Plant growth regulator and method for producing same

Abstract

To provide a novel plant growth regulator.SOLUTION: A plant growth regulator contains a culture medium of lactic acid bacteria containing a high concentration of corn steep liquor (CSL), as an active ingredient.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a plant growth regulator, a method for producing the same, and a lactic acid bacterium and a lactic acid bacterial culture that can be used to prepare the plant growth regulator. [Background technology]

[0002] In the agricultural field, controlling plant growth is an important technology for improving productivity. Currently, various types of plant growth regulators are in practical use, contributing to improving crop yields and product quality.

[0003] However, among plant growth regulators, those that promote root development are few in number and variety, and their effectiveness is insufficient. Furthermore, many plant growth regulators have undesirable effects. For example, among plant growth regulators, auxin compounds such as indole acetic acid, which are currently widely used as rooting agents, can have undesirable effects such as upward leaf growth, stem twisting, stem cracking, root galling, and even plant death, depending on the type and condition of the plant and the application concentration, which limits their use method and dosage. Furthermore, their root development promotion effect has not been fully satisfactory. In response to this, a plant growth regulator containing phenyllactic acid and tryptophan produced by lactic acid fermentation has been proposed as a root-inducing agent that can solve the above problems (Patent Document 1). A production method has also been disclosed. Meanwhile, corn steep liquor (hereinafter referred to as "CSL") is known as a by-product of corn starch production. CSL is a dried or concentrated soluble component obtained during the process of producing starch from corn. The production process of CSL includes a lactic acid fermentation step as part of the process (Non-Patent Document 1). Patent Document 1 describes that in this production process, by adjusting the type of lactic acid bacteria strain colonized in the fermenter performing lactic acid fermentation and the concentration conditions of the fermentation liquid that serves as the raw material for CSL, a plant growth regulator containing phenyllactic acid and tryptophan can be produced from CSL.

[0004] However, the concentration of phenyllactic acid contained in this CSL is low, at 30 mg / L or less, and CSL is not suitable for use as a plant growth regulator as is. On the other hand, CSL contains 6 to 7% free amino acids. Therefore, it is expected to be effective as an amino acid-containing material (Non-Patent Document 2), a type of biostimulant that has been used in agriculture worldwide in recent years. However, CSL that is generally available on the market has high viscosity and is prone to producing sediment, making it difficult to actually use as an amino acid-containing material. In order to improve the occurrence of sedimentation in CSL, Patent Document 2 describes CSL that does not produce sediment (precipitation) and a method for producing the same. This production method requires a special centrifuge and does not solve the viscosity problem. Another possible method is to dilute CSL and use it in an amino acid-containing material, but this would further reduce the concentrations of free amino acids and phenyllactic acid, resulting in a quality that is not suitable for use as a plant growth regulator.

[0005] Furthermore, by using a diluted CSL solution as a medium and carrying out lactic acid fermentation, it is possible to increase the concentration of phenyllactic acid, thereby preparing a plant growth regulator with high concentrations of both free amino acids and phenyllactic acid. However, it is known that lactic acid bacteria have difficulty growing in high concentrations of CSL. For this reason, in conventional technology, even if the CSL used as a fermentation raw material has been improved by citric acid treatment, the concentration of CSL used in the medium was only 7% (see Patent Document 3). Furthermore, CSL is known to have a growth-promoting effect on lactic acid bacteria, but when used for this purpose, it is currently added to the culture medium at only 0.005% (see Non-Patent Document 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5389677 [Patent Document 2] Japanese Patent Application Laid-Open No. 2001-204410 [Patent Document 3] Japanese Patent Application Publication No. 53-72889 [Non-patent literature]

[0007] [Non-Patent Document 1] Taizo Miwa, 1979, Corn Processing Industry By-products, "Lectures on Formulated Feed (Volume 2)", pp. 265-269, Chikusan Publishing [Non-patent document 2] Calvo et al.2014.Agricultural uses of plant biostimulants.Plant Soil 383:3-41. [Non-patent document 3] Johnson et al.1971.Characterization of growth stimulants in corn steep for lactic Streptococci. Appl. Microbil. 21:316-320. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a novel plant growth regulator. Another object of the present invention is to provide a method for producing this novel plant growth regulator and a lactic acid bacterium for use in this production method. [Means for solving the problem]

[0009] That is, the present invention comprises the following configurations. (1) A plant growth regulator containing a highly concentrated lactic acid bacteria culture solution as its active ingredient. (2) The plant growth regulator according to (1), wherein the lactic acid bacteria culture solution containing a high concentration of corn steep liquor (CSL) contains 20% by mass or more of solids derived from CSL. (3) The plant growth regulator according to (1) or (2), wherein the lactic acid bacteria culture solution contains 160 mg / L or more of phenyllactic acid. (4) The plant growth regulator according to any one of (1) to (3), wherein the lactic acid bacterium is selected from the group consisting of Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus rapi, and Lactobacillus diolivorans. (5) The plant growth regulator according to any one of (1) to (4), wherein the lactic acid bacterium is selected from Lactobacillus paracasei and Lactobacillus rhamnosus. (6) The plant growth regulator according to any one of (1) to (5), wherein the lactic acid bacterium is any one of Lactobacillus paracasei #720 strain (accession number NITE BP-03117), Lactobacillus rhamnosus #555 strain (accession number NITE ABP-03379), and Lactobacillus sp. #728 strain (accession number NITE ABP-03380). (7) A lactic acid bacterium that grows in a medium containing 20% ​​by mass or more of solids derived from corn steep liquor (CSL) and produces a plant growth regulator. (8) The lactic acid bacterium according to (7), wherein the lactic acid bacterium is selected from the group consisting of Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus rapi, and Lactobacillus diolivorans. (9) The lactic acid bacterium according to (7) or (8), which is either Lactobacillus paracasei or Lactobacillus rhamnosus. (10) A plant growth regulator containing as an active ingredient a lactic acid bacteria culture solution containing 20% ​​by mass or more of solids derived from corn steep liquor (CSL) and phenylalanine. (11) A plant growth regulator containing, as an active ingredient, a lactic acid bacteria culture solution containing 20% ​​by mass or more of solids derived from corn steep liquor (CSL) and EDTA iron (III valent) and / or EDTA zinc (II valent). (12) A method for producing a plant growth regulator according to any one of (1) to (6), characterized in that lactic acid bacteria are cultured in a medium containing 20% ​​by mass or more of a solid content derived from corn steep liquor (CSL). (13) A composition for plant rooting, soluble in isopropanol and butanol, obtained from a lactic acid bacteria culture medium containing a high concentration of corn steep liquor (CSL). [Effects of the Invention]

[0010] The plant growth regulator of the present invention has high root-promoting activity and extremely little side effect such as promoting leaf growth, so it can be used as a plant growth regulator, particularly as a root promoter, throughout the growing period. It is also effective as a root promoter during the seedling raising and transplanting stages. Furthermore, because the plant root-promoting activity is enhanced, the plant growth regulator of the present invention can also be used as an agent for inhibiting fatigue in crops that are continuously harvested in greenhouse cultivation, such as cucumbers, tomatoes, eggplants, bell peppers, and strawberries. Therefore, the plant growth regulator of the present invention can be used as a pesticide or fertilizer additive, or as a fertilizer itself. Furthermore, the lactic acid bacteria of the present invention can grow in a medium containing a high concentration of CSL, where conventional lactic acid bacteria have difficulty growing, and can be easily cultured in a culture medium containing CSL as the main component, producing a high concentration of phenyllactic acid in the culture solution.Furthermore, after completion of this culture, the culture solution becomes a highly active plant growth regulator. Furthermore, the method for producing a plant growth regulator of the present invention uses a simple culture device and an inexpensive culture medium, making it possible to produce a highly active plant growth regulator at extremely low cost. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a scatter plot showing the relationship between the amount of phenyllactic acid (PLA) produced and the amount of phenylalanine (Phe) added in Test Example 4. FIG. [Figure 2] This is a graph showing the time course of measurements of various metal compounds added during culture and their pH-lowering effects in Test Example 5. In the figure, EDTA (4H, free type) is represented as "H," tetrasodium EDTA as "Na," calcium EDTA (divalent) as "Ca," magnesium EDTA (divalent) as "Mg," zinc EDTA (divalent) as "Zn," and iron EDTA (trivalent) as "Fe." [Figure 3] 1 is a graph showing the results of measuring the change in culture medium pH over time in Production Example 1. [Figure 4] 1 is a graph showing the results of measuring the rooting activity of a plant growth regulator prepared by diluting the culture solution of Production Example 1 on cut parts of adzuki beans. DETAILED DESCRIPTION OF THE INVENTION

[0012] As a result of intensive research to solve the problem, the present inventors discovered lactic acid bacteria strains that can grow in liquid media containing high concentrations of CSL, and further discovered that lactic acid bacteria culture solutions obtained using these lactic acid bacteria strains are excellent plant growth regulators. This lactic acid bacteria culture solution contains high concentrations of phenyllactic acid and tryptophan, and also contains root-promoting substances extracted with n-butanol, which promotes plant rooting and makes it an excellent plant growth regulator.

[0013] Corn steep liquor (hereinafter referred to as "CSL") in the present invention refers to concentrated or dried soluble components obtained in the process of producing starch from corn. Furthermore, "lactic acid bacteria culture medium containing a high concentration of CSL" refers to a culture medium obtained by culturing lactic acid bacteria capable of growing in a CSL dilution having a concentration that makes it difficult for general lactic acid bacteria to grow, or in a CSL-rich medium containing 20% ​​or more CSL by mass in dry mass equivalent, in the dilution or medium. Furthermore, the term "plant growth regulator" as used herein refers to a chemical that has a regulating effect on the growth and development of plants and is used to regulate the growth of agricultural crops.

[0014] The plant growth regulator of the present invention is a culture medium obtained by culturing a CSL-resistant lactic acid bacterium in a culture medium containing a high concentration of CSL. This plant growth regulator contains a CSL-derived solids content of 20% by mass or more, preferably 20 to 25% by mass, and particularly preferably 20 to 23% by mass, and a phenyllactic acid content of 160 mg / L or more, preferably 160 to 200 mg / L, and more preferably 16 to 150 mg / L. The plant growth regulator also contains a free amino acid content of 3% by mass or more, preferably 3 to 10% by mass, and more preferably 3 to 5% by mass. The solid content of corn steep liquor can be measured by the dry weight measurement method generally used to measure the solid content of organic matter. For example, 1 mL of sample is placed in a container such as an aluminum cup that has been dried in a desiccator, accurately weighed, heated and dried on a hot plate until it becomes a viscous candy, then dried in an oven at 105°C for 3 hours, allowed to cool in the desiccator, and reweighed to calculate the solid content.

[0015] Furthermore, the plant growth regulator of the present invention contains phenyl lactic acid and tryptophan in a ratio of 0.01 to 600 parts by mass of tryptophan per 1 part by mass of phenyl lactic acid, preferably 0.1 to 100 parts by mass of tryptophan, and more preferably 0.5 to 5 parts by mass of tryptophan. Furthermore, when the tryptophan concentration in the culture medium after culturing the lactic acid bacteria is low, it is preferable to use the plant growth regulator of the present invention by adding tryptophan so that the above-mentioned tryptophan concentration is reached.

[0016] When the lactic acid bacteria culture solution is used as a plant growth regulator, it can be directly sprayed on plants or soil as a plant growth regulator, or it can be diluted 100 to 1000 times with water or the like before spraying.

[0017] The plant growth regulator of the present invention can be produced as a formulation by mixing, stirring, etc. the above-mentioned culture solution and other optional components according to a conventional method. The plant growth regulator of the present invention can be formulated by using the above culture solution as a carrier used in ordinary plant growth regulators, such as a wettable powder, emulsifiable concentrate, granules, or dust. There are no limitations on the shape of the formulation, and it can be formed into any formulation form such as dust, granules, powder, wettable powder, flowable, emulsifiable concentrate, and paste.

[0018] For example, solid carriers that can be used for formulation include mineral powders (kaolin, bentonite, clay, montmorillonite, talc, diatomaceous earth, mica, vermiculite, gypsum, calcium carbonate, lime phosphate, etc.), plant powders (soybean flour, wheat flour, wood flour, tobacco powder, starch, crystalline cellulose, etc.), polymeric compounds (petroleum resins, polyvinyl alcohol resins, polyvinyl acetate resins, polyvinyl chloride, ketone resins, etc.), alumina, waxes, etc. Furthermore, examples of liquid carriers that can be used for formulation include alcohols (methanol, ethanol, propanol, butanol, ethylene glycol, benzyl alcohol, etc.), aromatic hydrocarbons (toluene, benzene, xylene, etc.), chlorinated hydrocarbons (chloroform, carbon tetrachloride, monochlorobenzene, etc.), ethers (dioxane, tetrahydrofuran, etc.), ketones (acetone, methyl ethyl ketone, cyclohexane, etc.), esters (ethyl acetate, butyl acetate, etc.), acid amides (N,N-dimethylacetamide, etc.), ether alcohols (ethylene glycol ethyl ether, etc.), and water.

[0019] As surfactants used for purposes such as emulsification, dispersion, and diffusion, any of nonionic, anionic, cationic, and amphoteric surfactants can be used. Examples of surfactants that can be used in the present invention include polyoxyethylene alkyl ethers, polyoxyethylene alkylaryl ethers, polyoxyethylene fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitol fatty acid esters, glycerin fatty acid esters, oxyethylene polymers, oxypropylene polymers, polyoxyethylene alkyl phosphates, fatty acid salts, alkyl sulfates, alkyl sulfonates, alkylaryl sulfonates, alkyl phosphates, alkyl phosphate salts, polyoxyethylene alkyl sulfates, quaternary ammonium salts, oxyalkylamines, lecithin, and saponin. Furthermore, gelatin, casein, sodium alginate, starch, agar, polyvinyl alcohol, and the like can be used as adjuvants, if necessary.

[0020] The plant growth regulator of the present invention, when made into an aqueous solution or suspension, preferably has a pH (25°C) of 2 to 8. Examples of buffers used to adjust the pH include organic acid salts such as acetic acid, citric acid, fumaric acid, malic acid, lactic acid, gluconic acid, and tartaric acid; inorganic salts such as phosphoric acid, hydrochloric acid, and sulfuric acid; hydroxides such as sodium hydroxide; ammonia; and aqueous ammonia. These may be used alone or in combination of two or more, and may also be appropriately combined with other pH adjusters.

[0021] The plant growth regulator of the present invention has the effect of increasing the amount of roots and promoting the overall growth, and is particularly preferably used as a root promoter and a ripening promoter for grass plants. Furthermore, since the plant growth regulator composition of the present invention contains amino acids derived from CSL, it can be used as a fertilizer or biostimulant itself.

[0022] When the plant growth regulator of the present invention is applied to plants, it may be used directly as it is, or may be used after being appropriately diluted or suspended in water.

[0023] When applied to plants, they can be used as soil treatment agents, foliage treatment agents, seed treatment agents before sowing, treatment agents for plants before transplanting, treatment agents for plants at the time of transplanting, etc. In hydroponic cultivation, they may be used by mixing with a hydroponic solution, and in tissue culture, they may be used by suspending or dissolving in a medium.

[0024] When the plant growth regulator of the present invention is used as a formulation for spraying on soil or plants, the concentration used can be preferably 100 to 100,000 times diluted, more preferably 100 to 10,000 times diluted, and particularly preferably 500 to 1,000 times diluted. When used on seedlings in the nursery stage, it is desirable to spray 50 to 200 mL of the diluted solution at the above concentration per 1 L of culture soil. Furthermore, when used as a ripening enhancer for grass plants, it is desirable to spray 200 to 2,000 L per 1 hectare of land area. In these cases, a wetting agent may be used, and the type and amount of the wetting agent used are not particularly limited.

[0025] When the plant growth regulator of the present invention is applied directly to soil, including when mixed with fertilizer, the amount used is preferably 100 to 100,000 g per hectare, and particularly preferably 500 to 50,000 g. When used on seedlings in the raising stage, the amount used is preferably 0.001 to 100 g per liter of culture soil. In this case, the plant growth regulator may be mixed in advance with the culture soil before sowing, or may be sprayed during the seedling raising period.

[0026] When used for seed treatment before sowing, the compound can be diluted or suspended in a liquid carrier such as water, alcohols (e.g., methanol, ethanol), ketones (e.g., acetone), ethers (e.g., diethyl ether), or esters (e.g., ethyl acetate) at a dilution of 10 to 10,000 times and sprayed onto dry seeds, or the dry seeds can be immersed in the diluted solution for absorption into the seeds. The immersion time is not particularly limited, but is preferably 1 second to 30 minutes. The treated seeds may be air-dried, dried under reduced pressure, heated, or vacuum-dried to evaporate the liquid carrier. Alternatively, the compound can be formulated using a solid carrier such as a mineral powder, such as clay, and applied to the seed surface. It can also be mixed with a commonly used seed coating agent or seed coating film to coat the seeds.

[0027] When used in tissue culture or cell culture, the compound can be dissolved or suspended in a commonly used plant tissue culture medium (such as MS medium, White medium, or Gamborg's B5 medium) at a concentration of preferably 0.010 to 100,000 ppm, and particularly preferably 0.100 to 1,000 ppm, as the total concentration of phenyllactic acid or its salt and tryptophan or its salt. In this case, sugars (sucrose, glucose, etc.) as carbon sources, and various plant hormones such as cytokinins (benzyladenine, kinetin, etc.), auxins (indoleacetic acid, naphthaleneacetic acid, etc.), gibberellins (GA3, GA4, etc.), and abscisic acid can be added as appropriate, as is commonly done.

[0028] When directly absorbing the compound into plants before transplanting, the roots or the entire plant can be immersed in a solution diluted or suspended 10 to 10,000 times. For cuttings, sprouts, and rooted cuttings, the base or the entire plant can be immersed. The immersion time in this case is preferably 1 second to 1 week, and more preferably 1 minute to 3 days. Alternatively, a formulation prepared using a solid carrier of mineral powder can be applied to the roots. Alternatively, for cuttings, sprouts, and rooted cuttings, the compound can be applied to the base of the stem.

[0029] The plant growth regulator of the present invention can be administered at any time during the growing season, but when used as a root promoter, it is particularly effective before sowing, at the time of sowing, during seedling cultivation, before and after cultural root pruning such as transplanting, when root growth is inhibited or root damage occurs due to weather factors, etc. When used as a ripening promoter for grasses, the appropriate application period is from the flowering stage to the yellow-ripe stage.

[0030] When the plant growth regulator of the present invention is applied to plants as a rooting promoter, the number of roots such as lateral roots and adventitious roots is increased, thereby increasing root volume and root density, thereby providing effects such as improved root establishment rate at transplanting, healthy seedling cultivation, growth promotion, improved water absorption, improved fertilizer absorption, improved fertilizer component utilization rate, maintenance of green color, improved photosynthetic ability, improved water stress tolerance, lodging prevention, and increased yield. Furthermore, when applied to grasses as a ripening promoter, the grain weight per grain increases, resulting in effects such as increased yield.

[0031] Plants to which the plant growth regulator composition of the present invention can be applied include, but are not limited to, eggplants such as tomatoes, bell peppers, chili peppers, and eggplants; gourds such as cucumbers, pumpkins, melons, and watermelons; fresh and spicy vegetables such as celery, parsley, and lettuce; alliums such as leeks, onions, and garlic; beans such as soybeans, peanuts, kidney beans, peas, and adzuki beans; other fruit vegetables such as strawberries; taproots such as radishes, turnips, carrots, and burdock; potatoes such as taro, cassava, potato, sweet potato, and Chinese yam; asparagus; spinach; Examples of suitable crops include soft vegetables such as Japanese laurel and mitsuba, flowering plants such as lisianthus, stock, carnation and chrysanthemum, cereals such as rice, wheat, barley, oats and corn, turf grasses such as bentgrass and Zoysiagrass, oil crops such as rapeseed and sunflower, sugar crops such as sugarcane and sugar beet, fiber crops such as cotton and rush, forage crops such as clover, sorghum and dent corn, deciduous fruit trees such as apples, pears, grapes and peaches, citrus fruits such as Satsuma mandarins, lemons and grapefruit, and woody plants such as azalea, azalea and cedar.

[0032] Among these, when applied as a root promoter, it is particularly effective for plants that are transplanted during cultivation, such as tomatoes, bell peppers, chili peppers, eggplants, cucumbers, pumpkins, melons, watermelons, celery, parsley, lettuce, leeks, onions, asparagus, lisianthus, stock, rice, bentgrass, Zoysiagrass, and sugar beet rush, and for plants that are propagated by rooting from cuttings or scions, such as chrysanthemums, carnations, azaleas, azaleas, and grapes. Furthermore, when used as a ripening promoter for grass plants, it is particularly effective for plants whose grains are not covered with a periwinkle, such as wheat, rice, barley, and oats.

[0033] To enhance the effects of the present invention, the compound can be used in combination with other plant growth regulators, and in some cases, a synergistic effect can be expected. For example, when used as a root promoter, when raising seedlings under conditions that are highly prone to elongation, such as high planting density, high humidity, and insufficient sunlight, the compound can be used in combination with anti-gibberellins (paclobutrazol, uniconazole P, ancymidol, etc.) that have a strong stem elongation inhibitory effect, growth inhibitors (daminozide, etc.), and ethylene generators (ethephon, etc.) to cultivate high-quality seedlings with a low above-ground / underground weight ratio. Furthermore, when using cuttings, grafting, cuttings, and tissue culture, the compound can be used in combination with auxin compounds (indoleacetic acid, indolebutyric acid, naphthylacetamide, naphthaleneacetic acid, etc.) to enhance the root-promoting effect. Furthermore, when treating seeds before sowing, the compound can be used in combination with gibberellins that have a germination-promoting effect. Furthermore, when used as a ripening improver for grasses, it may be used in combination with other ripening improvers such as hydroxyisoxazole, isoprothiolane, etc. These are merely examples, and other plant growth regulators that can be used in combination with the plant growth regulator of the present invention are not limited to these.

[0034] The plant growth regulator of the present invention can also be mixed or used in combination with various insecticides, fungicides, microbial pesticides, fertilizers, etc. In particular, when used as a root promoter, it is effective to use it in combination with hydroxyisoxazole, metasulfocarb, metalaxyl, etc., which have been reported to have root-promoting effects in addition to fungicidal effects. Mixing it with an insecticide or fungicide used during the seedling raising period is particularly effective. When used in combination with a fertilizer, it is particularly effective to use it in combination with a seedling raising fertilizer aimed at growing healthy seedlings, or with a fertilizer applied immediately before transplanting aimed at promoting rooting. Mixing it with a slow-release fertilizer aimed at maintaining the efficacy of the plant growth regulator of the present invention for a long period of time and improving the utilization rate of fertilizer components is also particularly effective. In addition, when used as a ripening promoter for grasses, it is also effective to mix it with other foliar fertilizers such as urea, ammonium phosphate, and amino acids.

[0035] The lactic acid bacteria of the present invention grow in a medium containing 20% ​​by mass or more of CSL-derived solids, in which conventional lactic acid bacteria cannot grow, and when 0.5% L-phenylalanine is added to the medium, they produce 160 mg / L or more of phenyllactic acid and 3% by mass or more of free amino acids in the medium at the end of the culture. Furthermore, the lactic acid bacteria of the present invention can be selected from lactic acid bacteria growing in a medium containing a high concentration of CSL, which is used as a selection medium.

[0036] The CSL blended into the medium used for lactic acid bacteria selection has an osmotic pressure that is too high for the growth of both general lactic acid bacteria and the lactic acid bacteria of the present invention. Therefore, the CSL is diluted appropriately before use in the medium used for selection. The CSL is diluted to a final solid content of 20 to 30% (w / v), more preferably 20 to 24%, in the selection medium or the medium for producing the plant growth regulator. The CSL diluted solution alone can also be used as the selection medium.

[0037] As a medium for selecting lactic acid bacteria, various carbon and nitrogen sources can be added to CSL. Any carbon source can be used as long as it can be utilized by the target lactic acid bacteria. Among these, glucose, fructose, sucrose, lactose, etc. can be mentioned. Any nitrogen source can be used as long as it can be utilized by the target lactic acid bacteria, with wheat gluten enzymatic hydrolysate and soy protein hydrolysate being preferred. It is more preferable to further add metal elements such as zinc and vitamins.

[0038] The lactic acid bacteria used in the present invention can grow in the above-mentioned selective medium, and when cultured at 37°C for 3 days, produce 160 mg / L or more of phenyllactic acid per medium at the end of the culture. The production amount and growth ability can be used together as selection criteria to select lactic acid bacteria suitable for the present invention. The lactic acid bacteria to be selected may be either Lactococcus or Lactobacillus, but Lactobacillus is preferred, with Lactobacillus being particularly preferred.

[0039] Preferred examples of the lactic acid bacteria classified into the genus Lactobacillus according to the present invention include the following: The genus of lactic acid bacteria is preferably Lactobacillus (hereinafter sometimes abbreviated as "Lb."). Examples of species of lactic acid bacteria include Lactobacillus acetotolerans, Lactobacillus acidifarinae, Lactobacillus acidopiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, and Lactobacillus spp. Lactobacillus amylolyticus (Lb.amylolyticus), Lactobacillus amylophilus (Lb.amylophilus), Lactobacillus amylotrophicus (Lb.amylotrophicus), Lactobacillus amylovorus (Lb.amylovorus), Lactobacillus animalis (Lb.animalis), Lactobacillus antri (Lb.antri), Lactobacillus apis (Lb.apis), Lactobacillus apodemi (Lb.apodemi), Lactobacillus aquati Lactobacillus aquatile, Lactobacillus aquaticus, Lactobacillus aviarius, Lactobacillus backii, Lactobacillus bifermentans, Lactobacillus brantae, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus cacaonum, Lactobacillus Lactobacillus camelliae (Lb.camelliae), Lactobacillus capillatus (Lb.capillatus), Lactobacillus casei (Lb.casei), Lactobacillus ceti (Lb.ceti), Lactobacillus coleohominis (Lb.coleohominis), Lactobacillus collinoides (Lb.collinoides), Lactobacillus composti (Lb.composti), Lactobacillus concavus (Lb.concavus), Lactobacillus coliniformis (Lb.coryniformis, Lactobacillus crispatus, Lactobacillus crustrum, Lactobacillus curieae, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus equi , Lactobacillus equicursoris (Lb. equicursoris), Lactobacillus equigenerosi (Lb. equigenerosi), Lactobacillus fabifermentans (Lb. fabifermentans), Lactobacillus faecis (Lb. faecis), Lactobacillus farciminis (Lb. farciminis), Lactobacillus farraginis (Lb. farraginis), Lactobacillus fermentum (Lb. fermentum), Lactobacillus floricola (Lb. floric ola), Lactobacillus florum (Lb.florum), Lactobacillus fornicalis (Lb.fornicalis), Lactobacillus fructivorans (Lb.fructivorans), Lactobacillus frumenti (Lb.frumenti), Lactobacillus fuchuensis (Lb.fuchuensis), Lactobacillus futsaii (Lb.futsaii), Lactobacillus gallinarum (Lb.gallinarum), Lactobacillus gasseri (Lb.gasseri), Lactobacillus gastricus ( Lb.gastricus), Lactobacillus ghanensis (Lb.ghanensis), Lactobacillus gigeriorum (Lb.gigeriorum), Lactobacillus graminis (Lb.graminis), Lactobacillus hammesii (Lb.hammesii), Lactobacillus hamsteri (Lb.hamsteri), Lactobacillus harbinensis (Lb.harbinensis), Lactobacillus heilongjiangensis (Lb.heilongjiangensis), Lactobacillus hayakitensis (Lb.hayakitensis), Lactobacillus helveticus (Lb. helveticus), Lactobacillus hilgardii (Lb. hilgardii), Lactobacillus hokkaidonensis (Lb. hokkaidonensis), Lactobacillus hominis (Lb. hominis), Lactobacillus homohiokii (Lb. homohiochii), Lactobacillus hordei (Lb. hordei), Lactobacillus iners (Lb. iners), Lactobacillus ingluviei (Lb. ingluviei), Lactobacillus Lactobacillus intestinalis (Lb. intestinalis), Lactobacillus iwatensis (Lb. iwatensis), Lactobacillus jensenii (Lb. jensenii), Lactobacillus johnsonii (Lb. johnsonii), Lactobacillus kalixensis (Lb. kalixensis), Lactobacillus kefiranofaciens (Lb. kefiranofaciens), Lactobacillus kefiri (Lb. kefiri), Lactobacillus kimchicus (Lb. kimchicus), Lactobacillus kimchie Lactobacillus kimchiensis, Lactobacillus xonensis, Lactobacillus kitasatonis, Lactobacillus koreensis, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillus indneri, Lactobacillus malefermentans, Lactobacillus mali ), Lactobacillus manihotivorans (Lb. manihotivorans), Lactobacillus mindensis (Lb. mindensis), Lactobacillus mucosae (Lb. mucosae), Lactobacillus murinus (Lb. murinus), Lactobacillus nagelii (Lb. nagelii), Lactobacillus namurensis (Lb. namurensis), Lactobacillus nantensis (Lb. nantensis), Lactobacillus nasuensis (Lb. nasuensis), Lactobacillus nenjiangensis (Lb.nenjiangensis), Lactobacillus nodensis (Lb. nodensis), Lactobacillus odoratitofui (Lb. odoratitofui), Lactobacillus oeni (Lb. oeni), Lactobacillus oligofermentas (Lb. oligofermentans), Lactobacillus oris (Lb. oris), Lactobacillus oryzae (Lb. oryzae), Lactobacillus otakiensis (Lb. otakiensis), Lactobacillus ozensis (Lb. ozensis), Lactobacillus patens Lactobacillus panis (Lb. pantheris), Lactobacillus parabrevis (Lb. parabrevis), Lactobacillus parabuchneri (Lb. parabuchneri), Lactobacillus paracasei (Lb. paracasei), Lactobacillus paracollinoides (Lb. paracollinoides), Lactobacillus parakefiri (Lb. paraakefiri), Lactobacillus paralimentarius (Lb. paralimentarius), Lactobacillus para Lactobacillus plantarum (Lb. paraplantarum), Lactobacillus pasteurii (Lb. pasteurii), Lactobacillus paucivorans (Lb. paucivorans), Lactobacillus pentosus (Lb. pentosus), Lactobacillus perolens (Lb. perolens), Lactobacillus plantarum (Lb. plantarum), Lactobacillus pobzihii (Lb. pontis), Lactobacillus porcinae (Lb. porcinae) ), Lactobacillus psittaci (Lb. psittaci), Lactobacillus rapi (Lb. rapi), Lactobacillus rennini (Lb. rennini), Lactobacillus reuteri (Lb. reuteri), Lactobacillus rhamnosus (Lb. rhamnosus), Lactobacillus rodentium (Lb. rodentium), Lactobacillus rogosae (Lb. rogosae), Lactobacillus rossiae (Lb. rossiae), Lactobacillus ruminis (Lb. ruminis), Lactobacillus saerimuneri (Lb.saerimneri), Lactobacillus sakei (Lb. sakei), Lactobacillus salivarius (Lb. salivarius), Lactobacillus sanfranciscensis (Lb. sanfranciscensis), Lactobacillus saniviri (Lb. saniviri), Lactobacillus satsumensis (Lb. satsumensis), Lactobacillus secaliphilus (Lb. secaliphilus), Lactobacillus selangorensis (Lb. selangorensis), Lactobacillus senioro Lactobacillus senioris (Lb.senmaizukei), Lactobacillus sharpeae (Lb.sharpeae), Lactobacillus shenzhenensis (Lb.shenzhenensis), (Lactobacillus silagei (Lb.silagei), Lactobacillus siliginis (Lb.siliginis), Lactobacillus similis (Lb.similis), Lactobacillus songhuajiangensis (Lb.songhuajiangensis), Lactobacillus spicata (Lb.spicata) Lactobacillus spicheri, Lactobacillus sucicola, Lactobacillus suebicus, Lactobacillus sunkii, Lactobacillus taiwanensis, Lactobacillus thailandensis, Lactobacillus tucceti, Lactobacillus ultunensis, Lactobacillus uvarum, Lactobacillus Examples include Lactobacillus vaccinostercus (Lb. vaccinostercus), Lactobacillus vaginalis (Lb. vaginalis), Lactobacillus versmoldensis (Lb. versmoldensis), Lactobacillus vini (Lb. vini), Lactobacillus xiangfangensis (Lb. xiangfangensis), Lactobacillus yonginensis (Lb. yonginensis), Lactobacillus zeae (Lb. zeae), and Lactobacillus zymae (Lb. zymae).

[0040] Among these exemplified species, preferred lactic acid bacteria of the genus Lactobacillus include Lactobacillus paracasei (Lb. paracasei), Lactobacillus casei (Lb. casei), Lactobacillus rhamnosus (Lb. rhamnosus), Lactobacillus plantarum (Lb. plantarum), Lactobacillus buchneri (Lb. buchneri), Lactobacillus brevis (Lb. brevis), Lactobacillus rapi (Lb. rapi), and Lactobacillus diolivorans (Lb. diolivorans), and more preferred are lactic acid bacteria selected from Lactobacillus paracasei (Lb. paracasei), Lactobacillus rhamnosus, and Lactobacillus sp. Furthermore, any strain selected from Lactobacillus paracasei (Lb. paracasei) #720 strain (accession number NITE BP-03117), Lactobacillus rhamnosus (Lb. rhamnosus) #555 strain (accession number NITE ABP-03379), and Lactobacillus sp. #728 strain (accession number NITE ABP-03380), which have been deposited by the applicant at the Patent Microorganisms Deposit Center, Biotechnology Center, National Institute of Technology and Evaluation, an independent administrative institution, is particularly preferred.

[0041] To produce the plant growth regulator of the present invention, the above-mentioned lactic acid bacteria are appropriately selected and added to a production medium containing 20% ​​by mass or more of a solid content derived from CSL at a concentration of 1 × 10 6 The culture solution pre-cultured in an appropriate pre-culture medium is inoculated into the production medium at about 1% by mass to a density of about cpu / mL, and cultured. The culture temperature after ingestion of the lactic acid bacteria is preferably 25 to 37°C, more preferably 37°C, and the culture time is preferably 24 to 300 hours, more preferably 48 to 72 hours. As a medium for producing the plant growth regulator of the present invention, CSL is diluted with water to obtain a production medium in which the solid content derived from CSL is 20% by mass or more, preferably 20 to 30% by mass, and particularly preferably 20 to 25% by mass. CSL generally has a hydrogen ion concentration of around pH 4, which is too acidic for lactic acid bacteria to grow in. For this reason, it is desirable to add an agriculturally acceptable base in advance to raise the pH of the medium to pH 5 to 8, more preferably pH 5 to 7. Agriculturally acceptable bases include salts and bases of ammonia, potassium, calcium, magnesium, manganese, sodium, etc. Ammonia and potassium salts and bases are preferred. Furthermore, from the standpoint of ease of handling in manufacturing, aqueous ammonia and potassium hydroxide are preferred.

[0042] The CSL diluted and pH adjusted as described above can be used as is as a medium for lactic acid bacteria for carrying out the present invention. If necessary, various carbon and nitrogen sources can be further added to promote the growth of lactic acid bacteria. Any carbon source can be used as long as it can be utilized by the lactic acid bacteria to be cultured, and examples of such carbon sources include sugars such as glucose, fructose, sucrose, and lactose. Furthermore, cheese whey is a preferred lactose source. Any nitrogen source can be used as long as it can be utilized by the lactic acid bacteria to be cultured, but enzymatic hydrolysates of wheat gluten and soybean protein hydrolysates are preferred. Furthermore, to increase the phenyllactic acid concentration in the culture medium, it is preferable to add L-phenylalanine, which is a substrate for phenyllactic acid. The concentration of L-phenylalanine in the medium is preferably 0.1 to 1% by mass, more preferably 0.3 to 0.7% by mass. Furthermore, it is preferable to add iron and zinc to the medium as trace elements to promote the growth of lactic acid bacteria. The compound forms of the added iron and zinc may be any salt or compound as long as they dissolve in the medium, but chelate salts are preferred, and more preferably, iron EDTA and zinc EDTA are used for iron and zinc, respectively.

[0043] In the case of the plant growth regulator of the present invention, the culture solution is recovered after the culture is completed. If necessary, the culture solution can be filtered or centrifuged to remove the lactic acid bacteria by a known method, or can be sterilized without removing the lactic acid bacteria, and can be used as a plant regulator or as a raw material for a plant regulator formulation. [Example]

[0044] The present invention will now be described in more detail with reference to examples and test examples, but the present invention is not limited to the following examples and test examples.

[0045] <Test Example 1 (Relationship between CSL concentration and growth of lactic acid bacteria)> In the following test examples, "%" stands for weight / volume (w / v) % unless otherwise specified. 1. Test Method CSL (40.5% solids, w / v) from Showa Sangyo Co., Ltd. was adjusted to pH 6 with aqueous ammonia (Wako Pure Chemical Industries, Hokkaido). The mixture was then centrifuged at 8,000 G to obtain a supernatant. One mL of the supernatant was placed in an aluminum cup that had been dried in a desiccator, weighed, and heated on a hot plate until it became a viscous, syrup-like substance. The mixture was then dried in an oven at 105 °C for 3 hours, cooled in a desiccator, and reweighed to calculate the solids content, which was 40.5 w / v%. This supernatant was then diluted with water to concentrations of 30%, 40%, 50%, 60%, 70%, 80%, and 90%. The CSL-derived solids concentrations of the diluted solutions were 12.15%, 16.2%, 20.25%, 24.3%, 28.35%, 32.4%, and 36.45%, respectively. The amount of precipitate precipitated by centrifugation was so small that it did not affect the solid content. Next, 0.25% glucose (manufactured by Hokkaido Wako Pure Chemical Industries, Ltd.) was added to these diluted solutions and the supernatants after centrifugation and dissolved. This solution was dispensed into test tubes in 10 mL portions and sterilized in an autoclave at 121°C for 15 minutes to prepare the medium.

[0046] As lactic acid bacteria strains for testing, Lactobacillus paracasei SBS0003 strain, Lactobacillus diolivorans SBS0007 strain, and Lactobacillus buchneri NK01 strain, which are known to be capable of growing in a culture medium containing CSL, were selected as test strains. These lactic acid bacteria strains were pre-cultured in MRS (Becton Dickinson) medium, and the culture solution was inoculated at 1% into the above-mentioned CLS medium supplemented with glucose, and cultured at 37°C for 3 days. The pH of the culture medium before and after the culture was measured.

[0047] 2. Test Results The pH of the culture medium after the end of the culture and the amount of decrease in pH from the original culture medium are shown in Table 1 below (note that the (-) sign indicates a decrease in pH).

[0048] [Table 1]

[0049] When the CSL concentration was 30% (CSL-derived solid content 12.15%), the pH of the culture solution for all three test strains decreased by 0.1 or more, indicating that the lactic acid bacteria were able to grow sufficiently. On the other hand, when the CSL concentration was 40 to 50% (CSL-derived solid content 16.2 to 20.25%), the SBS0003 strain only reduced the pH by about 0.1. Furthermore, when the CSL concentration was 60% or higher (CSL-derived solids content 24.3%), the pH drop in the culture medium for all strains tested was 0.1 or less. In other words, it was found that the lactic acid bacteria did not substantially grow and fermentation did not progress. These test results confirmed that general lactic acid bacteria cannot grow when the CSL-derived solids content exceeds 20%.

[0050] <Test Example 2 (Selection of high-concentration CSL-resistant lactic acid bacteria)> 1. Test Method As in Test Example 1, ammonia water (manufactured by Hokkaido Wako Pure Chemical Industries, Ltd.) was added to CSL to adjust the pH to 7, and the mixture was centrifuged (8,000 G) to obtain a supernatant. This supernatant was diluted with water to a concentration of 60% (solids concentration 24.3%), and 2% cheese whey powder FC06 (whey powder, manufactured by Megmilk Snow Brand Co., Ltd.) was added. 10 mL of this solution was dispensed into test tubes and sterilized in an autoclave at 121°C for 15 minutes. This sterilized medium was used as the selection medium.

[0051] Among the lactic acid bacteria strains isolated and identified from pasture and silage, ten were selected that were confirmed in preliminary tests to be able to grow at high concentrations of CSL. Additionally, the standard strains of common lactic acid bacteria Lb. rhamnosus JCM1136, Lb. diolivorans JCM12183, and Lb. plantarum JCM1149 were selected. These lactic acid bacteria strains were pre-cultured in MRS medium (Becton Dickinson), and the culture solution was inoculated at 1% per test medium and cultured at 37°C for three days. After completion of the culture, the pH was measured. For comparison, the lactic acid bacteria strain selected in Test Example 1 was also cultured in the same manner, and the pH was measured.

[0052] 2. Test Results The pH and pH change after cultivation are shown in Table 2.

[0053] [Table 2]

[0054] The strain grown in Test Example 1 only reduced the pH by 0.15 to 0.31 in a medium with a CSL concentration of 60% (solid content 24.3%) compared to an uninoculated medium. In other words, the strain barely grew or only grew slightly. Furthermore, commercially available standard strains of lactic acid bacteria, Lb. rhamnosus JCM1136 and Lb. plantarum JCM1149, only reduced the pH by 0.31 and 0.25, respectively. No reduction in pH was observed with Lb. diolivorans JCM12183, suggesting that the lactic acid bacteria did not grow and fermentation was not progressing. On the other hand, 10 newly isolated strains (strains selected from the collected lactic acid bacteria) decreased the pH by 0.68 to 0.82. In other words, they were able to grow in a medium containing a high concentration of CSL. From these test results, it can be said that these 10 strains are strains that can grow in a high concentration of CSL. These 10 strains were determined to be lactic acid bacteria with properties that make them usable in the present invention.

[0055] <Test Example 3 (Effect of whey addition on lactic acid bacteria growth)> 1. Test Method The same CSL (solid content 40.5%, w / v) used in Test Example 1 was diluted to 60% with water. Next, glucose or cheese whey powder FC06 (whey powder, manufactured by Megmilk Snow Brand Co., Ltd.) was added at 0.25%, 1%, or 2% and dissolved. After that, the pH was adjusted to 7.0 with ammonia water and 100 mL was dispensed into Erlenmeyer flasks. This was sterilized in an autoclave at 121°C for 15 minutes to prepare the test medium. The lactic acid bacteria selected were Lb. paracasei #736 and Lb. sp. #768, which were newly isolated from the above-mentioned grass and silage. Furthermore, Lb. rhamnosus JCM1136, Lb. diolivorans JCM12183, and Lb. plantarum JCM1149 were pre-cultured in MRS medium as reference strains, and the cultures were inoculated at 1% per test medium and cultured at 37°C for 3 days. After incubation, the pH was measured.

[0056] 2. Test Results Table 3 shows the pH of each culture medium before and after culture and the difference in pH from the control group.

[0057] [Table 3]

[0058] In all test media, the pH decrease was greater in the cultures of Lb. paracasei #736 and Lb. sp. #768 than in the reference strain (JCM). Furthermore, when glucose was added, the pH of the culture medium of Lb. paracasei #736 and Lb. sp. #768 did not fall below 0.12 compared to the control group, and no substantial growth-promoting effect of the addition was observed. On the other hand, when whey was added, the pH of the culture medium inoculated with the reference strain (JCM strain) did not fall below 0.23 compared to the control group. Furthermore, in the culture medium of Lb. paracasei #736 and Lb. sp. #768, the pH of the culture medium in the treatment groups with 0.25-1% whey powder added fell by 0.45 or more compared to the control group. However, the effect was small when 2% whey powder was added. From the above test results, it was revealed that the addition of whey powder to the culture medium for Lb. paracasei #736 and Lb. sp. #768 strains promotes the proliferation of lactic acid bacteria and fermentation. It was thought that the preferred amount of whey powder added was 0.25-2%.

[0059] <Test Example 4 (Effect of Addition of Phenylalanine)> 1. Test Method The effect of adding phenylalanine on the amount of phenyllactic acid produced was examined. (1)Culture conditions The same CSL (40.5% solids, w / v) as in Test Example 1 was diluted directly with water to 50% (20.25% CSL-derived pore size). 1% cheese whey powder FC06 (whey powder, manufactured by Megmilk Snow Brand Co., Ltd.), 1% wheat gluten enzymatic hydrolyzate Solpro 505 (manufactured by Tereos Syral), and 1% EDTA iron were added. Furthermore, L-phenylalanine (Phe) was added at 0%, 0.1%, 0.3%, 0.5%, and 0.7%. The mixture was thoroughly dissolved and adjusted to pH 7.0 with aqueous ammonia. 500 mL of this mixture was dispensed into Erlenmeyer flasks to serve as test media. The Lb. paracasei #720 strain, which was separately selected and capable of growing in high-concentration CSL medium, was precultured in MRS medium (manufactured by #), and this culture solution was inoculated at 1% per test medium and cultured at 37°C for 12 days.

[0060] (2) Phenyllactic acid analysis method The concentration of phenyllactic acid in the culture medium was analyzed by the following method. Specifically, the culture medium was mixed with an equal volume of 20% methanol and passed through a SepPaktC18 (Waters Co.) acclimated with 10% methyl alcohol. The resulting effluent was concentrated and dissolved in 0.1% formic acid. The concentrate was then analyzed using a high-performance liquid chromatograph-triple quadrupole mass spectrometry system (LCMS-8050, Shimadzu Corporation). The HPLC column used for analysis was L-Column2 ODS 2.1 × 100 mm, particle size 2 μm (Chemicals Evaluation and Research Institute, Japan). The mobile phase for HPLC was acetonitrile mixed with 0.1% formic acid, and the acetonitrile concentration was increased from 5% to 95% over 15 minutes. The column oven temperature was set at 40°C. The interface conditions were nebulizer gas flow rate 3 L / min, heating gas flow rate 10 L / min, interface temperature 300 °C, DL temperature 250 °C, heat block temperature 400 °C, and drying gas flow rate 10 L / min. Ionization was performed by ESI. Quantitation was performed in MRM mode (precursor ion 165.1 m / z, product ion 147.0 m / z, collision energy 16.0 V). The phenyllactic acid standard was detected at a retention time of 5.96 minutes, and the phenyllactic acid, which was the substance to be measured in the sample, was detected at a retention time of 5.95-5.98 minutes.

[0061] (3) Tryptophan analysis The tryptophan concentration in the culture medium was analyzed as follows. That is, the culture medium was directly analyzed using a high performance liquid chromatograph-triple quadrupole mass spectrometry system (LCMS-8050, Shimadzu Corporation). The column used was L-Column2 ODS 2.1 x 100 mm, particle size 2 μm (Chemicals Evaluation and Research Institute, Japan). The mobile phase used was acetonitrile mixed with 0.1% formic acid, and the acetonitrile concentration was increased from 5% to 95% over 15 minutes. The column oven temperature was set to 40°C. The interface conditions were set to a nebulizer gas flow rate of 3 L / min, a heating gas flow rate of 10 L / min, an interface temperature of 300°C, a DL temperature of 250°C, a heat block temperature of 400°C, and a drying gas flow rate of 10 L / min, and ionization was performed by ESI+. Quantitation was performed in MRM mode (precursor ion 205.0 m / z, product ion 188.2 m / z, collision energy -10.0 V). The tryptophan standard was detected at a retention time of 4.02 minutes, and the tryptophan target substance in the sample was detected at a retention time of 3.94-4.00 minutes.

[0062] 2. Test Results (1) Phenylacetic acid lactate measurement results The concentration of phenyllactic acid (PLA) in each culture medium is shown in FIG. As shown in Figure 1, the production of phenyllactic acid improved when the phenylalanine (Phe) concentration was in the range of 0.3 to 0.7%. It was confirmed that the optimum concentration was around 0.5 to 0.6%.

[0063] (2) Tryptophan concentration The tryptophan concentration and phenylalanine amount in the culture medium are shown in Table 4.

[0064] [Table 4]

[0065] As shown in Table 4, it was found that the tryptophan concentration decreased as the amount of phenylalanine added increased, and then when the amount of phenylalanine added exceeded 0.5%, the tryptophan concentration increased again. For the strain and medium used in this study, phenylalanine was added to the medium, and the optimum concentration of phenylalanine to increase the concentrations of phenylacetic acid lactic acid and tryptophan was 0.5%.

[0066] Furthermore, as shown in Table 5 below, the ratio of phenyllactic acid to tryptophan in all of the culture solutions (test culture solutions) was in the range of 0.01 to 600 parts by mass per part by mass of phenyllactic acid (1:600 ​​to 99:1).

[0067] <Test Example 5 (Effect of Addition of Metal Ions)> 1. Test Method The same CSL (solid content 40.5%, w / v) as in Test Example 1 was used and diluted 50% in water without centrifugation. To this solution, 1% cheese whey powder FC06 (whey powder, manufactured by Megmilk Snow Brand Co., Ltd.), 1% wheat gluten enzymatic hydrolysate Solpro 505, and 0.5% L-phenylalanine were added. Trace metals were added to this medium: EDTA (4H, free) (H), tetrasodium EDTA (Na), calcium (divalent) EDTA (Ca), iron (III) EDTA (Fe), magnesium (divalent) EDTA (Mg), and zinc (divalent) EDTA (Zn) (all manufactured by Dojindo Laboratories). The solution was thoroughly dissolved, adjusted to pH 7.0 with ammonia water, and 100 mL of the solution was dispensed into an Erlenmeyer flask. This was used as the test medium. As in Test Example 5, the Lb. paracasei #720 strain was pre-cultured in MRS medium, and the culture solution was inoculated at 1% per test medium, followed by culturing at 37°C for 11 days.

[0068] 2. Test Results The results of measuring the culture medium pH over time are shown in Figure 2. The greatest changes were observed in the following order: EDTA (4H, free) (H), tetrasodium EDTA (Na), calcium (II) EDTA (Ca), magnesium (II) EDTA (Mg), zinc (II) EDTA (Zn), and iron (III) EDTA (Fe). Among these, zinc EDTA and iron EDTA were particularly effective. Comparing the results of the culture medium containing free EDTA with those containing iron (III) EDTA (Fe) or zinc (II) EDTA (Zn), the latter showed a rapid decrease in pH by day 4 of culture, whereas the decrease in pH with free EDTA was extremely gradual, reaching only a pH drop of about 1 by day 11 of culture. The addition of sodium EDTA (Na) was more effective than free EDTA, but the pH decrease plateaued by day 7. The addition of calcium EDTA (Ca) and magnesium EDTA (Mg) decreased the pH more than the decrease in sodium EDTA, but the degree of decrease in pH was small, and from the 7th day onwards, the pH plateaued at around pH 5.8. On the other hand, when EDTA iron (Fe) and EDTA zinc (Zn) were added, the final pH was reduced to around 5.0. From this graph of pH change, it was thought that the addition of EDTA iron (Fe) or EDTA zinc (Zn) promoted the growth of lactic acid bacteria, thereby enhancing the plant growth-regulating activity of the culture solution.

[0069] Of the lactic acid bacteria for which favorable results were confirmed in the above tests, three strains have been deposited by the applicant of the present invention at the Patent Microorganisms Depositary Center of the Biotechnology Center, National Institute of Technology and Evaluation, as follows, as lactic acid bacteria strains suitable for carrying out the present invention. Lactobacillus paracasei (Lb. paracasei) strain #720 (SBS-0010, accession number NITE BP-03117) Lactobacillus rhamnosus (Lb. rhamnosus) strain #555 (SBS-0012, accession number NITE ABP-03379) Lactobacillus sp. #728 strain (SBS-0013, accession number NITE ABP-03380)

[0070] <Production Example 1 (Production of plant growth regulator using a pilot plant)> 1. Manufacturing method The centrifuged CSL supernatant was diluted 50% with water (CSL-derived solid content: 20.25%). 1% FC10 (whey powder, manufactured by Megmilk Snow Brand Co., Ltd.), 1% powdered soy protein (Fujipro E, manufactured by Fuji Oil Co., Ltd.), 1% EDTA iron (III valence), and 0.5% L-phenylalanine were added and stirred. After dissolution, the pH was adjusted to 7.0 with aqueous ammonia. 200 L of this solution was heat sterilized and used as the production medium. As in Test Example 4, Lb. paracasei #720 strain was pre-cultured in MRS, inoculated into the above-mentioned production medium at 2% and subjected to static culture at 37°C for 9 days. The pH of the culture medium was measured every day during the culture period, and the changes in pH are shown in Figure 4. The pH of the culture solution dropped significantly after two days of culture, confirming the successful growth of the lactic acid bacteria. The pH change was small from the third day onwards, suggesting that lactic acid bacteria growth plateaued in about three days. The final pH of the culture solution was 4.7. This culture solution was collected, and the entire volume was used as the plant growth regulator of the present invention.

[0071] 2. Measurement of phenyllactic acid concentration in the plant growth regulator (culture solution) of Production Example 1 (1)Measurement method After the incubation period, the culture medium was mixed with an equal volume of 20% methanol and passed through a SepPakt C18 (Waters Co.) acclimated with 10% methanol. The resulting flow-through was then collected. The collected liquid was further concentrated, dissolved in 0.1% formic acid, and analyzed using a high-performance liquid chromatograph-triple quadrupole mass spectrometry system (LCMS-8050, Shimadzu Corporation). The analytical column used was an L-Column 2 ODS 2.1 × 100 mm column with a particle size of 2 μm (Chemicals Evaluation and Research Institute, Japan). Acetonitrile mixed with 0.1% formic acid was used as the mobile phase for HPLC, and the acetonitrile concentration was increased from 5% to 95% over 15 minutes. The column oven temperature was 40°C. The interface conditions were nebulizer gas flow rate 3 L / min, heating gas flow rate 1 L / min, interface temperature 300 °C, DL temperature 250 °C, heat block temperature 400 °C, drying gas flow rate 10 L / min, and ionization by ESI. Quantitation was performed in MRM mode (precursor ion 165 m / z, product ion 147 m / z, collision energy 16 V). The phenyllactic acid standard was detected at a retention time of 5.96 minutes, and phenyllactic acid in the sample was detected at a retention time of 5.95-5.98 minutes.

[0072] (2) Phenyllactic acid measurement results The content of phenyllactic acid (PLA) in the resulting culture medium was 195 mg / L. The tryptophan concentration in the culture medium of Production Example 1 was 29.3 mg / L.

[0073] <Production Example 2 (Production of plant growth regulators using a pilot plant) and comparison of the potency of a phenylalanine-tryptophan mixture> (1) Manufacturing method The centrifuged CSL was diluted 50% to adjust the CSL-derived solid content to 22.03%. 1% FC10 (whey powder, manufactured by Megmilk Snow Brand Co., Ltd.), 1% powdered soy protein (Fujipro E, manufactured by Fuji Oil Co., Ltd.), 1% EDTA iron (III), and 0.5% L-phenylalanine were added and stirred. After dissolution, the pH was adjusted to 7.0 with aqueous ammonia. 200 L of this lysate was used as the production medium. As in Test Example 4, Lb. paracasei #720 strain was pre-cultured in MRS, inoculated into the production medium at 2% and cultured statically at 37°C for 3 days. On the third day of culture, the pH of the culture medium was 5.4. At this point, the culture was terminated as the growth rate had almost reached a plateau. This confirmed that a culture period of 3 days was sufficient depending on the lot of corn steep liquor. The phenyllactic acid content in the resulting culture medium was measured in the same manner as in Production Example 1 and was found to be 686 mg / L. The tryptophan concentration was 14.523 mg / L. This culture solution was used as the plant growth regulator of Production Example 2.

[0074] <Test Example 6 (Rooting Assay of Adzuki Beans Using the Plant Regulator of Production Example 2)> 1. Test Method In order to evaluate the plant growth regulator obtained in Production Example 2, a test was carried out using rooting activity as an index. The culture medium obtained in Production Example 2 was adjusted to pH 6.8-7.0 and diluted 1000 times with distilled water to prepare a test solution. As comparative controls, a pre-culture medium and a solution containing 50 ppm of DL-phenyllactic acid (DL-PLA) and 50 ppm of L-tryptophan (L-Trp) (see Patent Document 1) were also tested in the same manner. Adzuki bean seeds (Snow Brand Seed Co., Ltd., variety: Elimo) were sown in a perforated tray filled with vermiculite No. 4 (Hokkaido Nozai Kogyo) and cultivated for 12 days. The grown adzuki bean seedlings were excised from the bottom 3 cm of the above-ground part to remove the growing point. The test solution was then placed in a 5 ml test tube and the excised part was immersed in this test solution. The test solution was replaced with fresh solution every day for three days. The plants were then immersed in aerated ultrapure water for four days. After the immersion treatment, the total number of roots that had developed at the cut site of the azuki bean seedlings was counted to determine rooting activity.

[0075] 2. Test Results Table 5 shows the results of measuring the rooting activity of each test solution.

[0076] [Table 5]

[0077] Compared to distilled water, the phenyllactic acid + tryptophan mixture showed a slight increase in the number of roots. Rooting activity was also observed in the pre-culture medium, but this was thought to be due to the combined effects of the phenyllactic acid contained in the corn steep liquor, as well as the nutrients, minerals, and vitamins contained in the whey powder, powdered soy protein, and soybeans that were added to the medium. On the other hand, it was revealed that the plant growth regulator of Production Example 2 (culture solution after 3 days of culture) exhibited overwhelmingly higher root-promoting activity than these comparative controls. From the above test results, it became clear that in order to obtain a highly active plant growth regulator by the method of the present invention, a culture period of 3 days is necessary.

[0078] <Test Example 7 (Rooting Assay of Butanol Extract Fraction)> Test Example 6 suggested that the rooting effect of the culture solution obtained in Production Example 2 was partly due to phenyllactic acid and tryptophan contained in the medium. In order to confirm that the plant growth regulator of the present invention contains root-inducing substances other than phenyllactic acid and tryptophan, the following test was carried out.

[0079] 1. Test Method (1) Removal of tryptophan 1 L each of the pre-culture medium used in Test Example 1 and the plant growth regulator obtained in Production Example 2 was adjusted to pH 3.0. Separately, a styrene-divinylbenzene synthetic adsorption resin, Diaion HP-20, was washed with methanol, and then packed into a column (inner diameter 45 mm×length 600 mm), and distilled water was passed through to adjust the amount of the resin. The above solution was passed through the column to adsorb the active ingredient, and after adsorption, the column was washed with distilled water. It was confirmed that under these column adsorption conditions, amino acids, including tryptophan, were not adsorbed to the carrier in the column. Therefore, tryptophan was not adsorbed to the column in this step and was washed away.

[0080] (2) Dissolution of active ingredients Next, the active ingredient adsorbed on the column was eluted with 1.0 L of 20% by volume isopropanol, and the eluate was collected.

[0081] (3) Removal of phenyllactic acid The collected eluate was concentrated to about 1.0 mL using an evaporator and adjusted to pH 8.0 with an aqueous sodium hydroxide solution. Next, solvent extraction was carried out three times with an appropriate amount of ethyl acetate, and the ethyl acetate layer was removed. The remaining aqueous phase was adjusted to pH 2.5 with hydrochloric acid, and extracted with ethyl acetate three times. The ethyl acetate fraction recovered after this extraction was confirmed to contain phenyllactic acid. The aqueous fraction (containing no phenyllactic acid) after ethyl acetate extraction was adjusted to pH 8.0 using aqueous sodium hydroxide, and then extracted three times with an appropriate amount of n-butanol. This n-butanol extract was stored as the neutral butanol fraction. After the n-butanol extraction, the aqueous phase was adjusted to pH 2.5 using hydrochloric acid and extracted three times with an appropriate amount of n-butanol. This n-butanol extract was stored as the acidic butanol fraction. The neutral butanol fraction and the acidic butanol fraction were each evaporated using an evaporator, and then passed through a Diaion HP-20 column again in the same manner as above to adsorb the active ingredient.Then, the fractions were eluted with 20% by volume of isopropanol and desalted. The collected isopropanol eluted fractions (neutral butanol fraction and acidic butanol fraction) were concentrated and dried using an evaporator to prepare test samples of the neutral butanol fraction and the acidic butanol fraction. The obtained test sample was diluted 1000 times with distilled water, and the adzuki bean rooting assay was carried out in the same manner as in Test Example 6 using this solution.

[0082] 2. Test Results The number of roots produced by each extract fraction (1,000-fold diluted solution) is shown in Table 6. The test results for the number of roots produced by the neutral butanol fraction and the acidic butanol fraction of the pre-culture medium extracted in the same manner are also shown.

[0083] [Table 6]

[0084] The neutral butanol fraction and acidic butanol fraction extracted from the culture solution of Production Example 2 increased the number of roots compared to the pre-culture solution. The number before culture was increased by 1.5 times and 1.7 times, respectively. From these test results, it was confirmed that the plant growth regulator of Production Example 2 contains a new substance that promotes rooting in addition to phenyllactic acid and tryptophan. It was found that the substance that promotes rooting is soluble in isopropanol and butanol.

[0085] <Test Example 8 (Broccoli seedling raising test)> 1. Test Method The plant growth regulator obtained in Production Example 1 was evaluated in a broccoli seedling raising test. An 8x8 cell tray was filled with Plug B soil (Hokkaido Nozai Kogyo Co., Ltd.), and two seeds of the broccoli cultivar "Pixel" were sown in each cell. When the cotyledons had expanded, the seedlings were thinned out to one seedling per cell. Next, the plant growth regulator obtained in Production Example 1 was diluted 500 times with water and sprayed twice, once at the true leaf development stage and once again one week later. The plant growth regulator used for spraying was a 500-fold diluted solution, and water was sprayed as a control. The amount of spray solution applied was 500 ml per tray. In the control area, only water was sprayed. After the seedling raising period (29 days after sowing), five plants with average appearance and growth were selected from each tray and collected, and the leaf area, total root length, and dry weight (above and below ground) of each plant were measured.

[0086] 2. Test Results The measurement results are shown in Table 7 as the total value of the measurement results for each individual (total value for 5 strains). In addition, the measured value for each item when only water was sprayed was set at 100, and the relative values ​​when the plant growth regulator of the present invention was sprayed were also shown.

[0087] [Table 7]

[0088] As shown in Table 7, the broccoli seedlings sprayed with the plant growth regulator (culture solution) of the present invention showed high values ​​for all measurement items. In particular, the increase in total root length was large, at 124, compared to 100 for the number sprayed with water alone. Furthermore, no individuals were found that showed excessive elongation. The test results in Table 6 show that when the plant growth regulator of the present invention is sprayed on broccoli seedlings, it promotes root elongation and helps the seedlings grow healthy without causing spindly growth. Furthermore, side effects such as leaf withering, which are seen with auxin treatment, did not occur.

[0089] <Test Example 9 (Rice seedling raising test using a plant growth regulator containing fertilizer components)> 1. Test Method Fertilizer components of 1 mass % of ammonia nitrogen, 1 mass % of nitrate nitrogen, 4 mass % of soluble phosphate, and 2 mass % of water-soluble potassium were added to the plant growth regulator obtained in Production Example 1 to prepare a test solution. The test was conducted at a rice farm in the Kamikawa region of Hokkaido. The test solution was further diluted 1000 times with water before irrigation, and this diluted solution was used as the irrigation treatment solution. For the irrigation treatment, 200 ml of the seed rice was sown per box, and 500 ml of the 1:1000 diluted solution was sprayed on mat seedlings of "Nanatsuboshi" that had germinated after sowing and were growing on the 8th and 17th days after sowing using a watering can or similar method, in the same way as for normal irrigation. Water for comparison was also sprayed in the same way. Seedling raising was completed 29 days after sowing, and sampling was carried out. Sampling was carried out by hollowing out areas of the mats that showed average growth with an 8-centimeter diameter hole cutter, and counting the number of rice seedlings contained in the hollowed-out areas. Furthermore, for 15 individuals showing average growth from the hollowed-out samples, the plant height, first leaf sheath height, number of leaves, and number of tillers were measured for each individual. The dry weight of the aboveground parts was also measured for all 15 individuals. The total root length was calculated by measuring the length of each root that had developed in the excavated area and then totaling the total. All roots were collected, dried, and weighed.

[0090] 2. Test Results The measurement results were tabulated and converted to one individual (average value) and displayed. The measurement results are shown in Table 8 below.

[0091] [Table 8]

[0092] As shown in Table 8, the treatment area sprayed with the test solution prepared from the plant growth regulator of Preparative Example 1 of the present invention was clearly superior in terms of plant height, total root length, and root dry weight. In other words, it is believed that spraying the plant growth regulator of the present invention significantly develops roots, resulting in increased plant height. Furthermore, in the treatment plots sprayed with the plant growth regulator of the present invention, yellowing and leaf withering, which occurred in the control plots, did not occur.

Claims

1. The solid content of corn steep liquor (CSL) is 20% by mass or more, and, One or more selected from phenylalanine, EDTA iron (III) and EDTA zinc (II) A plant growth regulator containing a component obtained by culturing a lactic acid bacterium selected from Lactobacillus paracasei #720 strain (accession number NITE BP-03117), Lactobacillus rhamnosus #555 strain (accession number NITE ABP-03379), and Lactobacillus sp. #728 strain (accession number NITE ABP-03380) in a lactic acid bacterium culture solution containing the above, and containing 160 mg / L or more of phenyllactic acid. (However, excluding microbial organic fertilizers containing 20 to 30% by weight of agricultural straw, 10% by weight of sake lees, and 45 to 62% by weight of corn syrup), Furthermore, 0.2 urokinase and uristatin were used to extract human urine (excluding fermentation liquid of lactic acid bacteria).

2. The solid content of corn steep liquor (CSL) is 20% by mass or more, and, One or more selected from phenylalanine, EDTA iron (III) and EDTA zinc (II) 2. The method for producing the plant growth regulator according to claim 1, wherein a lactic acid bacterium selected from Lactobacillus paracasei #720 strain (accession number NITE BP-03117), Lactobacillus rhamnosus #555 strain (accession number NITE ABP-03379), and Lactobacillus sp. #728 strain (accession number NITE ABP-03380) is cultured in a lactic acid bacterium culture solution containing

3. A composition for plant rooting, which is insoluble in ethyl acetate and soluble in isopropanol and butanol, obtained by the following manufacturing process: Corn steep liquor (CSL) solids content of 20% by mass or more and One or more selected from phenylalanine, EDTA iron (III) and EDTA zinc (II) A culture solution obtained by culturing a lactic acid bacterium selected from Lactobacillus paracasei #720 strain (accession number NITE BP-03117), Lactobacillus rhamnosus #555 strain (accession number NITE ABP-03379), and Lactobacillus sp. #728 strain (accession number NITE ABP-03380) in a lactic acid bacterium culture solution containing The liquid is passed through a column of styrene-divinylbenzene synthetic adsorption resin to adsorb the active ingredients. Next, the active ingredient adsorbed on the column was eluted with 1.0 L of 20% by volume isopropanol, and the eluate was subjected to solvent extraction three times with ethyl acetate. The ethyl acetate layer was removed. Ethyl acetate extraction was repeated three times. The aqueous phase fraction was extracted with an appropriate amount of n-butanol to obtain a neutral butanol fraction. After completion of the n-butanol extraction, the aqueous phase was adjusted to pH 2.5, and the extract obtained by extraction with n-butanol was used as an acidic butanol fraction. A manufacturing process in which the neutral butanol fraction and the acidic butanol fraction are adsorbed onto a styrene-divinylbenzene synthetic adsorption resin to obtain the active ingredient. (However, excluding microbial organic fertilizers containing 20 to 30% by weight of agricultural straw, 10% by weight of sake lees, and 45 to 62% by weight of corn syrup), Furthermore, 0.2 urokinase and uristatin were used to extract human urine (excluding fermentation liquid of lactic acid bacteria).

4. 4. The composition for rooting plants according to claim 3, which does not contain amino acids or phenyllactic acid.

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