Method for producing powder containing cyst nematode hatching promoter

Abstract

To provide a new method for producing a composition or powder containing a cyst nematode hatching promoter that can be used to commercialize a cyst nematode control agent.SOLUTION: The present invention relates to a method for producing a composition containing a cyst nematode hatching promoter, which comprises using a dialysis membrane or an ultrafiltration membrane to remove impurities from a culture solution containing a cyst nematode hatching promoter which is obtained by culturing a host plant for cyst nematodes in a liquid medium; a method for producing a powder containing a cyst nematode hatching promoter, which comprises powdering a culture solution containing a cyst nematode hatching promoter and which is obtained by culturing a host plant for cyst nematodes in a liquid medium, or a composition containing a cyst nematode hatching promoter from which impurities have been removed using a dialysis membrane or an ultrafiltration membrane from the culture solution by spray-drying; and a composition or powder containing a cyst nematode hatching promoter produced by these production methods.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing a composition or powder containing a substance that promotes hatching of cyst nematodes. [Background technology]

[0002] Generally, cyst nematodes parasitize the roots of host plants and grow within them. When the adult female reaches maturity, she leaves her body outside the root while keeping her head attached to the root, forming an enlarged cyst. The cyst contains many eggs, and large numbers of these cysts remain in the soil of fields contaminated with cyst nematodes. The following year, as the crop plants germinate, they parasitize the roots, causing severe damage to the crops. As an example, the potato cyst nematode (PCN), an internationally significant pest among cyst nematodes, is a soil-borne pest that parasitizes Solanaceae plants such as potatoes and tomatoes. It forms cysts during its life cycle. Cysts are formed after mating, and are hard-shelled and contain the next generation of eggs. Cysts also protect the dormant eggs contained within them from environmental changes in the soil (dryness and temperature), allowing them to survive for long periods in the soil. Furthermore, because cysts are resistant to pesticides, few pesticides are directly effective against them. Therefore, control measures must rely on soil disinfectants containing toxic active ingredients such as dilution agents. Furthermore, PCNs detect hatching factors (PCN-HFs) secreted by the roots of growing host plants, such as potatoes, and then hatch and parasitize the plants. Therefore, when PCN-HFs are not detected in the soil, i.e., when there are no host plants, the eggs remain dormant within the cysts. This allows them to remain dormant in the soil for long periods of time, which is one of the reasons why PCNs are difficult to eradicate. More than 30 years ago, one idea for controlling PCNs was proposed: spraying PCN-HF on fields before potato planting to force the PCNs to hatch and starve to death. However, a method for mass-producing PCN-HF had not yet been established, and this method has not yet been put to practical use at the field level.

[0003] Subsequently, Solanoeclepin A, a triterpenoid compound found in potato hydroponic culture solution, was discovered as one of the components of PCN-HF (Patent Document 1). However, its biosynthetic pathway remains unclear, making recombinant production impossible. Furthermore, its concentration in plants is low, making recovery and purification from plants difficult. Furthermore, while Solanoeclepin A can be chemically synthesized, a 52-step process is required (Non-Patent Document 1), resulting in low production efficiency and high costs, making chemical synthesis of Solanoeclepin A extremely difficult to implement. In addition to natural PCN-HF secreted from plants, compounds such as sodium metavanadate have been reported as non-natural hatching-promoting substances, but their activity is insufficient and they have not yet been put to practical use.

[0004] In addition to solanoeclepin A, steroid glycoalkaloids (SGAs, Non-Patent Document 2), solanoeclepin B (Non-Patent Document 3), lactones and lactams (Patent Document 2), and methionine (Patent Document 3) are also known as PCN-HFs. Among cyst nematodes other than the potato cyst nematode, glycinoeclepin A is known as a hatching promoter for the soybean cyst nematode. In addition to the hatching-promoting substances, secretions from cyst nematode host plants have been suggested to contain unidentified hatching-promoting substances, substances that enhance their activity (enhancing factors), and conversely, hatching-inhibiting substances that inhibit their activity. Sephadex G-10 column fractionation of root exudates of Solanaceae plants has also been attempted as a method for obtaining or isolating these hatching-promoting substances and related substances (enhancing factors and hatching-inhibiting substances) (Patent Document 4). Other methods that have been attempted include cultivating Solanaceae or legumes in a culture medium containing zeolite as a base material, allowing the hatching-promoting substances to be adsorbed onto the zeolite (Patent Document 5), and solid-phase extraction from potato root exudates (Non-Patent Document 4).

[0005] However, the production of PCN-HF using these existing technologies requires steps such as adsorption onto a carrier, elution from the carrier using an organic solvent, concentration by evaporation, and drying of the sample, which are time-consuming and labor-intensive. Issues include the high cost of the carrier and the use of organic solvents designated as hazardous substances for extraction, and the practical production of cyst nematode control agents has not yet been achieved. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 5,585,505 [Patent Document 2] Japanese Patent Publication No. 2022-128804 [Patent Document 3] International Publication No. 2019 / 004252 [Patent Document 4] European Patent No. 1085812 [Patent Document 5] Patent No. 5884118 [Non-patent literature]

[0007] [Non-Patent Document 1] K. Tanino et al., “Total synthesis of solanoeclepin A”, Nature Chemistry volume 3, pages 484-488 (2011) [Non-patent document 2] K. Shimizu et al., “Hatching stimulation activity of steroidal glycoalkaloids toward the potato cyst nematode, Globodera rostochiensis”, Plant Biotechnology 37, 319-325 (2020) [Non-patent document 3] K. Shimizu et al., “Solanoeclepin B, a hatching factor for potato cyst nematode”, Sci. Adv. 9, eadf4166 (2023) 15 March 2023 [Non-patent document 4] A. Guerrieri et al., “UPLC-MS / MS analysis and biological activity of the potato cyst nematode hatching stimulant, solanoeclepin A, in the root exudate of Solanum spp.”, Planta 254:112 (2021) Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors conducted research with the objectives of developing a method for inexpensively, safely, and efficiently recovering cyst nematode hatching promoters such as PCN-HF, and developing a method for producing naturally occurring cyst nematode hatching promoters on a commercial scale as cyst nematode control agents. As a result, the present inventors confirmed that by culturing cyst nematode host plants in a liquid medium containing sugars such as sucrose, it is possible to efficiently mass-produce a culture solution containing cyst nematode hatching promoters, and that this culture solution exhibits a high hatching-promoting effect. However, in commercializing the product, they faced an entirely new challenge: impurities such as sucrose posed problems in terms of use and quality control. Storing a liquid culture solution containing substances that promote cyst nematode hatching requires measures to prevent spoilage and maintain activity. When the culture solution contains sugars such as sucrose, spoilage is particularly likely to occur during storage, so it is necessary to maintain a sterile environment throughout the process from cultivation to collection. Low-temperature storage is also necessary to maintain activity, but refrigerated storage leads to a decrease in activity within a few weeks. Therefore, freezing is required for long-term storage of months to years. Furthermore, concentrated solutions from this solution have the following problems: (1) they are difficult to handle due to their high viscosity; (2) their high sugar content and high viscosity make them difficult to penetrate and mix into soil during soil treatment; and (3) mold grows after addition to soil, inhibiting cyst nematode hatching.

[0009] Furthermore, when commercializing a product, it is difficult to adjust the concentration of the active ingredient in the culture solution containing a cyst nematode hatching promoter, which is disadvantageous in terms of volume and weight during transportation. Therefore, the inventors attempted to solve this problem by powdering the culture solution containing a cyst nematode hatching promoter. However, it has been reported that Solanoeclepin A, a known hatching promoter, is unstable at temperatures above 35°C (Patent Document 1), and it has also been suggested that some hatching promoters are heat-sensitive. This presented a completely new challenge: the possibility that drying and powdering by heating might not be applicable. Therefore, the object of the present invention is to provide a method for producing a composition containing a cyst nematode hatching-promoting substance, which removes impurities such as sucrose while maintaining the high hatching-promoting activity contained in a culture solution containing the cyst nematode hatching-promoting substance, when producing and commercializing a cyst nematode control agent on a commercial scale; or to provide a method for producing a powder containing a cyst nematode hatching-promoting substance, which can be powdered from a culture solution containing the cyst nematode hatching-promoting substance while maintaining the high hatching-promoting activity, and used to commercialize a cyst nematode control agent, when producing and commercializing a cyst nematode control agent on a commercial scale. [Means for solving the problem]

[0010] In the course of intensive research to solve the above-mentioned problems, the inventors unexpectedly discovered that by using a dialysis membrane or ultrafiltration membrane, it is possible to remove impurities such as sucrose while maintaining the hatching-promoting activity of a culture medium containing a cyst nematode hatching-promoting substance. As a result of further research, they have completed the following invention.

[0011] Thus, in one aspect, the present invention relates to: [1] A method for producing a composition containing a cyst nematode hatching promoter, which comprises using a dialysis membrane or an ultrafiltration membrane to remove impurities from a culture solution containing a cyst nematode hatching promoter obtained by culturing a cyst nematode host plant in a liquid medium. [2] The method according to [1] above, wherein the host plant is a plant of the Solanaceae family. [3] The method according to [1] or [2] above, wherein the cyst nematode is a potato cyst nematode. [4] The method according to any one of [1] to [3] above, wherein the liquid medium contains sucrose. [5] The method according to any one of [1] to [4] above, wherein the contaminants to be removed include sucrose. [6] The method for producing the present invention according to any one of [1] to [5] above, wherein a dialysis membrane is used, and the pore size of the dialysis membrane is 0.5 kDa to 1 kDa. [7] The production method according to any one of [1] to [5] above, wherein an ultrafiltration membrane is used, and the pore size of the ultrafiltration membrane is 1 kDa to 3 kDa. [8] A composition containing a cyst nematode hatching promoter, produced by the production method described in any one of [1] to [7] above. [9] The composition containing the cyst nematode hatching promoter described in [8] above, which is further powdered by spray drying.

[0012] Furthermore, in the course of intensive research to solve the above-mentioned problems, the inventors unexpectedly discovered that by using a spray-drying method, it was possible to powderize a culture solution containing a cyst nematode hatching-promoting substance while maintaining its hatching-promoting activity. As a result of further research, they were able to complete the following invention.

[0013] Thus, in one aspect, the present invention relates to: (1) A method for producing a powder containing a cyst nematode hatching promoter, which comprises spray-drying a culture solution containing a cyst nematode hatching promoter obtained by culturing a cyst nematode host plant in a liquid medium, or a composition containing a cyst nematode hatching promoter obtained by removing impurities from the culture solution using a dialysis membrane or an ultrafiltration membrane. (2) The method according to (1) above, wherein the host plant is a plant of the Solanaceae family. (3) The method for producing according to (1) or (2) above, wherein the cyst nematode is a potato cyst nematode. (4) The method according to any one of (1) to (3), wherein the liquid medium contains sucrose. (5) The method according to any one of (1) to (4), wherein the contaminants to be removed include sucrose. (6) The method according to any one of (1) to (5) above, wherein an excipient is added and then powdered by spray drying. (7) The method according to (6), wherein the excipient content is 1 to 2 times the sucrose content. (8) The method according to any one of (1) to (7), wherein the spray drying is carried out by setting the exhaust temperature to be in the range of 35 to 70% of the hot air temperature, with the upper limit of the exhaust temperature being 100°C. (9) A powder containing a cyst nematode hatching promoter, produced by the production method according to any one of (1) to (8) above. [Effects of the Invention]

[0014] According to the method for producing a composition containing a cyst nematode hatching-promoting substance of the present invention, by using a dialysis membrane or ultrafiltration membrane, which has not previously been reported to be used for a composition containing a cyst nematode hatching-promoting substance, it is possible to remove impurities such as sucrose while maintaining the hatching-promoting activity of the culture solution containing the cyst nematode hatching-promoting substance.

[0015] The present invention solves the above problems by removing contaminants such as sucrose while maintaining the hatching-promoting activity. Furthermore, powdering by spray drying improves storage stability, solving the problem of long-term storage, which is difficult with liquids.

[0016] In particular, one would not consider using a dialysis membrane or ultrafiltration membrane to separate substances with similar molecular weights from a culture medium containing contaminants such as sucrose (molecular weight 342), which has a similar molecular weight to Solanoeclepin A (molecular weight 498) and Solanoeclepin B (molecular weight 500), known to be cyst nematode hatching promoters. Furthermore, since existing research suggests that culture medium containing cyst nematode hatching promoters contains hatching promoters other than Solanoeclepin A, as well as hatching inhibitors, the ability to remove contaminants while maintaining hatching-promoting activity is also a particularly effective method.

[0017] According to the present invention, since dialysis and ultrafiltration are used, the test solution is fed sequentially and continuously treated based on established parameters (such as membrane pore size), so the operation itself is simple and large-scale treatment is possible, making it possible to produce and commercialize cyst nematode control agents on a commercial scale. Furthermore, since no organic solvents are used, there is no problem with the discharge of organic solvents designated as deleterious substances. Furthermore, when ultrafiltration is used, it is possible to remove impurities and simultaneously concentrate the culture solution containing the cyst nematode hatching promoter.

[0018] According to the method for producing powder containing cyst nematode hatching-promoting substances of the present invention, by using a spray-drying method that has not previously been reported to be used on culture solutions containing cyst nematode hatching-promoting substances, it is possible to powder the culture solutions containing cyst nematode hatching-promoting substances while maintaining their hatching-promoting activity.

[0019] In particular, it has been reported that Solanoeclepin A, which is known as one of the substances that promotes cyst nematode hatching, is unstable at temperatures above 35°C (Patent Document 1). Despite the use of the spray-drying method, in which the test solution (component) is exposed to high heat, albeit only momentarily, the fact that it can be powdered relatively easily while maintaining its hatching-promoting activity is an effect that would not generally be predicted. Existing research has suggested that culture medium containing cyst nematode hatching-promoting substances contains hatching-promoting substances other than Solanoeclepin A, as well as hatching-inhibiting components, so the ability to powder the substance while maintaining its hatching-promoting activity is also a special advantage.

[0020] According to the present invention, the spray-drying method is used, and the test solution is sequentially fed into the spray-drying device under established parameters for continuous processing, making the process simple and enabling large-scale processing. Furthermore, the formulation can be simultaneously prepared by directly powdering the liquid, enabling commercial-scale production and commercialization of cyst nematode control agents. Furthermore, because no organic solvents are used, there is no need to worry about the discharge of organic solvents designated as hazardous substances. Furthermore, if a concentrated solution is prepared using centrifugal thin-film evaporation or ultrafiltration and then spray-dried, it is possible to reduce the processing volume, remove impurities, and simultaneously powderize the culture solution containing the cyst nematode hatching promoter.

[0021] Furthermore, it is known that sugars are often difficult to dry using the spray-drying method, and while an auxiliary agent (excipient) is often added to liquids containing sugars, until now, there has been no information on excipients that do not affect the activity of hatching promoters. In contrast, by adding an excipient and adjusting the spray-drying conditions as in the present invention, it is possible to powder the substance while maintaining its hatching-promoting activity, regardless of the presence or absence of sugar. In addition, when powdered by spray drying, it has the advantages of excellent storage stability, ease of transport, and ease of adjusting the amount of cyst nematode hatching promoter used in the field. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a graph showing the hatching-promoting activity of tangential flow ultrafiltration treated solution (pore size 1 kDa). [Figure 2] FIG. 2 is a graph showing the hatching-promoting activity of the tangential flow ultrafiltration treatment solution (pore size: 2.5 kDa). [Figure 3] FIG. 3 is a graph showing the activity evaluation of powders in spray drying studies (small scale). [Figure 4] FIG. 4 is a graph showing the hatching-promoting activity of powders in a spray-drying study (improving hot air temperature and liquid delivery rate). [Figure 5] FIG. 5 is a graph showing the activity evaluation of powders in a spray-drying study (improvement of hot air temperature and liquid delivery rate). [Figure 6] FIG. 6 is a graph showing the evaluation of hatching-promoting activity in a spray-drying study of the concentrate. [Figure 7] FIG. 7 is a graph showing the hatching-promoting activity of the concentrate obtained by ultrafiltration. [Figure 8]FIG. 8 is a graph showing the evaluation of hatching-promoting activity in a spray-drying study using a potato culture broth concentrate after ultrafiltration. [Figure 9] FIG. 9 is a graph showing the evaluation of hatching-promoting activity in a spray-drying study using a potato culture broth concentrate after dialysis. [Figure 10] FIG. 10 is a graph showing the results of storage stability of the spray-dried powder. DETAILED DESCRIPTION OF THE INVENTION

[0023] In one aspect, the present invention relates to a method for producing a composition containing a cyst nematode hatching promoter, which comprises using a dialysis membrane or an ultrafiltration membrane to remove impurities from a culture solution containing a cyst nematode hatching promoter obtained by culturing a cyst nematode host plant in a liquid medium.

[0024] Examples of cyst nematodes include plant-parasitic cyst nematodes of the genus Globodera, such as the potato cyst nematode (Globodera rostochiensis), the potato white cyst nematode (Globodera pallida), Globodera ellingtonae, the tobacco cyst nematode (Globodera tabaccum), and the artemisia cyst nematode (Globodera hypolysi), as well as plant-parasitic cyst nematodes of the genus Heterodera, such as the soybean cyst nematode (Heterodera glycines), the clover cyst nematode (Heterodera trifolii), the sugar beet cyst nematode (Heterodera schachtii), the rice cyst nematode (Heterodera oryzae), and the okabo cyst nematode (Heterodera elachista). In the present invention, the potato cyst nematode (Globodera rostochiensis) (Potato Cyst Nematode: PCN) is preferred.

[0025] The host plants for cyst nematodes are not particularly limited as long as they are host plants for each cyst nematode. For example, host plants for potato cyst nematodes include plants of the Solanaceae family, such as potato, eggplant, and tomato.

[0026] The culture solution containing the cyst nematode hatching promoter can be obtained as a root exudate, a hydroponic nutrient solution, a microtuber culture solution, etc. For example, a PCN-HF-containing solution can be obtained by the following methods (a) to (c). (a) Conventional method Host plant seedlings are planted in pots or cultivation tanks filled with cultivation soil or plant support (e.g., sand, zeolite, peat moss, etc.), and then fertilizer is irrigated. The wastewater (root exudate) that passes through the cultivation tank is collected as a PCN-HF-containing solution. When cultivating in pots or other containers, the plants are irrigated periodically, left to stand for a certain period of time (approximately 24 hours), and the irrigation water is then collected from the drainage holes. (b) Culture medium from plant culture of the host plant Host plants (potato, tomato, tobacco, etc.) that produce PCN-HF are cultured (aseptically), and the culture medium is recovered. More specifically, the plants are cultured in a liquid medium prepared by adding a predetermined amount of sugar (sucrose) to MS medium, a commonly used medium for plant culture. Sugars added include glucose, fructose, galactose, maltose, trehalose, and other sugars, depending on the host plant being cultured. The sugar addition rate ranges from 0.1 to 10% depending on the host plant, but may be 10% or more. After a certain period of culture, the liquid medium is recovered, and the culture is continued by adding more liquid medium. (c) Culture medium from hairy root culture The host plant is infected with Agrobacterium sp. (currently classified as Rhizobium sp.), and the resulting hairy roots are cultured in liquid, and the culture solution is collected as a PCN-HF-containing solution (I. Ashikawa et al., Plant Tissue Culture Letters, 9(2), 123-125 (1992)).

[0027] The cyst nematode hatching factor (HF) of the present invention is not particularly limited as long as it is a hatching promoter obtained from the host plant of each cyst nematode, such as solanoeclepin A (SEA), solanoeclepin B, glycinoeclepin A, steroid glycoalkaloids (SGAs), methionine, lactones, and lactams. Examples of the potato cyst nematode hatching promoter (PCN-HF) include solanoeclepin A, solanoeclepin B, steroid glycoalkaloids (SGAs), etc. Solanoeclepin A is particularly preferred.

[0028] The liquid medium used in the present invention is not particularly limited as long as it is a medium that can generally be used for culturing plants. Examples include MS medium, Gamborg B5 medium, and N6 medium. It is preferable to add a sugar such as sucrose to the liquid medium, as this allows for efficient mass production of a culture solution containing a cyst nematode hatching promoter. The amount of sugar added is not particularly limited as long as it does not affect the growth of the host plant, but 1 to 10% is preferred.

[0029] The method for producing a composition containing a cyst nematode hatching promoter of the present invention includes removing impurities using a dialysis membrane or an ultrafiltration membrane. The impurities herein refer to substances that inhibit cyst nematode hatching or that make the culture solution containing the cyst nematode hatching promoter difficult to handle, and may be components derived from the culture medium or the plant. The impurities are typically sugars such as sucrose.

[0030] The dialysis membrane or ultrafiltration membrane that can be used in the method for producing a composition containing a cyst nematode hatching promoter of the present invention is not particularly limited, as long as it can remove impurities such as sucrose while maintaining the hatching-promoting activity of the culture solution containing the cyst nematode hatching promoter. The dialysis membrane is not particularly limited, but examples thereof include cellulose-based membranes such as cellulose acetate, and synthetic polymer membranes such as ethylene vinyl alcohol, polyacrylonitrile, polymethyl methacrylate, polysulfone, polyethersulfone, etc. When a dialysis membrane is used, the pore size of the dialysis membrane is preferably 0.5 kDa to 1 kDa. The ultrafiltration membrane is not particularly limited, but examples thereof include hollow fiber membranes, spiral membranes, tubular membranes, and flat membranes. The material of the ultrafiltration membrane is not particularly limited, but examples thereof include cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, and ceramics. When using an ultrafiltration membrane, the pore size of the ultrafiltration membrane is preferably 1 kDa to 3 kDa.

[0031] In one aspect, the present invention relates to a composition containing a cyst nematode hatching promoter, which is produced by the above-mentioned production method. The composition containing a cyst nematode hatching promoter of the present invention can be powdered by spray drying.

[0032] In one aspect, the present invention relates to a method for producing a powder containing a cyst nematode hatching promoter, which comprises spray-drying a culture solution containing a cyst nematode hatching promoter obtained by culturing a cyst nematode host plant in a liquid medium, or a culture solution or concentrated solution from which impurities have been removed using a dialysis membrane or ultrafiltration membrane. Spray drying (atomization drying) can convert a test solution into a powder by heating the atomized test solution and drying it instantly. In the present invention, a conventional spray drying device can be used for spray drying. Alternatively, an excipient may be added and then the test solution may be powdered by spray drying. The excipient is not particularly limited as long as it does not affect the activity of the hatching promoter, and examples thereof include dextrin, cyclodextrin, trehalose, gum arabic, and sugar alcohols (xylitol, mannitol). Dextrin is particularly preferred. Among dextrins, dextrin derived from cornstarch produced for food additive use, and dextrin derived from cornstarch, potato starch, or tapioca starch produced for industrial use are more preferred.

[0033] The addition rate of the excipient may be about 1 to 3 times the blending rate of sucrose in the liquid to be spray-dried, preferably 1 to 2 times, and more preferably the same. Spray drying can be performed more efficiently by appropriately setting conditions such as hot air temperature, exhaust temperature, and processing speed (liquid feed rate). As an example, conditions can be set using the ratio of exhaust temperature to hot air temperature as a guide. Setting the upper limit of the exhaust temperature to 100°C and setting the exhaust temperature to a range of 35% to 70% of the hot air temperature tends to increase the powder recovery rate and improve the powder quality. Furthermore, when the hot air temperature is less than 200°C, setting the exhaust temperature to a range of 50% to 70% of the hot air temperature, and when the hot air temperature is 200°C or higher, setting the exhaust temperature to a range of 35% to 45% of the hot air temperature, tends to increase the powder recovery rate.

[0034] Examples and application examples of the present invention will be specifically described below, but the scope of the present invention is not limited to these examples. [Example]

[0035] 1. Preparation of test materials such as a solution containing a potato cyst nematode hatching promoter (PCN-HF) and evaluation method for hatching promotion activity 1) Production of PCN-HF containing liquid Potato plants were cultured (aseptically) in MS medium (liquid medium) containing 3% sucrose for 3 to 10 weeks. The liquid medium was periodically collected and replaced with fresh medium during the culture period. The collected culture medium was used as a PCN-HF-containing solution.

[0036] 2) Preparation of concentrated PCN-HF-containing solution The PCN-HF-containing liquid was concentrated by vacuum distillation using a rotary evaporator or a centrifugal thin-film vacuum evaporator to prepare a concentrated liquid.

[0037] 3) Evaluation of hatching-promoting activity The hatching-promoting activity of the test solution was evaluated by measuring the hatching rate of PCN eggs. First, cysts were obtained from soil collected from potato cyst nematode-infested fields using a standard method (Fenwick method) (DW Fenwick, Journal of Helminthology, 18(4), 155-172 (1940)) or a method for obtaining axenic cyst nematodes (Patent No. 7254061). The cysts were immersed in sterilized water at 20°C or room temperature for 7 to 10 days, after which the cyst shells were crushed and the eggs were extracted. Sterile water was added to prepare a PCN egg suspension (approximately 1,000 eggs / mL), and 100 μL of this was dispensed into each well of a 96-well microplate (approximately 100 eggs / well). 100 μL of a test solution, such as a PCN-HF-containing solution, was added to the wells to which the PCN egg suspension had been dispensed. After the addition of the test solution, the number of PCN eggs and larvae (which had already hatched within the cysts) in each well was measured.

[0038] In addition, a positive control group was prepared for each microplate treated with sodium metavanadate (NaVO3 treatment concentration 1 mM), which is known to have hatching-promoting activity. The microplates were covered with plastic wrap to prevent evaporation of the test solution and left at 20°C to room temperature. Ten days after the test solution was added (day 0), the numbers of unhatched PCN eggs and hatched larvae in each well of the microplate were counted. The hatching rate (%) of PCN eggs was calculated by subtracting the number of hatched larvae on day 0 from the number of hatched larvae in each well measured 10 days after the test solution was added, and dividing the result by the number of PCN eggs. The hatching rate of the sodium metavanadate-treated group, the positive control group, was set to 1, and the relative hatching rate for each experimental group was calculated.

[0039] 2. Removal of impurities by dialysis 1) Small-scale testing Potato culture broth (sugar content: 3.4°Bx) was used as the PCN-HF-containing solution. Dialysis tests were performed using four dialysis membranes (Spectra / PorFloat-A-Lyzer G2, manufactured by REPLIGEN) with pore sizes of 100-500 Da, 500-1,000 Da, 3.5-5 kDa, and 8-10 kDa. 10 mL of culture broth was treated with the dialysis membrane for 72 hours. Distilled water was used as the external dialysis solution. 10 mL of culture broth was added to the dialysis membrane, and 5 L of external dialysis solution was used for treatment with three dialysis membranes (i.e., 30 mL of culture broth). The external dialysis solution was changed every two hours for the first eight hours of treatment. The external dialysis solution was then changed after 24 and 48 hours. The sugar content of the test solution was measured every 24 hours from the start of treatment as an indicator of impurity removal. After 72 hours of treatment, the hatching-promoting activity of the test solution was measured.

[0040] The results are shown in Table 1. Dialysis treatment resulted in a decrease in sugar content compared to before treatment for all pore sizes. On the other hand, after 72 hours of treatment, the hatching-promoting activity (relative hatching rate) was 0.43 (38% of before treatment) and 0.38 (33% of before treatment) for the 3.5k-5kDa and 8k-10kDa pore size treatment solutions, respectively, demonstrating a decrease. These results suggest that pore sizes of 100-500Da and 500-1,000Da are suitable for removing impurities (such as sugars) from PCN-HF-containing solutions.

[0041] [Table 1]

[0042] 2) Dynamic dialysis test of 30x concentrated solution Potato culture broth (57.7°Bx) concentrated 30-fold by centrifugal thin-film evaporation was used as the test solution. Dialysis tests were performed using a dynamic dialysis device (SpectraFlo, manufactured by REPLIGEN). Three types of dialysis membranes (Biotech, manufactured by REPLIGEN) with pore sizes of 100-500 Da, 500-1,000 Da, and 3.5 kDa-5 kDa were used. Four tubes (310 mL each) of potato culture broth per pore size were installed in the device, and dialysis was performed for 4 hours. Distilled water was used as the external dialysis solution at a flow rate of 100 mL / min. Due to the 10 L capacity of the dialysis cell, the flow rate, and treatment time, 2.5 L of external dialysis solution was used for every 100 mL of test solution.

[0043] The results are shown in Table 2. Dialysis treatment resulted in a decrease in sugar content compared to before treatment for all pore sizes. On the other hand, the hatching-promoting activity (relative hatching rate) after 4 hours of treatment was found to be 0.69 (approximately 56% of the value before treatment) in the 3.5k-5kDa pore size treatment solution. These results indicate that pore sizes of 100-500 Da and 500-1,000 Da are suitable for removing impurities from PCN-HF-containing solutions by dialysis treatment.

[0044] [Table 2]

[0045] 3. Removal of impurities by ultrafiltration 1) Small-scale testing Potato culture broth (sugar content: 3.6°Bx) was used as the PCN-HF-containing solution. Ultrafiltration tests were performed using four centrifugal ultrafiltration cartridges (Amicon Ultra-15, manufactured by Merck Millipore) with pore sizes of 1 kDa, 3 kDa, and 10 kDa. 15 mL of culture broth was centrifuged at 5,000 × g through the centrifugal ultrafiltration cartridges. Each centrifugation yielded 1.5 mL of concentrate, which remained after filtration, and 13.5 mL of permeate, which passed through the membrane. An additional 13.5 mL of sterile water was added to the concentrate, and the concentrate was collected after a second centrifugation. The concentrate was reconstituted with sterile water (diluted to the same concentration as the pre-treatment solution), and the hatching-promoting activity was measured before and after ultrafiltration.

[0046] The results are shown in Table 3. Ultrafiltration treatment resulted in a decrease in sugar content for all pore sizes compared to before treatment. On the other hand, the hatching-promoting activity (relative hatching rate) was 0.67 (63% of before treatment) in the 10 kDa pore size treatment solution, indicating a decrease. The 1 kDa pore size was equivalent to before treatment, and the 3 kDa pore size was 0.83 (80% of before treatment), maintaining the hatching-promoting activity of the original solution before treatment. These results suggest that pore sizes of 1 kDa and 3 kDa are suitable for removing impurities from PCN-HF-containing solutions by ultrafiltration.

[0047] [Table 3]

[0048] 2) Ultrafiltration test using dead-end filtration Potato culture broth (sugar content 3.2°Bx) was used as the PCN-HF-containing solution. Dead-end ultrafiltration tests were conducted using four types of ultrafiltration membranes (flat membranes) with pore sizes of 150-300 Da (Synder NFX series), 300-500 Da (Synder NFX series), 1 kDa (SUEZ GE series), and 2.5 kDa (SUES GH series). 300 mL of culture broth was filtered through the ultrafiltration membrane at 1 MPa pressure, yielding 30 mL of concentrate remaining on the membrane side and 270 mL of permeate. The concentrate was diluted to the same concentration as the unfiltered solution by adding sterilized water, and the hatching-promoting activity was measured before and after ultrafiltration.

[0049] The results are shown in Table 4. Compared to the untreated solution, the concentrated and reduced solutions with pore sizes of 1 kDa and 2.5 kDa showed a decrease in sugar content, while the 150-300 Da and 300-500 Da solutions showed no significant decrease in sugar content. A comparison of hatching-promoting activity (relative hatching rate) showed that the relative hatching rate for all pore sizes was 0.83-1.00, approximately 80-100% of the value for the untreated solution (1.00), maintaining hatching-promoting activity. These results suggest that pore sizes of 1 kDa and 2.5 kDa are suitable for removing impurities from PCN-HF-containing solutions by ultrafiltration.

[0050] [Table 4]

[0051] 3) Ultrafiltration by tangential flow filtration The test material was potato culture broth (sugar content: 2.7-2.8°Bx) containing PCN-HF. The pore size was 1 kDa (GE2540F30, manufactured by SUEZ Co., Ltd., effective membrane area: 2.6 m). 2 ), 2.5 kDa (SUEZ GH2540F30, effective membrane area 2.6 m 2A tangential flow ultrafiltration test was conducted using two types of ultrafiltration membranes (spiral membranes). 48 L of culture medium was subjected to ultrafiltration membrane treatment at a pressure of 1 to 1.8 MPa. The treatment was continued until the medium was concentrated approximately 30 times, and the concentrate was recovered. The permeate was further subjected to ultrafiltration, and the concentrate was recovered. This process was repeated twice. The recovered concentrates from the three treatments were mixed together to obtain the post-treatment concentrate.

[0052] The progress of the ultrafiltration process is shown in Table 5. 4.9 L of concentrated liquid was recovered in the 1 kDa pore size process, and 5.3 L in the 2.5 kDa pore size process. The sugar removal rate calculated by comparing the sugar content (concentrated and reduced) of the test solution (raw solution) before ultrafiltration and the concentrated solution after treatment (mixing of the first to third times) was 63% for 1 kDa and 71% for 2.5 kDa, showing a tendency for the 2.5 kDa to be higher. The processing speed calculated from the time required for the concentrated solution after treatment (mixing of the first to third times) was 4.10 L / m for 1 kDa. 2 / h, 5.20 L / m at 2.5 kDa 2 / h, with the 2.5kDa being faster.

[0053] The concentrate was diluted with sterile water to a concentration equivalent to the untreated solution, and the hatching-promoting activity was measured before and after ultrafiltration. The hatching-promoting activity was confirmed in 2-, 10-, 50-, and 100-fold dilutions of the untreated and treated concentrates (concentrated and reduced). The results are shown in Figure 1 (pore size 1 kDa) and Figure 2 (pore size 2.5 kDa). The hatching-promoting activity of 50- to 100-fold diluted concentrates (concentrated and reduced solutions) obtained after ultrafiltration was compared to the activity of the untreated solution (calculated with the relative hatching rate of the untreated solution as 100%). The values for 1 kDa and 2.5 kDa were 39-42% and 32-37%, respectively, demonstrating a tendency for decline. On the other hand, the hatching-promoting activity of 2- to 10-fold diluted solutions was 112% and 96-136%, respectively, of the untreated solution, demonstrating equal or even greater activity. The hatching-promoting activity of the permeate after ultrafiltration decreased to less than 20% at 2- to 10-fold dilutions compared to the untreated solution. At 50- to 100-fold dilutions, the 1 kDa activity was 34-64% and the 2.5 kDa activity was 43-59%. The hatching-promoting activity of the control MS medium (containing 3% sucrose) was 1% of the untreated solution at 2- to 10-fold dilutions, but 8% at 50-fold dilution and 19% at 100-fold dilution. Since the values increased with increasing dilution ratio, the medium components possessed minimal hatching-promoting activity. Furthermore, the dilution series and hatching-promoting activity (relative hatching rate) showed similar trends between the permeate and MS medium. The hatching-promoting activity observed in the permeate may be due to some PCN-HF from the potato culture medium remaining in the permeate, or it may be due to MS medium components migrating to the permeate. These results suggest that it is possible to concentrate PCN-HF-containing solutions by ultrafiltration, and that pore sizes of 1 kDa and 2.5 kDa are suitable for removing impurities from PCN-HF-containing solutions by ultrafiltration.

[0054] [Table 5]

[0055] 4. Powderization by spray drying 1) Preliminary study: Powdering of potato culture solution only A preliminary test was conducted to determine whether potato culture solution alone could be powdered by spray drying. A spray dryer GB22 (Yamato Scientific Co., Ltd., evaporation capacity 1.3 kg / h) was used, with a drying air volume of 0.45 m 3 / h, spray pressure 1.5-2.0kgf / m 3 After fixing the temperature at 80°C, 50 mL of potato culture solution (sugar content 3.4°Bx) was treated under 50 conditions: hot air temperature 80-120°C (in 10°C increments) and solution flow rate 1-10 mL / min (in 1 mL increments). Under all conditions, the test solution adhered to the collection container of the spray dryer in a sticky form and could not be collected because it did not turn into powder. It was thought that the addition of an excipient as an auxiliary agent was necessary to apply potato culture solution containing sugar to the spray drying method.

[0056] 2) Selection of excipients It is widely known that adding excipients to test solutions that are difficult to powder using the spray-drying method can make them possible. However, there have been no cases or reports of spray-drying solutions containing PCN-HF, and it was necessary to identify excipients that do not affect the activity of PCN-HF in advance. Therefore, we prepared a suspension of potato culture solution containing excipients and tested it on PCN eggs to confirm the hatching rate. The excipient concentrations ranged from 5 to 50 mg / mL, and the hatching rate was compared with that of potato culture solution containing no excipient (0 mg / mL). The excipients tested were dextrin or gum arabic, which are commonly used in spray-drying. Food additive, industrial product, and reagent grades were used to examine their effect on hatching-promoting activity, i.e., hatching-promoting activity inhibition tests. Example results are shown in Table 6. Excipients that showed no decrease in hatching-promoting activity at any treatment concentration, i.e., no hatching-inhibiting activity, were deemed applicable as excipients for spray-drying PCN-HF-containing solutions and rated "OK." Excipients that showed hatching-inhibiting activity were rated "NG." The DE value in the remarks column in the table is the dextrin equivalent (dextrose equivalent), which indicates the saccharification rate of starch, and is listed as a characteristic of the dextrin tested. No correlation was observed between the DE value and hatching-promoting inhibitory activity within the range of dextrins tested. In the following examples, food additive-grade Pine Index #100 (manufactured by Matsutani Chemical Industry Co., Ltd.), which showed no hatching-inhibiting activity and was therefore applicable, was used as the excipient.

[0057] [Table 6]

[0058] 3) Spray drying study (small scale) A suspension containing 3% dextrin as an excipient was added to potato culture broth (sugar content 3.4°Bx) as a PCN-HF-containing solution, and the resulting suspension was subjected to spray drying. The dextrin addition ratio was set at 1:1 relative to the sugar content, based on the report by Wrzosek et al. (Acta Chimica Slovaca, 2013). In other words, for a potato culture broth with a sugar content of 3.4°Bx, dextrin was added at 3% (w / v) of the potato culture broth volume. The spray dryer used was a GB22 (Yamato Scientific Co., Ltd., evaporation capacity 1.3 kg / h). The drying air volume was 0.45 m 3 / h, spray pressure 1.5-2.0kgf / m 3 After fixing the temperature at 80°C to 120°C (in 10°C increments), 50 mL of 3% dextrin-added potato culture solution was treated under 40 conditions: hot air temperature 80 to 120°C (in 10°C increments) and liquid flow rate 3 to 10 mL / min (in 1 mL increments). 3 The results in / h are shown in Table 7. Also, spray pressure 1.5 to 2.0 kgf / m 3 The dry air volume was fixed at 0.55 m 3 The flow rate was increased to 1000 kJ / h, and 50 mL of potato nutrient solution containing 3% dextrin was treated under 26 conditions with the hot air temperature set to 80-120°C (in 10°C increments) and the liquid feed rate set to 3-10 mL / min (in 1 mL increments). 3 The test results, increased to / h, are shown in Table 8. The table shows the exhaust gas temperature (top row) for each treatment condition, the recovery rate (middle row) calculated from the amount of powder recovered relative to the solids content of the treatment solution, which is the sum of the solids content estimated from the sugar content of the potato culture solution and the amount of added excipient (dextrin), and the quality of the recovered powder, ranked on a three-point scale. Powder quality was rated "good" if it was about the same as the added dextrin, "fair" if it was slightly sticky, and "poor" if it was significantly sticky and stuck to the tube or other surface, making recovery difficult. Treatments that did not turn into powder but instead stuck to the tube wall of the collection container in a candy-like manner were rated "unrecoverable," and treatments where the treatment solution did not dry and condensed were rated "condensed." Powder quality was also evaluated in the following spray-drying study.

[0059] [Table 7]

[0060] As shown in Table 7, the dry air volume is 0.45 m 3 At a setting of 1-2 mL / min / h, powder with good powder quality could be recovered regardless of the hot air temperature. At a setting of 3-5 mL / min, deterioration of powder quality was observed at hot air temperatures of 80°C and 100°C. At a setting of 6 mL / min or more, deterioration of powder quality was observed at all hot air temperatures. Next, as shown in Table 8, when the drying air volume was set to 0.55 m 3 By increasing the spray-drying rate to 1 / h, high-quality powder recovery was possible even at a hot air temperature of 110–120°C and a liquid feed rate of 7 mL / min. The exhaust temperature was approximately 64°C at a hot air temperature of 120°C and a total liquid flow rate of 7 mL / min. Therefore, to confirm the effect of heating during spray drying on hatching-promoting activity, we evaluated the hatching-promoting activity of powder under 11 conditions (liquid feed rate of 5 mL / min and hot air temperature of 100°C, liquid feed rate of 6 mL / min and hot air temperature of 80–120°C, liquid feed rate of 7 mL / min and hot air temperature of 100–120°C, liquid feed rate of 8 mL / min and hot air temperature of 120°C, and liquid feed rate of 9 mL / min and hot air temperature of 120°C). (Treatment concentration: 1 mg / mL, equivalent to a 64-fold dilution of the pre-treatment solution.) The results are shown in Figure 3.

[0061] [Table 8]

[0062] As shown in Figure 3, the hatching-promoting activity of the powders under all treatment conditions was equivalent to that of the undiluted solution before spray-drying. Specifically, when the relative hatching rate, which indicates the hatching-promoting activity of the undiluted solution, was set at 100%, the relative hatching rates of the powders were compared. The hatching rates were 96% at a hot air temperature of 80°C and a liquid flow rate of 6 mL / min, and at a hot air temperature of 100°C and a liquid flow rate of 7 mL / min, and 100% (some even exceeding 100%) at other conditions. Thus, the hatching-promoting activity did not decrease during the spray-drying process. The spray-drying method involves spraying the test solution into fine mist droplets into a drying chamber, where it is instantly dried and powdered by the hot air inside the chamber. Because the drying process is instantaneous, the thermal load on the sample is low, but the temperature rises to at least the exhaust temperature. Solanoeclepin A, identified as PCN-HF, was reported in U.S. Patent No. 5,585,505 (Patent Document 1) to be unstable at temperatures above 35°C. Therefore, in this test, the powder obtained under the treatment conditions in which the exhaust temperature was 35°C or higher had the potential to have significantly reduced hatching-promoting activity compared to the untreated stock solution. However, contrary to expectations, even when the exhaust temperature was 35°C or higher, the powder was produced while maintaining the activity of the stock solution.

[0063] 4) Reduction of excipient addition amount In a previous test, 3% of an excipient (dextrin, Pine Index #100 (PD100, manufactured by Matsutani Chemical Industry Co., Ltd.) was added to potato culture solution (sugar content 3.2°Bx). To confirm whether this addition rate could be reduced, powderization studies were conducted at addition rates of 1% and 2%. A GB22 spray dryer (manufactured by Yamato Scientific Co., Ltd., evaporation capacity 1.3 kg / h) was used. The hot air temperature was 120°C, and the drying air volume was 0.55 m. 3 / h, spray pressure 1.5-2.0kgf / m 3The flow rate was fixed at 6 mL / min, and the flow rate was varied from 6 to 9 mL / min (four conditions, in 1 mL increments). The 3% dextrin-added solution was used as a control, with the test solution volume at 50 mL. The results are shown in Table 9. The recovery rate of the powder with a 2% dextrin addition rate decreased to 59.26% at a flow rate of 6 mL / min, 57.84% at a flow rate of 7 mL / min, 37.5% at a flow rate of 8 mL / min, and 25.66% at a flow rate of 9 mL / min, and the powder quality was "unacceptable" at all flow rates. In the test with a 1% dextrin addition rate, at a flow rate of 9 mL / min, the powder adhered to the drying chamber of the spray dryer and the collection container in a syrup-like form, making it impossible to collect as a powder. Furthermore, at flow rates of 6 to 8 mL / min, the recovery rate decreased to 7.25 to 14.07%, and the powder quality was "unacceptable" at all levels. From the above results, since sugar content can be converted into sucrose content, it is desirable that the addition rate of excipient (dextrin) be at least equal to the sugar content (sugar:excipient = 1:1).

[0064] [Table 9]

[0065] 5) Consider spray drying (increasing the amount of excipients) Approximately 10 L of PCN-HF-containing solution was prepared by adding 6.5% dextrin as an excipient to potato culture broth (sugar content 3.4°Bx) to prepare a suspension, which was then spray-dried. The spray dryer used was the L-8 (Okawahara Chemical Engineering Co., Ltd., evaporation capacity 5.5 kg / h). To achieve the same processing conditions as the GB22 spray dryer, where the hot air temperature was 90°C and the liquid flow rate was 6 mL / min, resulting in an exhaust temperature of 50°C, the liquid flow rate was checked using water operation, and the liquid flow rate was set to approximately 20 mL / min. The test was repeated twice, and the results are shown in Table 10. The liquid feed rate was adjusted based on the exhaust temperature, and the actual values for exhaust temperature and liquid feed rate were an exhaust temperature of 51-53°C and a liquid feed rate of 16 mL / min, and an exhaust temperature of 47°C and a liquid feed rate of 23 mL / min. The recovered powder was suspended in the equivalent pre-treatment liquid and the hatching-promoting activity was evaluated, and it maintained the same activity as before spray-drying.

[0066] [Table 10]

[0067] 6) Consider spray drying (improving hot air temperature and liquid delivery volume) A suspension containing 3% dextrin as an excipient was added to potato culture broth (sugar content 3.4°Bx) as the PCN-HF-containing solution, and the suspension was subjected to spray drying. The spray dryer used was a GB22 (manufactured by Yamato Scientific Co., Ltd., evaporation capacity 1.3 kg / h). The volume of drying air was 0.55 m 3 / h, spray pressure 1.5-2.0kgf / m 3 After fixing the temperature at 200°C, 50 mL of 3% dextrin-added potato culture solution was treated under five conditions: a hot air temperature of 200°C and a liquid flow rate of 16 to 20 mL / min (in 1 mL increments). The results are shown in Table 11. Powder was recovered from the treatments with liquid flow rates of 19 and 20 mL / min, but deterioration of powder quality was observed. Therefore, the hatching-promoting activity of the powders treated with liquid flow rates of 16 and 18 mL / min was evaluated. The powder from the treatment with 20 mL / min was also used for the evaluation of hatching-promoting activity as a comparative example of powder quality. A suspension equivalent to the pre-treatment solution was prepared from each powder based on the solid content calculated from the sugar content of the potato culture solution tested and the amount of excipient added. The suspension was diluted to a concentration corresponding to the dilution ratio of the pre-treatment solution. The results are shown in Figure 4. The hatching-promoting activity of the powder prepared with any of the liquid feed rates was 80% or more of the relative hatching rate, which indicates the hatching-promoting activity of the pre-treatment solution, at all treatment concentrations (dilution ratios), maintaining the activity of the pre-treatment solution. Furthermore, although the powder quality was rated "fair," no effect on the hatching-promoting activity was observed.

[0068] [Table 11]

[0069] 7) Consider spray drying (improving hot air temperature and liquid delivery volume) A suspension containing 6% dextrin as an excipient was added to potato culture broth (sugar content 6.0°Bx) as the PCN-HF-containing solution, and the suspension was subjected to spray drying. The spray dryer used was a DL410 (Yamato Scientific Co., Ltd., evaporation capacity 3.0 kg / h). The volume of drying air was 0.85 to 1.0 m 3 2,000 mL of 6% dextrin-supplemented potato broth was treated under 12 conditions: a hot air temperature of 200–230°C (four levels, in 10°C increments) and a broth flow rate of 2.6 L / h, 2.8 L / h, and 3.0 L / h (three levels), with the air temperature adjusted for each broth flow rate and a fixed spray pressure of 0.15 MPa. The results are shown in Table 12. At a broth flow rate of 3.0 L / h, the powder recovery rate was below 70%, and the powder quality was graded "fair," indicating a tendency toward deterioration. Therefore, the hatching-promoting activity was evaluated using powder from eight treatments with a recovery rate of 70% or higher. The recovered powder was suspended in sterilized water to the same concentration as the pretreatment solution. The results are shown in Figure 5. The hatching-promoting activity of the powders adjusted with any of the liquid delivery volumes was 90% or more of the relative hatching rate, which indicates the hatching-promoting activity of the pre-treatment liquid, at all treatment concentrations (dilution ratios), and maintained activity equivalent to that of the pre-treatment liquid.

[0070] [Table 12]

[0071] 8) Spray drying study of concentrated solution (1) A suspension of potato culture broth concentrate (equivalent to a 15x concentrate, sugar content 30.6°Bx) concentrated by centrifugal thin film evaporation, to which 30% dextrin was added as an excipient, was subjected to spray drying. The spray dryer used was a DL410 (Yamato Scientific Co., Ltd., evaporation capacity 3.0 kg / h). The volume of drying air was 0.85 m 3The spray pressure was fixed at 0.2 MPa and the hot air temperature was 130°C with a liquid flow rate of 0.9 L / h, 200°C with a liquid flow rate of 1.3 L / h, 220°C with a liquid flow rate of 2.0 L / h, and 220°C with a liquid flow rate of 3.0 L / h. The results are shown in Table 13. All treatments resulted in powder recovery with good powder quality. The recovery rate was highest at 83.54% for the treatment with a hot air temperature of 130°C and a liquid flow rate of 0.9 L / h. Furthermore, the exhaust temperature exceeded 90°C at both the hot air temperature of 220°C with a liquid flow rate of 2.0 L / h and the hot air temperature of 200°C with a liquid flow rate of 1.3 L / h. The recovered powder was evaluated for hatching-promoting activity. The recovered powder was adjusted with sterilized water to the same concentration as the pre-spray-drying solution. To evaluate the hatching-promoting activity, the powder was diluted (concentrated and reduced) to the same level as the original solution before concentration. The results are shown in Figure 6. The hatching-promoting activity of the powder prepared at any given volume of solution was 85% or more of the relative hatching rate, which indicates the hatching-promoting activity of the pre-treatment solution, at all treatment concentrations (dilution ratios), maintaining activity equivalent to that of the pre-treatment solution.

[0072] [Table 13]

[0073] 9) Spray drying study of concentrated solution (2) A suspension of potato culture broth concentrate (equivalent to a 7x concentrate, sugar content 14°Bx) concentrated by centrifugal thin film evaporation, to which 14% dextrin was added as an excipient, was subjected to spray drying. The spray dryer used was a DL410 (Yamato Scientific Co., Ltd., evaporation capacity 3.0 kg / h). The volume of drying air was 0.60 m 3The test was conducted under a total of 42 conditions: a hot air temperature of 200–250°C (6 levels, in 10°C increments) and a liquid feed rate of 1.0–2.2 L / h (7 levels), with the temperature and spray pressure fixed at 0.15 MPa. The results are shown in Table 14. At a hot air temperature of 250°C and a liquid feed rate of 1.0 L / h and 250°C and 1.3 L / h, the exhaust temperature exceeded 100°C, causing overheating, and the test was discontinued. At a hot air temperature of 200°C and a liquid feed rate of 2.20 L / h and 210°C and 2.20 L / h, the test was discontinued due to the possibility of condensation. In the test group with a hot air temperature of 220°C and a liquid feed rate of 1.3 to 1.6 L / h, the recovery rate was 94.60% at a liquid feed rate of 1.0 L / h, 95.85% at a liquid feed rate of 1.3 L / h, and a highest recovery rate of 98.97% at 1.5 L / h. Compared to the recovery rates at other hot air temperatures, the recovery rate tended to be consistently higher in the range of liquid feed rates of 1.0 to 1.5 L / h. At liquid feed rates of 1.6 L / h or higher, with the exception of 90.41% at a hot air temperature of 200°C and 95.87% at 220°C, the recovery rate tended to decrease overall, and a tendency for powder quality to deteriorate was also observed.

[0074] [Table 14]

[0075] 10) Spray drying study using potato nutrient concentrate after tangential flow ultrafiltration Potato culture solution (sugar content 2.7°Bx) was used as the PCN-HF-containing solution. 2An ultrafiltration test was conducted using an ultrafiltration membrane (spiral membrane) of the same size as the 109 L culture medium. A pressure of 1.4 to 1.8 MPa was applied to the ultrafiltration membrane. The culture medium was concentrated approximately 30 times, and the concentrate was collected. The permeate was further ultrafiltered, and the concentrate was collected. This was then filtered twice, for a total of three times. The collected concentrates from the three times were combined to form the post-treatment concentrate. The progress of the ultrafiltration process is shown in Table 15. After approximately 9.5 hours, the sugar removal rate was 52% (calculated by comparing the sugar content (concentrated and reduced) of the test solution (raw solution) before ultrafiltration and the post-treatment concentrate (mixed first to third times). Furthermore, the concentrate was diluted with sterilized water (to the same concentration as the pre-treatment solution). The pre-treatment solution was used as a control, and the post-treatment concentrate and the third permeate were used to measure the hatching-promoting activity before and after ultrafiltration. The hatching-promoting activity of the untreated solution and the post-treatment concentrated solution (concentrated and reconstituted) was confirmed in 2-, 10-, 50-, and 100-fold diluted solutions. The results are shown in Figure 7. The hatching-promoting activity of the 50- to 100-fold diluted solutions was 30-58% of that of the untreated solution (calculated with the relative hatching rate of the untreated solution as 100%), indicating a tendency for decline. On the other hand, the hatching-promoting activity of the 2- to 10-fold diluted solutions was 108-125% higher than that of the untreated solution. The hatching-promoting activity of the permeate after ultrafiltration treatment decreased to 9-18% of that of the untreated solution at 2- to 10-fold dilutions and 36-43% at 50- to 100-fold dilutions. The hatching-promoting activity of the concentrated solution was equivalent to that of the permeate at 100-fold dilution, but exceeded that of the other permeates, and even exceeded that of the untreated solution at 2- to 10-fold dilutions. From the above, it was possible to concentrate the PCN-HF-containing liquid by ultrafiltration.

[0076] [Table 15]

[0077] The concentrate (sugar content 15.4°Bx) after the membrane ultrafiltration described above was mixed with 15% dextrin as an excipient to form a suspension, which was then subjected to spray drying. The spray dryer used was a DL410 (manufactured by Yamato Scientific Co., Ltd., evaporation capacity 3.0 kg / h). The volume of drying air was 0.60 m 3The spray pressure was fixed at 0.15 MPa, and the treatment was performed under nine conditions: a hot air temperature of 220–240°C (in 10°C increments, three levels) and a liquid delivery rate of 1.3–1.6 L / h. The results are shown in Table 16. At a hot air temperature of 240°C and a liquid delivery rate of 1.3 L / h, the test was discontinued due to overheating caused by the exhaust temperature exceeding 100°C. Therefore, an alternative test was performed with a hot air temperature of 240°C and a liquid delivery rate of 1.9 L / h. Powder was recovered with a recovery rate of 85% or higher and a powder quality of "good" in all treatments. At hot air temperatures of 230°C or higher, the exhaust temperature exceeded 90°C at all liquid delivery rates. The recovered powder was suspended in sterilized water to the same concentration as the pre-spray-drying solution, and then diluted (concentrated and reduced) to the same concentration as the original solution for evaluation of hatching-promoting activity. The results are shown in Figure 8. The hatching-promoting activity of the 2-fold diluted powder, when compared with the relative hatching rate of the pre-treatment solution (defined as 100%), was 100% (some even higher) at all treatment speeds at a hot air temperature of 220°C, equivalent to or higher than that of the pre-treatment solution. At hot air temperatures of 230°C or higher, the activity was approximately 90% of that of the pre-treatment solution at all treatment speeds, but slightly lower than that at 220°C. At 10-fold diluted powder, the activity was 90-95% of that of the pre-treatment solution at a hot air temperature of 220°C, approximately 80% at 230°C, and 75-84% at 240°C, at all treatment speeds. Compared to the activity at 220°C, there was a slight tendency for the activity to decrease at hot air temperatures of 230°C or higher. At 50-fold diluted powder, the activity was 60-70% for all treatments (hot air temperature, liquid volume), indicating a slight tendency for a decrease. From the above, it was possible to powder the pre-treatment solution without losing its hatching-promoting activity.

[0078] [Table 16]

[0079] 11) Spray drying study using concentrated potato nutrient solution after dialysis Potato culture broth (sugar content: 3.2 Bx) was used as the PCN-HF-containing solution. A 24x concentrated solution of potato culture broth (sugar content: 53.4°Bx) was prepared by centrifugal thin-film evaporation using a centrifugal thin-film vacuum evaporator (Okawahara Seisakusho CEP-Lab). A dialysis test was performed using 4,000 mL of the 24x concentrated solution with a dynamic dialysis device (REPLIGEN SpectraFlo). Dialysis was performed for approximately 4 hours using a dialysis membrane with a pore size of 100-500 Da (REPLIGEN Biotech). The concentrate was recovered after dialysis, and its sugar content was measured, revealing a drop to 8.5°Bx. After dialysis, 9% dextrin was added as an excipient to the concentrate, and the resulting suspension was spray-dried. A DL410 spray dryer (Yamato Scientific Co., Ltd., evaporation capacity: 3.0 kg / h) was used. The drying air volume was 0.85 m 3The spray pressure was fixed at 0.15 MPa, with a hot air temperature of 220°C and a liquid delivery rate of 2.6 L / h. The exhaust temperature was approximately 87°C, and the powder recovery rate was approximately 92%, resulting in a good powder with low stickiness. The recovered powder was suspended in sterilized water to the same concentration as the solution before spray drying and diluted (reduced) to the same concentration as the original solution before concentration of hatching-promoting activity. Furthermore, the concentrated solution before and after dialysis were also diluted (reduced) to the same concentration as the original solution before concentration, and the hatching-promoting activity was evaluated. The results are shown in Figure 9. The hatching-promoting activity of the dialyzed concentrate was compared to the relative hatching rate of the concentrate before dialysis, which was set at 100%. The results were 100% at a 3.125-fold dilution, 76% at a 6.25-fold dilution, 64% at a 12.5-fold dilution, 22% at a 25-fold dilution, 41% at a 50-fold dilution, 25% at a 100-fold dilution, and 40% at 200- to 800-fold dilutions. While some dilutions tended to result in lower activity than the pre-dialysis concentrate, the sugars were successfully removed while maintaining the activity of the pre-dialysis concentrate. The hatching-promoting activity of the spray-dried powder of the dialyzed concentrate, compared to the pre-spray-drying solution (i.e., the dialyzed concentrate) was higher at 100- to 800-fold dilutions, exceeding 100%. Furthermore, the hatching rate was approximately 70% at a 25- to 50-fold dilution and over 80% at a 3.125- to 12.5-fold dilution. From the above, it was possible to obtain powder by spray drying without impairing the hatching-promoting activity of the solution before spray drying.

[0080] 12) Storage stability of spray-dried powder Approximately 10 L of PCN-HF-containing solution was prepared by adding 6.5% dextrin as an excipient to potato culture broth (sugar content 3.4°Bx) and spray-drying the suspension. The spray dryer used was an L-8 (Okawahara Chemical Engineering Co., Ltd., evaporation capacity 5.5 kg / h) with a hot air temperature of 90°C and a liquid flow rate of 16 mL / min. The collected powder was stored in a refrigerator (4°C). The hatching-promoting activity of the powder at the time of spray-drying and after three years of refrigerated storage was compared in terms of relative hatching rate. The results are shown in Figure 10. The relative hatching rate at the time of spray-drying (labeled "at the time of spray-drying") was set to 100%, and the hatching-promoting activity after three years of storage (labeled "after 3 years") was compared. The hatching-promoting activity was 30-50% at treatment concentrations of 0.001-0.1 mg / mL. However, at this treatment concentration, the hatching-promoting activity itself was low during spray-drying, and no decrease in activity was observed during storage. On the other hand, at treatment concentrations of 0.5 to 50 mg / mL, the hatching-promoting activity was over 100% of that at the time of spray-drying, and no decrease in hatching-promoting activity was observed during storage. Furthermore, at a treatment concentration of 100 mg / mL, the hatching-promoting activity was 86%, equivalent to that at the time of treatment, and no decrease in hatching-promoting activity was observed during storage. However, the potato culture solution used for the powder before treatment spoiled approximately one month after storage in a refrigerator, making it impossible to evaluate its hatching-promoting activity. These results demonstrate that the spray-dried powder offers superior storage stability compared to the culture solution in terms of maintaining the hatching-promoting activity of PCN-HF.

Claims

1. A method for producing a powder containing a cyst nematode hatching promoter, which comprises spray-drying a culture solution containing a cyst nematode hatching promoter obtained by culturing a cyst nematode host plant in a liquid medium, or a composition containing a cyst nematode hatching promoter obtained by removing impurities from the culture solution using a dialysis membrane or an ultrafiltration membrane.

2. The method according to claim 1 , wherein the host plant is a Solanaceae plant.

3. 2. The method according to claim 1, wherein the cyst nematode is a potato cyst nematode.

4. The method of claim 1 , wherein the liquid medium contains sucrose.

5. The method of claim 1 , wherein the contaminants to be removed include sucrose.

6. The method according to claim 1, wherein the excipient is added and then the mixture is powdered by spray drying.

7. The method according to claim 6, wherein the excipient is added in an amount 1 to 2 times the amount of sucrose.

8. The method according to claim 1, wherein the spray drying is carried out by setting the exhaust temperature to be in the range of 35 to 70% of the hot air temperature, with the upper limit of the exhaust temperature being 100°C.

9. A powder containing a substance that promotes hatching of cyst nematodes, produced by the method according to any one of claims 1 to 8.

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