001] This invention relates to the field of edible fungi cultivation technology, specifically to a method for accurately analyzing the substrate utilization rate of Boletus thunbergii. Background Technology

[0002] Dark brown stalk Boletus ( Phlebopus portentosus ), also known as black boletus or giant boletus, belongs to the order Boletales ( Boletales ), Boletaceae ( Boletinelaceae ), Boletus genus ( Phlebopus *Boletus davidii*, the first edible bolete to be successfully cultivated artificially, is now cultivated on a large scale in factories. *Boletus davidii* has weak saprophytic properties and lacks a complete cellulase and ligninase system, making it unable to effectively utilize lignocellulose. In artificial cultivation, it primarily relies on its complete starch-hydrolyzing enzyme system to degrade grain starch and obtain nutrients. Anatomical observations of grains at various cultivation stages revealed that *Boletus davidii* mycelia first invade from the opening, gradually decomposing and utilizing the rice grain starch granules. After cultivation, the starch granules are usually not completely utilized. Therefore, improving the utilization rate of grain starch has become a pressing scientific problem in artificial cultivation, and one of the foundations for solving this problem is the ability to accurately analyze the substrate utilization rate of *Boletus davidii*.

[0003] The main nutrient in the cultivation substrate of *Boletus pumilus* is grains, accounting for about 30%. These grains consist of white rice grains encased in a tightly packed seed coat, bran, and hard husk. The rice grains are rich in nutrients, primarily starch and protein, with a starch content as high as 80%. The seed coat and bran contain abundant protein, vitamins, and minerals, while the husk is composed of coarse fiber and silica. Dissection and microscopic observation of grains at different cultivation stages revealed that *Boletus pumilus* hyphae utilize the starch in the grains through degradation, absorption, and transformation. After soaking and sterilization, the grains only have small openings, and the rice grains remain encased in the husk. After inoculation, *Boletus pumilus* hyphae grow into the grains through these openings and gradually decompose and utilize the starch. Throughout the cultivation cycle, the rice grains remain encased in the husk; apart from being decomposed and absorbed by the hyphae, the starch cannot naturally detach from the husk, and the contents do not seep out during later grain separation. Furthermore, sawdust, brown clay, and wood chips constitute the majority of the volume and weight of the Boletus thunbergii cultivation substrate, and these components are not effectively utilized during cultivation, resulting in minimal overall weight loss. Therefore, the bioavailability of the Boletus thunbergii cultivation substrate is low.

[0004] In the cultivation of Boletus thunbergii, substrate utilization efficiency has traditionally been assessed by directly weighing or analyzing the remaining nutrients in the mixed substrate. However, post-fruiting biological efficiency assessments are unsuitable for Boletus thunbergii because it primarily utilizes single components, and the most abundant component is not lignocellulose. Furthermore, differences in starch and protein content among substrate materials from different sources, as well as variations between different strains, lead to significant differences in cultivation results for Boletus thunbergii substrates from different origins. This makes it difficult to obtain universally effective and efficient cultivation techniques for Boletus thunbergii, and the component contents of the culture medium need to be adjusted according to specific conditions.

[0005] In summary, there is a need for an accurate method to analyze the substrate utilization rate of Boletus thunbergii, which can promptly analyze the substrate utilization of the currently cultivated Boletus thunbergii and enable accurate adjustments to the substrate and cultivation methods during the cultivation process. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for accurately analyzing the substrate utilization rate of Boletus thunbergii. This method enables accurate analysis of substrate utilization during the cultivation of Boletus thunbergii, providing timely and accurate adjustment reference data for subsequent artificial cultivation of Boletus thunbergii.

[0007] The objective of this invention is achieved through the following technical solution: On the one hand, this application provides a method for accurately analyzing the matrix utilization rate of Boletus thunbergii, comprising the following steps: S1. The *Boletus edulis* was conventionally cultivated using a cultivation substrate containing grains as nutrients, and substrate samples were taken at different cultivation stages. S2. After crushing the substrate samples from different cultivation stages, separate the grains and dry them, remove the shriveled grains, and obtain grain samples from different cultivation stages. S3. Randomly select 100 grains from the grain sample and weigh them to obtain the dry weight of 100 grains; S4. Dehull 100 grain samples selected from different cultivation stages, weigh the dehulled grains to obtain the dry weight of 100 grains of hulled grains, calculate the dry weight of 100 grains of rice, and then calculate the percentage of weight loss of rice grains and the cumulative percentage of weight loss. S5. Analyze the average and standard deviation of the weight, the percentage of weight loss of the rice grains and the cumulative percentage of weight loss to obtain the utilization of the substrate by the dark brown stalked Boletus at different cultivation stages.

[0008] Furthermore, in addition to grains as the main nutrient, the cultivation substrate also includes sawdust as a skeletal support, sawdust for filling micro-pores and slow-release water retention, and red soil that must be added but whose function is not yet clear.

[0009] Furthermore, the cultivation stages are as follows: the initial inoculation stage after sterilization; the half-bag stage where dark brown stamen Boletus mycelium appears in half of the bag; the full-bag stage where the entire bag is filled with dark brown stamen Boletus mycelium; the primordia formation stage where dark brown stamen Boletus buds just appear; the young mushroom stage I (approximately 1.0 g in weight and 20 mm in length); the young mushroom stage II (approximately 10.0 g in weight and 40 mm in length); the young mushroom stage III (approximately 40.0 g in weight and 60 mm in length); the young mushroom stage IV (approximately 60.0 g in weight and 80 mm in length); the finished mushroom (approximately 120.0 g in weight and 100 mm in length); and the mature mushroom (approximately 125.0 g in weight and 105 mm in length). mm), the inter-flush period (referring to the period between the harvest of mature mushrooms and the formation of the second primordia); among which, the sampling time point for the inter-flush period is the 7th day after the harvest of mature mushrooms and the continued cultivation of the substrate.

[0010] Furthermore, the substrate samples used in this application are the substrate remaining after cross-sectioning of the culture bags at each stage and removing the top 2 cm thick substrate layer.

[0011] Furthermore, the calculation method for the dry weight of 100 rice grains is: Dry weight of 100 rice grains = Dry weight of 100 grains of rice - Dry weight of 100 grains of rice husks.

[0012] Furthermore, the drying process involves drying the grains at 60°C until they are completely dry, thereby preventing residual moisture from interfering with the data obtained when the balance is used to weigh the grains and husks in subsequent analysis.

[0013] Furthermore, the steps for separating the grains include: first, using a sieve with a 5 mm aperture to remove sawdust, then using a 2 mm sieve to remove sawdust, and then rinsing off the red soil and mycelium on the surface of the grains with water.

[0014] On the other hand, this application provides the application of the above-mentioned method for accurately analyzing the substrate utilization rate of Boletus thunbergii in the high-efficiency cultivation technology of Boletus thunbergii.

[0015] It is worth noting that the rice added to the substrate meets the accuracy requirements of the analysis in terms of nutritional composition. The openings in the sterilized rice husks allow mycelia to grow in and degrade the starch, while the chemical components of the husks cannot be degraded and utilized by the mycelia. This ensures that the rice grains are protected throughout the entire cultivation process, allowing for complete separation of the grains at each stage. This application utilizes these characteristics to establish a method for evaluating material utilization efficiency through grain utilization rate analysis, providing a basis for parameter adjustment in the subsequent development of high-efficiency cultivation technologies. The separation process not only ensures the elimination of interference from large quantities of undegradable components but also ensures that the main variable (residual starch) is not lost during the separation process. The established method is convenient, accurate, reliable, and reproducible.

[0016] The beneficial effects of this invention are: 1. The method of this application is based on the dry weight measurement of the isolated grains, which can more accurately reflect the utilization pattern of grain nutrients by mycelium; 2. The method of this application analyzes the dry weight of 100 grains of rice and 100 grains of wheat, which reduces the interference of non-target variables, has a smaller standard deviation, more stable data, and can clearly show the consumption dynamics of grains at different stages, and can even detect slight fluctuations. 3. This application uses "percentage weight loss per 100 grains" to directly reflect the proportion of grains consumed by mycelium, which can reflect the actual utilization efficiency of mycelium on grains. Attached Figure Description

[0017] Figure 1 Dry weight of 100 grains of rice at different stages; Figure 2 This is a graph showing the weight loss analysis of rice grains. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0019] Source of experimental materials: Strain: Boletus davidii 15018, obtained from wild fruiting bodies collected on August 23, 2015, and domesticated through tissue isolation. Fruiting occurred after 16–20 days of soil covering, with an average single mushroom weight ≥102.0 g. The maturity rate in the experimental mushroom house was ≥78.5%, and there was no earthy smell. It is deposited in the strain preservation room of the Plant Protection and Microbial Utilization Research Center, Yunnan Tropical Crops Research Institute.

[0020] Example 1 Sampling Using grains, sawdust, and red soil as cultivation substrates, *Boletus glomeratus* strain 15018 was conventionally cultivated. Three biological replicates were set up. The cultivation process included the following stages: the initial inoculation stage immediately after sterilization; the half-bag stage (mycelium of *Boletus glomeratus* appears in half the bag); the full-bag stage (mycelium is evenly distributed throughout the bag); the primordia formation stage (newly emerging buds of *Boletus glomeratus*); the young mushroom stage I (approximately 1.0 g, approximately 20 mm in length); the young mushroom stage II (approximately 10.0 g, approximately 40 mm in length); the young mushroom stage III (approximately 40.0 g, approximately 60 mm in length); the young mushroom stage IV (approximately 60.0 g, approximately 80 mm in length); the mature mushroom stage (approximately 120.0 g, approximately 100 mm in length); and the fully mature mushroom stage (approximately 125.0 g, approximately 105 mm in length). The cultivation substrate was sampled at 11 stages, including 1 mm), 7 days after the mature mushrooms were harvested, and 2 cm of substrate layer was removed during sampling.

[0021] Example 2: Determination of dry weight of 100 grains: Grain separation: The substrate samples obtained from different cultivation stages are crushed, sawdust is removed by sieving through a 5 mm sieve, and sawdust is removed by sieving through a 2 mm sieve. Then, the surface red soil and mycelium are washed away with water to obtain clean grains.

[0022] Grain drying: Grains at different cultivation stages are dried at 60℃ until completely dry, and shriveled grains are carefully blown away with a hair dryer.

[0023] Determination of 100-grain dry weight: Dry grains from different stages were mixed separately and then randomly divided into 5 groups. 100 grains were randomly selected from each group and weighed using a Mettler ME204E analytical balance. The mean and standard deviation of the 100-grain dry weight were calculated.

[0024] Rice grain dry weight determination: After the rice grain dry weight determination is completed, the rice grains are gently pressed and the husks are separated from the remaining rice grains with tweezers. Then the weight of the husks is weighed. According to the formula rice grain dry weight = rice grain dry weight - rice husk dry weight, the average value of rice grain dry weight is obtained. Then the percentage of rice grain weight loss at each stage and the cumulative percentage of rice grain weight loss are calculated.

[0025] Then, 100 plump, fresh grains were randomly selected from 5 groups for drying and weighing. The average and standard deviation of the dry weight of 100 freshly dried grains were obtained. After organizing all the above data, Tables 1 and 2 were obtained, showing the dry weight of 100 grains and standard deviation, the percentage of weight loss / cumulative weight loss and standard deviation of rice grains, and so on. Figure 1 and Figure 2 The graph shows the analysis of 100 grain dry weight and grain weight loss at different stages.

[0026] Table 1. Dry weight of 100 grains of rice / grain and standard deviation (unit: g) Table 2. Percentage of weight loss per grain / cumulative weight loss and standard deviation like Figure 1 As shown, the dry weight of 100 grains generally decreased throughout the cultivation cycle, with a total decrease of 1.2869 g. The largest decrease occurred during the mycelial growth stage (from the bag-forming stage to the full-coverage stage), with a decrease of 0.9962 g. From the primordia formation stage to the young mushroom stage (stage IV), the dry weight of 100 grains continued to decrease, with significant differences between different stages, a decrease of 0.2274 g. From stage IV to the mature mushroom stage, the fruiting bodies grew rapidly, but the decrease in dry weight of 100 grains was not significant. After harvesting the fruiting bodies, during the inter-flush stage, the difference in dry weight of 100 grains became significant, with a decrease of 0.0929 g.

[0027] like Figure 2 As shown: ① During the mycelial growth stage (from the bag stage to the stage of full mycelial coverage), the weight of rice grains decreased by 56.41%, accounting for 74.75% of the total weight loss. The rice grains were utilized rapidly, resulting in the largest weight loss. ② During the mushroom cultivation stage (from the primordia formation stage to the mature mushroom stage), the weight of rice grains decreased by 14.43%, accounting for 19.12% of the total weight loss. The cumulative weight loss increased to 70.84%. The dry weight of rice grains continued to decrease, but the rate of decrease was relatively small. ③ During the inter-hydatid period, the weight loss was 4.61%. The rate of decrease increased, and the utilization of rice grains was accelerated, accumulating substances and energy for the second flush of mushrooms.

[0028] Comparative Example 1: Cultivation Substrate Drying and Weighing Method To highlight the effectiveness of the proposed solution, this application uses the direct drying of the entire substrate bag and the measurement of the total substrate dry weight (including grains and other substrate components) as a comparative example, and calculates the cumulative weight loss and weight loss percentage, resulting in the substrate change table for different cultivation stages shown in Table 3.

[0029] Table 3. Substrate Changes at Different Cultivation Stages As can be seen from the data in Tables 1-3, the comparative example measures the dry weight of the entire mushroom bag, including grains, lignocellulose, and mycelium. The weight loss in this example may be due to the consumption of multiple components, failing to accurately reflect the utilization of the grains themselves. In contrast, the method in this application directly separates and measures the dry weight of the grains, more accurately reflecting the mycelium's utilization of grain nutrients. Secondly, the dry weight of the mushroom bag in the comparative example is significantly affected by other components (such as moisture, impurities, and mycelial biomass), resulting in a relatively high standard deviation. This application, by separating the grains and using a hundred-grain counting method, reduces the interference of non-target variables, thus lowering the standard deviation. Smaller dimensions (e.g., the standard deviation of grain dry weight in the "sterilization kit" stage is only 0.0174) result in more stable data. Furthermore, the comparative method is affected by the overall matrix, and the stage changes are relatively gradual, making it impossible to capture subtle changes. In contrast, this application can clearly show the consumption dynamics of grains at different stages and can even detect slight fluctuations. In addition, the "cumulative weight loss percentage" in the comparative method includes the consumption of nutrient matrix from non-grain sources (such as lignocellulose), which is highly misleading. In contrast, the "cumulative weight loss percentage of rice grains" in this application directly reflects the proportion of grains consumed by mycelium, which is more in line with the research objective (the law of nutrient utilization).

[0030] In summary, the method of separating grains first, drying them, and then counting 100 grains used in this application can accurately anchor the nutritional components of grains, eliminate interference from other matrix components, and produce data with a smaller standard deviation and better repeatability when studying the utilization pattern of grains by Boletus thunbergii. It can also sensitively capture the dynamics of grain consumption at each cultivation stage and more directly reflect the actual utilization efficiency of mycelium on grains.

[0031] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. An accurate method for analyzing Boletus thunbergii (Brown stalk Boletus) Phlebopus portentosus The method for improving matrix utilization is characterized by, Includes the following steps: S1. The *Boletus edulis* was conventionally cultivated using a cultivation substrate containing grains as nutrients, and substrate samples were taken at different cultivation stages. S2. After crushing the substrate samples from different cultivation stages, separate the grains and dry them, remove the shriveled grains, and obtain grain samples from different cultivation stages. S3. Randomly select 100 grains from the grain sample and weigh them to obtain the dry weight of 100 grains; S4. Dehull 100 grain samples selected from different cultivation stages, weigh the dehulled grains to obtain the dry weight of 100 grains of hulled grains, calculate the dry weight of 100 grains of rice, and then calculate the percentage of weight loss of rice grains and the cumulative percentage of weight loss. S5. Analyze the average and standard deviation of the weight, the percentage of weight loss of the rice grains and the cumulative percentage of weight loss to obtain the utilization of the substrate by the dark brown stalked Boletus at different cultivation stages.

2. The method according to claim 1, characterized in that, The cultivation substrate also includes wood chips, sawdust, and red soil.

3. The method according to claim 1, characterized in that, The cultivation stages include the spawn bag stage, the half-bag stage, the full-bag stage, the primordia formation stage, the young mushroom stage I, the young mushroom stage II, the young mushroom stage III, the young mushroom stage IV, the finished mushroom, the mature mushroom, and the inter-flush stage. The inoculum period refers to the time point after substrate sterilization when inoculated with the inoculum. The half-bag period of mycelium refers to the time point at which dark brown veined Boletus mycelium appears in half of the bag. The period of full mycelial coverage is the time when the entire bag space is evenly covered with dark brown Boletus stalk mycelium. The primordium formation period is the period when the dark brown stalked Boletus buds have just appeared. The young mushrooms in stage I weighed approximately 1.0 g and were approximately 20 mm long. The young mushrooms in stage II weighed approximately 10.0 g and were approximately 40 mm long. The mushrooms in stage III of the young mushroom stage weighed approximately 40.0 g and were approximately 60 mm long. The young mushroom at stage IV weighed approximately 60.0 g and was approximately 80 mm long. The finished mushroom weighs approximately 120.0 g and is approximately 100 mm long; The mature mushroom weighs approximately 125.0 g and is approximately 105 mm long; The sampling time point for the interphase of the *Pleurotus ostreatus* was the 7th day after the mature mushrooms were harvested and the cultivation substrate was continued.

4. The method according to claim 1, characterized in that, The substrate samples were selected from the substrate remaining after the culture bags at each stage were cut crosswise and the top 2 cm thick substrate layer was removed.

5. The method according to claim 1, characterized in that, The method for calculating the dry weight of 100 rice grains is: Dry weight of 100 rice grains = Dry weight of 100 grains of rice - Dry weight of 100 grains of rice husks.

6. The method according to claim 1, characterized in that, The drying temperature is 60°C, and the grains are dried to an absolute dryness. And / or, the device used for weighing is an analytical balance.

7. The method according to claim 2, characterized in that, The steps for separating the grains include: first, using a sieve with a 5 mm aperture to remove sawdust, then using a 2 mm sieve to remove sawdust, and then rinsing off the red soil and mycelium on the surface of the grains with water.

8. The application of the method described in any one of claims 1 to 7 in the high-efficiency cultivation technology of Boletus thunbergii.

Images