Enzymology method for accurately monitoring maturity of lentinus edodes fungus bags

Abstract

The invention discloses an enzymology method for accurately monitoring the maturity of a mushroom bag, which comprises the following steps: (1) aiming at a studied strain; collecting fungus bags in different color changing stages according to the fungus age range in production practice, wherein the fungus bags are used for morphological characteristic observation, fruiting rate detection, cellobiohydrolase activity determination and yield determination of the first flush of mushrooms in a plurality of color changing stages with the fruiting rate of 100% before and after the fungus age; (2) establishing a relation between a cellobiohydrolase sudden rising node and a yield peak period; and (3) determining the fungus bags which are physiologically mature and can be used for bag removal and fruiting by monitoring the sudden rising nodes of cellobiohydrolase of mycelia in the fungus bags. According to the method for monitoring the maturity of the shiitake mushroom bags, the strains are cultivated in an industrialized mode, the maturity of the shiitake mushroom bags can be dynamically monitored by measuring the dynamic change rule of the activity of cellobiohydrolase in culture materials in different color changing periods, and the method has good market application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus cultivation, and in particular to an enzymatic method for accurately monitoring the maturity of shiitake mushroom spawn. Background Art

[0002] The physiological maturity of mycelium refers to the period from when the mycelium fills the container to before the mushrooms are produced, marking the transition of the mycelium from vegetative growth to reproductive growth (Jingyunge 2018, Du et al. 2019). Accurately judging the maturity of shiitake mushroom bags is the key to ensuring the smooth and high-quality completion of the shiitake mushroom cultivation process. If the bags are physiologically immature, they will not form fruiting bodies even under the induction of suitable environmental factors; if the bags are physiologically overmature, the fruiting body yield will decrease, and the cultivation cycle is too long, resulting in excessive production costs. Only in the appropriate physiological maturity state can the bags produce high-yield and high-quality fruiting bodies under low-temperature induction. At present, there is no clear definition of the different stages of the physiological maturity of shiitake mushroom bags, nor is there a matching quantifiable indicator for accurately monitoring the maturity of shiitake mushroom bags.

[0003] At present, in production practice, front-line production personnel generally roughly judge the maturity of mushroom bags based on practical experience such as the degree of color change, the hardness of the mushroom bags, the age of the mushrooms, and whether mushroom buds appear. For example, reddish brown is the most ideal, and soft and elastic mushroom bags are better (Huang Nianlai 1994, Li Ju and Zhou Wei 2002). However, due to the influence of factors such as the formula of the cultivation material, the stability of the raw materials, the accuracy of the environmental factors, and the judgment experience of the front-line production personnel, it is difficult to control the physiological maturity of the mushroom bags suitable for fruiting.

[0004] Based on the changes in the appearance and morphological characteristics of the mushroom bags, Wang Wenyan (2021), Si Zhenyu (2021), Wu Shumin (2024) and Wang Mingzhen (2024) used deep learning, software development and other technologies to establish an identification system and APP that can dynamically and quickly monitor the color change of the mushroom bags. This technology is suitable for factory-based cultivation enterprises with relatively stable strains, culture material formulas, and environmental control. Once a certain factor changes, the law of changes in the appearance and morphology of the mushroom bags is affected, and the system is no longer applicable and needs to be re-established, which is costly.

[0005] Many scholars start from the low-cost physicochemical indicators that can dynamically reveal the physiological state of mycelium inside the mushroom bag to explore suitable monitoring indicators or technologies. Zhang Zhenyue et al. (2017) believed that when the sugar content of the supernatant squeezed out of the mushroom bag is greater than 11 and the weight percentage of dry matter consumed is greater than 26.5%, the mushroom bag has reached physiological maturity. The activity of lignin-degrading enzymes is also often used as a monitoring indicator, but due to factors such as strains, culture formulas, cultivation conditions, and sampling time, the results of the same enzyme activity in different research reports are different.

[0006] However, a careful review will reveal that cellulase activity has an acute upward trend when entering reproductive growth, but the stage of reaching the peak varies slightly in different articles: some are in the primordium stage (Huang Kefu et al. 1985; Pan Yingjie et al. 1991; Zhang Xiaoyu 1995; Wang Weike et al. 2014), and some are in the fruiting body maturity stage (Liu Bin and Li Zhengxiang 1991; Zhang Quan 2016). Ohga et al. (2000) found that cellulase mRNA transcripts began to accumulate during the primordium formation stage and were expressed at the highest level when the fungus curtain broke. The biggest reason why different articles produce different nodes for the acute rise of cellulase activity is that there is no clear standard for measuring the maturity of the mushroom bag.

[0007] Many studies have found that before the formation of fruiting bodies, there is a significant accumulation of soluble carbohydrates (such as glycogen, trehalose and mannitol) inside the mycelium (Herman & Bleichrodt 2022; Nagy et al. 2023). Among them, trehalose and mannitol are synthesized from glycogen and fructose in the mycelium, respectively, and then transported to different parts of the fruiting body, providing carbohydrate substrates and osmotic agents for the growth and development of the fruiting body, respectively (Deveau et al. 2008; Patyshakuliyeva et al. 2013; Zhou et al. 2016; Nagy et al. 2023). From the metabolic pathways of trehalose, glycogen and mannitol ( Figure 1 ), they are all storage forms of excess glucose, are related to the glycolysis pathway, and can directly provide hexose phosphate precursors for cell wall polymer synthesis (Deveau et al. 2008; Ceccaroli et al. 2010; Patyshakuliyeva et al. 2013).

[0008] Cellulose is the main glucose supplier in shiitake mushroom sawdust culture medium, and is mainly degraded by three types of hydrolases: first, endoglucanases (EGs: EC 3.2.1.4, also known as endo-β-1,4-glucanase, carboxymethyl cellulase) cut long-chain cellulose into shorter oligosaccharides, then cellobiohydrolases (CBHs: EC 3.2.1.91, also known as exo-β-1,4-glucanase or cellobiosidase) degrade oligosaccharides into cellobiose, and finally cellobiose is decomposed into glucose (glucosidase) by β-glucosidase (BGLs: EC 3.2.1.21). Figure 1 ;Baldrian& 2008; Wang et al. 2020; Ren et al. 2024). In the existing studies on the detection of cellulase activity, endoglucanase, β-glucosidase and filter paper cellulase (a cellulose complex enzyme) have been studied more (Huang Kefu et al. 1985; Liu Bin and Li Zhengxiang 1991; Wang Weike et al. 2014; Guan Wan et al. 2021). The activity of cellobiohydrolase has only been detected on plates (Carvalho et al. 2016), and there are no related reports during the color change period.

[0009] Therefore, it is necessary to research and develop a more scientific method for monitoring the maturity of shiitake mushroom bags.

[0010] Technical content

[0011] The present invention provides an enzymatic method for accurately monitoring the maturity of shiitake mushroom spawn, comprising the following steps:

[0012] (1) For the strain under study, fungus bags at different color change stages were collected according to the range of fungus age in production practice: starting from the time when the mycelium filled the fungus bag, 20-30 fungus bags were collected every 10 days for morphological observation, fruiting rate detection, cellobiohydrolase activity determination, and multiple color change period first-wave mushroom yield determination with a fruiting rate of 100% before and after the fungus age;

[0013] (2) After clarifying the different stages of physiological maturity of the strain, the dynamic changes of cellobiohydrolase activity at different color change stages were linked to establish the connection between the sudden increase node of cellobiohydrolase and the peak period of production;

[0014] (3) By monitoring the sudden increase of cellobiohydrolase in the mycelium inside the bag, it can be determined that the bag has reached physiological maturity and can be used for bagging and fruiting.

[0015] The present invention mainly uses factory-cultivated strains to construct a method for measuring the physiological maturity of shiitake mushroom mycelium, and accurately monitors the maturity of shiitake mushroom spawn by measuring the dynamic change law of cellobiohydrolase activity in the culture medium at different color change stages, which has the following advantages and innovations:

[0016] 1) Rapidly identify the physiological maturity changes of the strain from the internal and external characteristics and the first wave of mushroom production;

[0017] 2) The physiological maturity of the culture bag can be quickly determined by quantitatively monitoring the changes in the activity of cellobiohydrolase in the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 、Partial Glucose Metabolism Pathway in Lentinus edodes Mycelium

[0019] Figure 2Specific technical route of the method for monitoring the maturity of mushroom spawn bags of the present invention

[0020] Figure 3 Comparison of appearance and fruiting rate of KS11 mushroom bags at different stages

[0021] A. Appearance comparison B. Mushroom yield comparison

[0022] Figure 4 Comparison of the yield of KS11 fungus bags at different stages

[0023] Figure 5 , the changing trend of cellobiohydrolase activity of KS11 bacterial bag

[0024] Figure 6 , seven The fruiting rate of He No. 9 at different color change stages

[0025] Figure 7 , seven (A) Average yield per pack of the first batch of He No. 9 mushrooms; (B) Dynamic changes in cellobiohydrolase activity

[0026] law DETAILED DESCRIPTION

[0027] The following embodiments and drawings are only used to explain and illustrate the contents of the present invention, and are not intended to limit the technical contents of the present invention.

[0028] The following examples are all based on Figure 2 The flowchart shown is used to monitor the maturity of mushroom bags:

[0029] (1) For the strain under study, fungus bags at different color change stages were collected according to the range of fungus age in production practice: starting from the time when the mycelium filled the fungus bag, 20-30 fungus bags were collected every 10 days for morphological observation, fruiting rate detection, cellobiohydrolase activity determination, and multiple color change period first-wave mushroom yield determination with a fruiting rate of 100% before and after the fungus age;

[0030] (2) After clarifying the different stages of physiological maturity of the strain, the dynamic changes of cellobiohydrolase activity at different color change stages were linked to establish the connection between the sudden increase node of cellobiohydrolase and the peak period of production;

[0031] (3) By monitoring the sudden increase of cellobiohydrolase in the mycelium inside the bag, it can be determined that the bag has reached physiological maturity and can be used for bagging and fruiting.

[0032] Example 1

[0033] Two batches of short fungus bags at different color change stages (labeled as fungus bag Ⅰ and fungus bag Ⅱ, inoculated on 2023-2-6 and 2023-3-28, respectively) were purchased from Shanghai Chengying Agricultural Development Co., Ltd. The strain was KS11 (age 90-95 days), 160 in each batch. From the same batch, 20 fungus bags were taken at 35 days, 45 days, 55 days, 65 days, 75 days, 85 days, 95 days and 105 days respectively: 5 of them were used for observation of fungus bag appearance characteristics and determination of cellobiohydrolase activity, and 15 were used for statistics of mushroom fruiting traits.

[0034] Break the bacteria bag from the middle part, use sterile tweezers to draw a "cross" on one section, take the culture medium at the four endpoints, mix and put it in a sterile sealable bag, take 5 bacteria bags at each color change stage as 5 replicates. All samples were freeze-dried, 0.15g of each sample was weighed in a 2mL centrifuge tube, 1.5mL of distilled water was added, let stand for 1h, centrifuged at 10000r / min and 4℃ for 20min, and the supernatant obtained was the crude enzyme solution. Take 10μL of crude enzyme solution for cellobiohydrolase activity determination. For specific steps, refer to the instructions of the soil β-1,4-glucanase / cellobiosidase (S-C1) activity detection kit of Sangon Biotech (Shanghai) Co., Ltd.

[0035] The 15 breathable bags at each color change stage were fruited with the same measures and conditions. The specific process was as follows: after 3 days of day and night temperature stimulation (22°C during the day and 14°C at night), the bags were opened for fruiting. The first-wave fruiting rate of the 15 bags at different color change stages was recorded (fruiting rate = number of fruiting bags / total number of bags × 100%), and the average fruiting yield per bag of all bags at the first wave at 85 days, 95 days and 105 days was calculated respectively.

[0036] Experimental results: In the two batches of KS11 fungus bags (Ⅰ and Ⅱ), the mycelium grew all over the fungus bag at 35 days, which was defined as the physiological maturity period of KS11 fungus bags - the initial stage, at which the mushroom yield rate was 0%. After entering the physiological maturity period, the mycelium on the surface of the KS11 fungus bag began to change color and form brown fungus films of different colors. As the culture time increases, the area of ​​the brown fungus film gradually spreads to the entire fungus bag, and the color gradually changes from yellow-brown and red-brown to black-brown ( Figure 3 A), the fruiting rate gradually became 100% ( Figure 3 B). At 65 days, the entire fungus bag has completed color change, which is defined as the physiological maturity period - color change completion stage, and the fruiting rate is greater than 50%; at 75 days, the fruiting rate reaches 100%, which is defined as the physiological maturity period - full fruiting stage; at 95 days, the yield is the highest, which is defined as the physiological maturity period - peak yield stage ( Figure 3, 4). The cellobiohydrolase activity of the two batches of KS11 bacteria bags showed the same trend of change, remaining stable from 35 to 85 days, and then rising sharply at 95 days (physiological maturity-peak yield period), with no difference between 95 days and 105 days ( Figure 5 ). From the above, it can be seen that the cellobiohydrolase activity increases sharply during the peak period of production during the physiological maturity period, which can be used as a quantifiable indicator to accurately monitor the maturity of shiitake mushroom spawn.

[0037] Example 2

[0038] The fungus bags at different color change stages were purchased from Shandong Qihe Biotechnology Co., Ltd. The strain was Qihe No. 9 (110-120 days old). Since the main purpose was to verify whether the sudden increase point of cellobiohydrolase activity corresponded to the peak period of production, only fungus bags at the late color change stage were purchased, 25 each of 80 days, 90 days, 100 days, 110 days and 120 days old. Among them, 5 of the 25 fungus bags at each color change stage were used for observation of fungus bag appearance characteristics and determination of cellobiohydrolase activity, and 20 were used for statistics of mushroom fruiting traits.

[0039] Each bacterial bag has 4 inoculation ports. The bacterial bag is divided into 3 parts by breaking it apart from the middle of the two inoculation ports. Use sterile tweezers to draw a "cross" on the 3 cross sections, take the culture medium at the 4 endpoints, mix them and put them in a sterile sealing bag. Take 5 bacterial bags at each color change stage as 5 replicates. The preparation of crude enzyme solution and the determination of cellobiohydrolase activity are the same as in Example 1.

[0040] The 20 spawn bags of Qihe No. 9 at each color change stage were fruited with the same measures and conditions. The specific process was as follows: the spawn bags were placed in a 4°C cold storage for stimulation and then opened for fruiting. The fruiting rate of the spawn bags used in each color change stage was recorded, and the average fruiting yield per bag in the first wave before and after the age of the spawn (90d, 100d, 110d and 120d) was recorded.

[0041] Experimental results:

[0042] Qihe No. 9 had completed color change at 80 days, and the fruiting rate reached about 80%; at 90-120 days, the fruiting rate reached 100% ( Figure 6 At 110 days, the average yield of the first-wave mushrooms per package can reach about 600g, which is the highest peak ( Figure 7 A). At the same time, the cellobiohydrolase activity also showed a sudden increase trend at 110 days ( Figure 7 B).

Claims

1. An enzymatic method for accurately monitoring the maturity of shiitake mushroom spawn, characterized in that The steps include: (1) For the strains under study, fungus bags at different color change stages were collected according to the range of fungus age in production practice for morphological observation, fruiting rate detection, cellobiohydrolase activity determination, and the first-wave mushroom yield determination during the color change period with a fruiting rate of 100% before and after the age of the fungus; (2) After clarifying the different stages of physiological maturity of the strain, the dynamic changes of cellobiohydrolase activity at different color change stages were linked to establish the connection between the sudden increase node of cellobiohydrolase and the peak period of production; (3) By monitoring the sudden increase of cellobiohydrolase in the mycelium inside the bag, it can be determined that the bag has reached physiological maturity and can be used for bagging and fruiting.

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