Extract and method for producing same

Abstract

This invention enables expression of resistance-inducing active genes against pathogens in order to inhibit diseases without relying on synthetic agrochemicals.　A resin mixture obtained by compounding thermoplastic resin and a mushroom waste fungal bed is mixed with water, and the resultant is heated and filtered to obtain an extract. By spraying the extract to a plant, expression of resistance-inducing active genes is increased in the plant, and a plant that is highly resistant to diseases can be obtained.

Description

Extract and its manufacturing method

[0001] The present invention relates to an extract and a method for producing the same.

[0002] A method using non-volatile synthetic pesticides has been known to suppress disease in agricultural crops (Patent Document 1). Meanwhile, the existence of volatile components from mushrooms that are effective against mold and bacteria has been known, and it has been proposed to utilize these volatile components as a means of efficiently controlling mold and bacteria in air (Patent Document 2). Additionally, a method has been investigated in which components extracted from waste mushroom beds are used to induce and activate inherent plant resistance. This is because mushroom cell wall components are known to be recognized by plants as elicitors, inducing and activating the plant's resistance (Patent Document 3).

[0003] JP 2009-161472 A JP 2011-167073 A JP 2011-140463 A

[0004] As described above, methods have been proposed for exerting antibacterial effects against mold and bacteria and utilizing such antibacterial effects in plant growth. However, chemically synthesized pesticides such as those described in Patent Document 1 remain in the environment after use, potentially affecting the surrounding biological environment and aquatic fauna. Furthermore, many currently used fungicides are characterized by a narrow antibacterial spectrum and high selectivity, which can lead to problems such as the emergence of resistant bacteria. Furthermore, the volatile components disclosed in Patent Document 2 are highly safe, but due to their volatility, their effectiveness is limited, being effective only in the vicinity of the target mold or fungi. Furthermore, the method using waste mushroom beds disclosed in Patent Document 3 involves obtaining an extract directly from waste mushroom beds, which decay and degenerate over time. Therefore, steps such as sterilizing the waste mushroom beds are required each time an extract is to be obtained to obtain an extract of consistent quality.

[0005] The extract according to the present invention is an extract from a resin mixture, characterized in that the resin mixture is a composite of waste mushroom bed and a thermoplastic resin. A resin mixture is a mixture of a resin and another substance, which are combined into one, and the resin and the other substance are adhered to each other so that they do not easily separate. A composite is a mixture in which at least a portion of the surface of the waste mushroom bed is intimately mixed with the thermoplastic resin, bonding the two together. The bonded state occurs when at least a portion of the thermoplastic resin melts, and the molten portion adheres to the waste mushroom bed and then solidifies.

[0006] The resin mixture is preferably in a granular form, where the individual constituent units are identifiable with the naked eye and generally have a diameter (longest part) of about 0.1 mm to 15 mm; smaller diameters are called powders, and larger diameters are called chunks.

[0007] The particulate form is preferably a pellet form, a crushed form, or a granular form, and the longest part thereof is preferably 0.1 mm or more and 10 mm or less. A pellet form is one in which the longest part is 2 mm or more and 10 mm or less, a crushed form is one in which the longest part is 0.1 mm or more and less than 1 mm, and a granular form is one in which the longest part is 1 mm or more and less than 2 mm.

[0008] It is preferable that the extract is extracted with water.

[0009] It is preferable that the waste mushroom bed is a waste shiitake mushroom bed, and the thermoplastic resin is polyethylene, polypropylene, or a polyester-based resin.

[0010] The polyester resin is preferably any one of polylactic acid, polybutylene adipate terephthalate, polyhydroxybutyrate polyhydroxyvalerate, polyhydroxybutyrate polyhydroxyhexanoate, polybutylene succinate, and polybutylene succinate adipate.

[0011] It is preferable that the weight ratio of the waste mushroom bed in the resin mixture is 10% or more and 70% or less.

[0012] The method for producing an extract according to the present invention includes the steps of: compounding a waste mushroom bed with a thermoplastic resin; granulating the compound; adding water to the granulated material and heating the granulated material; and filtering the heated water to obtain an extract.

[0013] The extract of the present invention can be produced by compounding a thermoplastic resin with a waste mushroom bed, pelleting the compound, adding water to the pellets, heating the mixture of the pellets and water, and filtering the heated mixture. The extract can be sprayed onto plants with a spray bottle or the like to activate the induction of plant disease resistance.

[0014] 1 shows the appearance of lesions caused by black sooty mildew on cabbage in Example 2. FIG. 2 shows the appearance of lesions caused by black sooty mildew on cabbage in Comparative Example 2.

[0015] Hereinafter, embodiments of the present invention will be described in detail.

[0016] The present inventors investigated a novel method using waste mushroom beds to activate plant resistance induction. One known mechanism by which plants are deterred from infection by pathogenic fungi is that fungal cell wall components β-1,3-glucan and chitin are degraded by substances induced by the plant, and the oligosaccharides released by the degradation are recognized by the plant as elicitors, resulting in the development of systemic induced resistance. The substances induced by the plant are thought to be chitinase and β-1,3-glucanase substances. After extensive investigation, we decided to use fungal cell wall components contained in waste mushroom beds as raw materials, and further to utilize elicitors contained in extracts from waste mushroom beds complexed with thermoplastic resin components.

[0017] The above-mentioned extract is not a chemically synthesized pesticide as in Patent Document 1, and is therefore safe for the environment and human body. Furthermore, since an extract liquid containing the extract is used, there is no need to treat volatile components in a vaporized state, eliminating the spatial limitations as disclosed in Patent Document 2. Furthermore, since the waste mushroom bed is composited with resin at high temperatures, it is sterilized and becomes a composite in which the waste mushroom bed is isolated from the outside air. From the waste mushroom bed composited with resin, an extract liquid of consistent quality can be obtained reliably without sterilization treatment as in Patent Document 3. Furthermore, the extract liquid was confirmed to act as a resistance inducer against diseases in agricultural crops such as tomatoes and cabbage, leading to the completion of the present invention.

[0018] In one example of the process for producing an extract in this embodiment, a thermoplastic resin pellet is first physically mixed with waste mushroom beds to produce a physical mixture. This physical mixture is then heated and kneaded using a device capable of thermally melting the thermoplastic resin and mixing and kneading it with the waste mushroom beds, such as a single-screw kneader, a twin-screw kneader, or a Banbury mixer, to produce a resin mixture. The resin mixture is a mixture of a resin component and another component (here, waste mushroom beds) that are kneaded while at least a portion of the resin component is thermally melted, resulting in adhesion and bonding. This resin mixture is then pelletized. The pellets are mixed with water, heated in an autoclave, or the like, and then filtered to obtain an extract. Spraying the extract onto, for example, tomato seedlings with a spray bottle can cause disease resistance-related genes to be expressed in the tomatoes.

[0019] (1) Raw Materials (Thermoplastic Resin) The thermoplastic resin used as the raw material in this embodiment is not limited to a specific resin, but may be any resin that can be thermally melted by heating and bonded to the waste mushroom bed. A mixture of multiple thermoplastic resins may also be used. Examples of thermoplastic resins include polyethylene, polyvinyl chloride, polypropylene, polystyrene, acrylonitrile-butadiene-styrene copolymer, acrylonitrile-styrene copolymer, polymethyl methacrylate, polybutylene terephthalate, polyethylene terephthalate, polylactic acid, polybutylene adipate terephthalate, polyhydroxybutyrate polyhydroxyvalerate, polyhydroxybutyrate polyhydroxyhexanoate, polybutylene succinate, polybutylene succinate adipate, polyamide, polyoxymethylene, polyvinyl alcohol, polyphenylene ether, polycarbonate, polyphenylene sulfide, aromatic polyether ketone, and polyimide. In particular, thermoplastic resins with a softening temperature of 150°C or higher and 250°C or lower are preferred in terms of ease of thermal melting during production and thermal stability during use. Among these, polypropylene and polyethylene are preferred from the viewpoints of general-purpose use and excellent moldability. Furthermore, from the viewpoints of biodegradability and low environmental impact, polylactic acid, polybutylene adipate terephthalate, polyhydroxybutyrate polyhydroxyvalerate, polyhydroxybutyrate polyhydroxyhexanoate, polybutylene succinate, polybutylene succinate adipate, etc. can be suitably used. Furthermore, a mixture of multiple types of these resins may be used.

[0020] (Waste mushroom bed) Waste mushroom bed refers to a used mushroom bed after cultivating mushrooms by planting mushroom spawn on a bed such as a hardwood log and harvesting the mushrooms. The waste mushroom bed used in this embodiment is not limited to a particular type, and waste mushroom beds used after cultivating edible mushrooms can be used. Examples of waste mushroom beds include trees themselves and sawdust (wood powder) mixed with rice bran or the like. The type of mushroom is not particularly limited, and may be shiitake mushrooms, maitake mushrooms, or the like, and waste shiitake mushroom beds (bed logs used after cultivating shiitake mushrooms) can be preferably used from the viewpoint of general availability.

[0021] (Water) As the water to be mixed with the resin mixture and used for extraction, tap water, distilled water, ion-exchanged water, etc. can be used from the viewpoint that water that is not contaminated with bacteria is preferable. Furthermore, distilled water and ion-exchanged water are more preferable from the viewpoint that they contain fewer impurities and can suppress variation in the quality of the extract from one production to another.

[0022] (2) Manufacturing Method (Method for Manufacturing Resin Composite) An example of a method for manufacturing a resin composite in an embodiment is a method in which a specified amount of thermoplastic resin and waste mushroom bed are mixed to form a mixture, the thermoplastic resin is heat-melted, kneaded, and cooled.

[0023] Before mixing, the waste mushroom bed is dried in a drying oven at 80°C to 100°C for approximately 48 hours until its moisture content reaches 60% or less (first waste mushroom bed drying process). If the moisture content is higher than 60%, the waste mushroom bed has a strong cohesive force, making it difficult to crush in the waste mushroom bed crushing process described below. Temperatures lower than 80°C take too long to dry, resulting in reduced productivity, while temperatures higher than 100°C are undesirable because they produce a strong odor during drying and worsen the working environment. The moisture content can be measured using a weight loss method or the like, and to ensure uniform drying, it is preferable to manually stir the waste mushroom bed during drying every 12 hours.

[0024] In the waste mushroom bed crushing process, the dried waste mushroom bed is crushed using a known crusher and classified using a 3 mm or less screen, and waste mushroom bed with a longest portion greater than 3 mm is removed. The crusher is not limited to any particular type, and known crushers such as a roller mill, jet mill, hammer mill, pin mill, rotary mill, vibration mill, planetary mill, and wonder crusher can be used. If waste mushroom bed of 3 mm or greater remains, large clumps of waste mushroom bed will be mixed into the resin during the composite process described below, hindering uniform composite formation and increasing the amount of waste mushroom bed exposed to the pellets upon pelletization. If a large amount of waste mushroom bed is exposed to the pellets, the pellets will be exposed to moisture, oxygen, and bacteria in the air during long-term storage, making the waste mushroom bed more susceptible to deterioration.

[0025] The classified waste mushroom bed is further dried at 80°C to 100°C until the moisture content reaches 5% (second waste mushroom bed drying step). If the moisture content is greater than 5%, the error in the blend weight of the waste mushroom bed increases in the kneading step, making it difficult to blend the specified amount, and the cohesive force between the particles of the waste mushroom bed increases, making it difficult to form a uniform composite, which is undesirable.

[0026] To mix the waste mushroom bed that has gone through the drying and crushing steps with the thermoplastic resin, the components can be weighed into a container or bag and mixed manually using a spatula or a rod-shaped jig that can be used for stirring, or mechanically mixed to a nearly uniform state using a rotary mixer such as a Henschel mixer (mixing step).The thermoplastic resin and waste mushroom bed that are prepared for mixing are preferably both in powder form, or the thermoplastic resin in pellet form, as this simplifies the mixing process.

[0027] To perform the hot melt mixing and kneading, the substantially uniformly mixed mixture can be mixed using a single-screw kneader, a twin-screw kneader, a roll kneader, a kneader, a Banbury mixer, or the like, or a combination of these can be used to perform the hot melt mixing and kneading (composite process). The temperature during the hot melt mixing should be equal to or higher than the melting point of the resin used, and for example, for polypropylene, it can be set to 190°C or higher. The upper limit of the temperature is preferably not higher than 250°C. Temperatures above 250°C are undesirable because they cause oxidation or thermal degradation of the organic components contained in the waste mushroom bed.

[0028] By combining the waste mushroom beds in this way, the temperature of the waste mushroom beds rises to kill unwanted bacteria, and most of the surface of the waste mushroom beds is covered with thermoplastic resin, blocking out air, so the condition of the waste mushroom beds (moisture content, sterility, etc.) can be maintained constant for a long period of time.

[0029] When pelletizing the resin mixture after hot melt mixing, the resin immediately after melt mixing is pelletized or granulated before cooling, and then cooled to produce the resin mixture (composite) of this embodiment.

[0030] As an example of a specific method for producing pellets of a resin composite, the molten mixture is stretched before cooling to form a string-like strand, which is then appropriately cut using a rotary strand cutter or the like to form granules while being cooled, thereby producing pellets or granules. The diameter of the strand can be 0.1 mm or more and 10 mm or less. In particular, strands of 1 mm or more but less than 2 mm are called granules, and those of 2 mm or more and 10 mm or less are called pellets. Pellets or granules refer to granular materials. In addition, in this embodiment, a material containing crushed mushroom waste bed with at least a portion of its surface in close contact with a resin, and in which the crushed mushroom waste bed and resin are mixed in close contact, is called a composite. In other words, in this embodiment, the above composite in a granular form of 2 mm or more and 10 mm or less is called a pellet.

[0031] The longest part of the granular material is preferably 0.1 mm or more and 10 mm or less. If it is smaller than 0.1 mm, the granular material will contain a large amount of waste mushroom bed with exposed parts, which will be exposed to moisture, oxygen, and germs in the air during long-term storage, making the waste mushroom bed more likely to deteriorate. If the longest part exceeds 10 mm, the area that comes into contact with water when mixed with water during the production of the extract solution described below will be small, which is undesirable as it reduces the amount of extract extracted into the extract solution. Within the brown granular material in which the waste mushroom bed is composited, the waste mushroom bed is composited almost uniformly, and at least a portion of the waste mushroom bed is in close contact with the thermoplastic resin.

[0032] In addition, other additives such as pigments and flame retardants can be added to the mixture of thermoplastic resin and waste mushroom bed fed into the hot melt mixing machine, depending on the application. The weight ratio of waste mushroom bed in the resin mixture can be 10% by mass or more and 70% by mass or less. If it is less than 10% by mass, the extract described below will not have sufficient resistance-inducing activity, and if it is more than 70% by mass, it will be difficult to produce a uniform resin mixture due to aggregation of the waste mushroom bed during the resin mixture production process. From the perspective of excellent productivity and the ability to contain a higher concentration of waste mushroom bed, it is even more preferable that the weight ratio of waste mushroom bed in the resin mixture be 30% by mass or more and 60% by mass or less.

[0033] The heating temperature during production should be equal to or higher than the melting point of the resin used. Furthermore, by setting the heating temperature to approximately 100°C or higher, preferably 120°C or higher, the waste mushroom bed can be sterilized at the same time, and the waste mushroom bed is coated with a thermoplastic resin at the same time as sterilization, which makes it possible to prevent subsequent adhesion of unwanted bacteria to the waste mushroom bed, oxidation deterioration, and deterioration due to water absorption.

[0034] (Method for Producing Extract) An example of a method for producing an extract is to prepare a mixture of a resin mixture and water (extraction mixture), heat the mixture, and then filter the mixture using a filter such as gauze. Regarding the mixture of the resin mixture and water, the weight ratio of the resin mixture to the water can be 1 to 100 parts by weight per 1 part by weight of the resin mixture. If the amount of water is less than 1 part by weight, the amount of extract (described below) obtained relative to the amount of resin mixture produced is too small, which is undesirable from the standpoint of productivity. If the amount of water is more than 100 parts by weight, the concentration of the extract as an aqueous solution becomes too low, which is undesirable because sufficient resistance-inducing activity cannot be obtained.

[0035] The resin composite and water mixture can be heated by any known method, such as by placing the extraction mixture in a glassware such as a beaker and heating it in a water bath, oil bath, or fuel combustion. However, heating by sealed heating using an autoclave or the like is preferred, as it can suppress water evaporation while also reliably sterilizing trace amounts of bacteria that may be mixed in the mixture. The heating temperature can be 80°C or higher and 100°C or lower. When using an autoclave, it is preferable to set the temperature to 100°C or higher but lower than the melting point of the thermoplastic resin. For example, a temperature of 100°C or higher but lower than 125°C is preferred, as this is below the melting point of many thermoplastic resins and ensures reliable sterilization. The heating time can be 5 minutes or higher and 60 minutes or lower. Heating for shorter than 5 minutes will not result in a sufficient extract concentration to exert its efficacy, while heating for longer than 60 minutes will result in saturation of the extract concentration, may lead to deterioration of the extract due to heat, and is undesirable from the perspective of reduced productivity.

[0036] The examples will be specifically described below.

[0037] Example 1 <Production of Resin Composite> A resin composite was produced in the following manner using polylactic acid as the thermoplastic resin and waste mushroom beds used for cultivating shiitake mushrooms as the waste mushroom beds.

[0038] The polylactic acid used was TE-2000, manufactured by Unitika Ltd. Shiitake mushroom waste beds (bed logs) were used as waste mushroom beds. These were crushed to a particle size of 3 mm or less using a universal crusher (SF-1, manufactured by Sanriki Seisakusho Co., Ltd.) and dried until the moisture content reached 4%. 90 g and 110 g of the polylactic acid and Shiitake mushroom waste beds were blended in a container and manually stirred and mixed using a spatula. The stirred mixture was melt-mixed and kneaded. A twin-screw kneader (KRC Kneader, manufactured by Kurimoto Iron Works Co., Ltd.) was used for melt-mixing and kneading, with a temperature setting of 200°C and a rotation speed of 50 rpm. The strand-like resin mixture obtained from the discharge outlet was cut to a length of approximately 5 mm to obtain a pellet-like resin mixture (composite) consisting of Shiitake mushroom waste beds and polylactic acid.

[0039] <Production of Extract> A mixture of 2 g of the resin composite pellets obtained in the above step and 10 ml of distilled water was placed in an autoclave and treated at 121°C for 10 minutes, and then filtered through gauze to obtain the extract in this example.

[0040] <Expression Analysis of Disease Resistance-Related Genes> Expression analysis of disease resistance-related genes in tomato seedlings was performed using the following procedure. Flower and vegetable soil: Tomato seeds were sown in black pots filled with a vermiculite-mixed soil (1:1). Five tomato seedlings one month after growth were sprayed with 1 mL of the above extract per plant. One day after treatment, two leaves were removed from each of three seedlings and stored at -80°C. RNA was extracted from the collected leaf samples. The RNA concentration was adjusted, and cDNA was synthesized by reverse transcription. Real-time PCR was performed using specific primers for each of the resistance-related genes (Glu-A, Glu-B, CHI3, and CHI9), and the expression level of each gene was analyzed.

[0041] (Example 2) The production of the resin mixture and the production of the extract were the same as in Example 1, and the following resistance development was verified.

[0042] <Verification of Resistance Expression> Verification of resistance expression was carried out for cabbage (variety: Hatsuaki) using the following procedure. Soil for flowers and vegetables: Cabbages were grown for 4 weeks in a vermiculite-mixed soil (1:1). Five cabbages were prepared in each test plot, and 5 ml of the extract was sprayed and inoculated per test plot. Next, the cabbages were placed in a plastic container filled with water, and after 24 or 48 hours, a spore suspension of the O264 strain of Cabbage Black Sooty Rot Fungus (5 x 10 ５ The cabbages inoculated with the O264 strain spore suspension were then sprayed in a plastic container filled with water, and the cabbages were then left to stand for 24 hours, after which the formation of lesions on the leaves was confirmed. The leaf area was calculated using graph paper, and the area of ​​1 cm ２ The number of lesions per cm was calculated using the following formula: Number of lesions (lesions / cm ２ ) = Number of lesions on one leaf ÷ Leaf area (cm ２ )

[0043] (Example 3) In Example 3, the production of a resin composite (composite), the production of an extract, and the expression analysis of disease resistance-related genes were carried out in the same manner as in Example 1, except that waste mushroom beds were replaced with waste nameko beds.

[0044] Comparative Example 1 In Comparative Example 1, expression analysis of disease resistance-related genes was carried out on tomato seedlings in the same manner as in Example 1, except that the extract was not sprayed.

[0045] Comparative Example 2 In Comparative Example 2, the expression of resistance in cabbage was verified in the same manner as in Example 2, except that the extract was not sprayed.

[0046] Table 1 shows the analysis results of the resistance-related genes Glu-A, Glu-B, CHI3, and CHI9 in tomatoes in each of Examples 1 and 3 and Comparative Example 1. Table 1 shows whether or not a significant difference was observed at the 5%, 1%, or 0.01% level in the t-test compared to Comparative Example 1.

[0047] FIG. 1 shows the lesions of sooty mildew on cabbages sprayed with the extract in Example 2, and FIG. 2 shows the lesions of sooty mildew on cabbages not sprayed with the extract in Comparative Example 2.

[0048] From Table 1, it can be seen that, for the Glu-A gene, when an extract obtained from a resin composite combined with waste Lentinula edodes mushroom beds was sprayed onto tomato seedlings, i.e., in Example 1, a significant difference in expression level was observed at a 5% level compared to Comparative Example 1. It can also be seen that in Example 3, a significant difference in expression level was observed at a 1% level compared to Comparative Example 1.

[0049] Furthermore, it can be seen that for the Glu-B gene, a significant difference in expression level was observed at the 5% level only in Example 1. No significant difference was observed in Example 3, but this does not mean that there was no effect on the expression level.

[0050] It can be seen that for the CHI3 gene, a significant difference in expression level was observed at the 1% level only in Example 1, and for the CHI9 gene, a significant difference in expression level was observed at the remarkable level of 0.01% in Example 1.

[0051] 1 and 2, it can be seen that the number of lesions caused by cabbage sooty mold on the cabbage leaves sprayed with the extract, i.e., Example 2, was small, while the number of lesions caused by cabbage sooty mold on the cabbage leaves not sprayed with the extract, i.e., Comparative Example 2, was large. The disease inhibition rate when the extract was sprayed was estimated from the number of lesions in each case, and was 78.9%. This can be said to be a result indicating the expression of resistance-related genes in the cabbages sprayed with the extract.

[0052] From the above, in the disease suppression verification in tomato, only in Example 1, a significant difference was observed in the expression level of all of the resistance-related genes Glu-A, Glu-B, CHI3, and CHI9 compared to Comparative Example 1, where no spraying or other treatment was performed. It is also clear that the extract obtained from the resin composite combined with shiitake mushroom waste bed is effective in expressing disease resistance-related genes. Note that in Example 3, a significant difference was observed in the expression level of the resistance-related gene Glu-A compared to Comparative Example 1, where no spraying or other treatment was performed. In the disease suppression verification in cabbage, it is clear from Example 2 and Comparative Example 2 that the extract obtained from the resin composite combined with shiitake mushroom waste bed is effective in suppressing disease.

[0053] (Other Embodiments) The above-described embodiments are merely examples of the present invention, and the present invention is not limited to these examples. These examples may be combined with well-known, commonly used, or publicly known technologies, or may be partially replaced. Modified inventions that would be easily conceived by a person skilled in the art are also included in the present invention.

Claims

1. An extract from a resin mixture, characterized in that the resin mixture is a composite of waste mushroom bed and a thermoplastic resin.

2. The extract of claim 1, wherein said resin composite is in granular form.

3. The extract according to claim 2, wherein the particulate form is a pellet, a crushed or a granular form, the longest part of which is 1 mm or more and 10 mm or less.

4. The extract according to claim 1 or 2, wherein the extract is extracted with water.

5. The extract according to claim 1 or 2, wherein the waste mushroom bed is a waste shiitake mushroom bed and the thermoplastic resin is polyethylene, polypropylene or a polyester-based resin.

6. The extract according to claim 5, wherein the polyester resin is any one of polylactic acid, polybutylene adipate terephthalate, polyhydroxybutyrate polyhydroxyvalerate, polyhydroxybutyrate polyhydroxyhexanoate, polybutylene succinate, and polybutylene succinate adipate.

7. The extract according to claim 1 or 2, wherein the weight ratio of the waste mushroom bed in the resin mixture is 10% or more and 70% or less.

8. A method for producing an extract, comprising the steps of: compounding waste mushroom beds with a thermoplastic resin; granulating the compound; adding water to the granulated material and heating the granulated material; and filtering the heated water to obtain an extract.

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