Biostimulant manufacturing method, biostimulant, plant cultivation method using the same, and waste milk recycling method

Abstract

To provide a technique capable of realizing regionally recycling persistent production centering on effective utilization of waste milk and activation of crop and soil by biostimulant, and is economically excellent.SOLUTION: Provided is a method for producing biostimulant in which a microorganism group including lactic acid bacteria and yeast is inoculated into a liquid raw material containing milk and / or dairy product, and the material is fermented at room temperature for 24 hours or more. Provided is biostimulant that contains a microorganism group including lactic acid bacteria and yeast, 40 mg or more of amino acid per 100 g of biostimulant, and has lactic acid, pH of 5 or less, and Brix value of 7% or less. Further, provided is a plant cultivation method in which the biostimulant is given to plant or soil.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for producing a biostimulant, a biostimulant and a method for cultivating a plant using the same, and more specifically, to a biostimulant obtained by fermenting a liquid raw material containing milk and / or a dairy product with Lactobacillus lactic acid bacteria and Pichia yeast. [Background technology]

[0002] Traditionally, in order to improve agricultural productivity, technological developments such as breeding, improving nutrition (fertilizer), and controlling pests and weeds (pesticides) have been actively carried out. However, while conventional farming methods that rely on pesticides and chemical fertilizers can increase yields in the short term, they have the problem of placing a heavy burden on the environment.

[0003] In recent years, therefore, "biostimulants," which are neither fertilizers nor pesticides, have been attracting attention as one of the means to reduce the environmental burden and realize sustainable agriculture. Biostimulants are agricultural materials that stimulate the inherent physiological processes of crops by applying them to soil, thereby reducing abiotic stress. Specific effects of biostimulants include improving metabolic efficiency, enhancing stress resistance, and promoting assimilation, translocation, and use of nutrients.

[0004] Known examples of such biostimulants include a plant stimulant containing chitin oligosaccharides and cellooligosaccharides (see Patent Document 1), and a plant stimulant containing amino acids, fulvic acid, and choline (see Patent Document 2). In addition, EM bacteria, which are commercially available as soil improving microbial materials containing useful microorganisms such as yeast, lactic acid bacteria, and photosynthetic bacteria, are also a type of biostimulant.

[0005] Meanwhile, in large dairy manufacturing plants, the amount of products that are discarded after quality control inspections can reach 1-2 tons per day. In recent years, the disposal of surplus raw milk has also become a problem. Disposal of such raw milk and dairy products is not only environmentally hazardous, but also costly, so there has been a demand for the development of technology to effectively utilize discarded milk.

[0006] An example of a technology for recycling waste milk is disclosed in Patent Document 3. In Patent Document 3, egg white and lactic acid bacteria are added to milk and lactic acid fermentation is carried out to produce a substance containing amino acid components and low molecular weight peptide components. It is said that the use of this substance in soil can have effects such as promoting plant growth, improving the soil microbial environment, and eradicating pests. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2022-34181 [Patent Document 2] JP 2013-82640 A [Patent Document 3] JP 2007-308358 A Summary of the Invention [Problem to be solved by the invention]

[0008] However, the above biostimulants all have the problem of not being sufficiently effective. In addition, the technology of Patent Document 3 requires the addition of egg white to milk, which is costly for treating the large amount of waste milk that is generated every day.

[0009] The object of the present invention is to provide a technology that enables economically superior, locally-circulated, sustainable production centered on the effective utilization of waste milk and the revitalization of crops and soil with biostimulants. [Means for solving the problem]

[0010] That is, the present disclosure provides a method for producing a biostimulant, which comprises inoculating a liquid raw material containing milk and / or a dairy product with a microbial group including lactic acid bacteria and yeast, and fermenting the liquid raw material at room temperature for 24 hours or more.

[0011] Here, the microbial community may further include acetic acid bacteria.

[0012] The liquid ingredient may further include fruit juice and / or vegetable juice. The liquid ingredient may further include coffee.

[0013] The present disclosure also provides a biostimulant that contains a microbial group including lactic acid bacteria and yeast, 40 mg or more of amino acids per 100 g of biostimulant, and lactic acid, and is characterized by having a pH of 5 or less and a Brix value of 7% or less.

[0014] Here, the biostimulant may further contain acetic acid bacteria and acetic acid, and the total content of the lactic acid and the acetic acid may be 0.5 mass % or more.

[0015] The present disclosure also provides a method for cultivating a plant, comprising applying the biostimulant to the plant or soil, wherein the plant may be one or more species selected from the family Rosaceae, Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Solanaceae, Amaranthaceae, and Nymphaeaceae. Effect of the Invention

[0016] In the present disclosure, the action of various amino acids and organic acids produced by the microbial community reduces crop stress, resulting in excellent effects such as increased yield and improved quality. In addition, in the present disclosure, biostimulants can be produced by fermenting only industrial waste such as waste milk as a raw material, thereby reducing waste disposal costs.

[0017] Therefore, according to the present disclosure, it is possible to provide technology that enables realization of sustainable regional production centered on the effective utilization of waste milk and the revitalization of crops and soil through biostimulants. [Brief description of the drawings]

[0018] [Figure 1] FIG. 2 is a schematic diagram showing the fermentation mechanism in this embodiment. [Diagram 2] FIG. 1 shows the results of metagenomic analysis in Example 1. [Diagram 3] 1 is a graph showing the change in free amino acid content during storage (Example 2). In the figure, the bar graphs show the free amino acid concentration (unit: mg / 100 g), and for each amino acid, from the left, the bar graphs show the storage periods of 0 days, 2 weeks, 6 weeks, and 8 weeks. [Figure 4] Photographs showing the growth of Komatsuna in black soil (Example 4). In the figure, (a) shows the aboveground part 7 days after sowing, (b) shows the aboveground part 21 days after sowing (at the time of the final inspection), and (c) shows the root part 21 days after sowing (at the time of the final inspection). [Diagram 5] Photographs showing the growth of Komatsuna in gray lowland soil (Example 4), in which (a) shows the aboveground part 7 days after sowing, (b) shows the aboveground part 21 days after sowing (at the time of final inspection), and (c) shows the root part 21 days after sowing (at the time of final inspection). [Figure 6] 1 is a photographic image showing the growth of Komatsuna (roots) in gray lowland soil (Example 4). In the figure, the dilution rates of 10x, 50x, and 100x are shown from the left in each column, and the control, spray, and irrigation plots are shown from the top in each row. [Figure 7] This is a diagram showing the results of a rice cultivation test (Example 5). In the figure, the vertical axis represents the weight of brown rice per plant (unit: g / plant), and the horizontal axis represents each test area. There was a significant difference in the treatment areas that did not share a common alphabet (Tukey-Kramer test, P<0.05). The values ​​represent the average value ± standard deviation. [Figure 8] 8A and 8B are diagrams showing strawberry yields (Example 6). Fig. 8(A) is a graph showing the progress of cumulative yields, with the solid line indicating the irrigation area, the dashed line indicating the spraying area, and the dotted line indicating the control area. The vertical axis indicates the yield (g / plant), and the horizontal axis indicates the harvest date. Fig. 8(B) is a graph showing the average yield, with the vertical axis indicating the yield (g / plant), the horizontal axis indicating the test area, and the values ​​indicating the average value ± standard deviation. [Figure 9]This is a scatter diagram showing the relationship between the weight of one fruit and the number of fruits harvested per harvest (Example 6). In the figure, (A) shows the control group, (B) shows the foliar spray group, and (C) shows the irrigation group. The vertical axis shows the weight of one fruit (g / fruit), and the horizontal axis shows the number of fruits harvested per harvest. [Figure 10] 1 is a graph showing the plant weight at the end of the test (Example 6). In the figure, the vertical axis shows the plant weight (g / plant fresh weight), and the horizontal axis shows the treatment group. In the bar graph, the diagonal line shows the weight of the aboveground part, and the white line shows the weight of the underground part, and the values ​​are the average value ± standard deviation. [Figure 11] 11 shows the results of the petri dish test on cucumber (Example 7). Fig. 11(A) is a graph comparing stem length, with the vertical axis representing stem length (unit: cm) and the horizontal axis representing each test plot. Fig. 11(B) is a graph comparing root length, with the vertical axis representing root length (unit: cm) and the horizontal axis representing each test plot. In the figure, NC stands for negative control (distilled water), and there was a significant difference in the treatment sections that did not contain a common alphabet (Tukey-Kramer test, P<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The embodiments of the present disclosure are described in detail as follows.

[0020] The method for producing a biostimulant according to this embodiment is characterized in that a liquid raw material containing milk and / or dairy products is inoculated with a microorganism group including lactic acid bacteria and yeast, and fermented at room temperature for 24 hours or more.

[0021] [Liquid raw materials] The liquid raw material used in this embodiment is capable of growing a microorganism described below, and contains milk proteins and sugars that serve as fermentation raw materials for producing amino acids and organic acids. Specifically, the liquid raw material can contain at least milk and / or dairy products.

[0022] Examples of milk include, but are not limited to, cow's milk, milk with adjusted ingredients, low-fat milk, non-fat milk, processed milk, raw milk, animal milk, etc. Examples of dairy products include, but are not limited to, milk drinks, lactic acid bacteria drinks, fermented milk, concentrated milk, condensed milk, whole milk powder, skim milk powder, whey, whey powder, ice cream, ice milk, lacto ice cream, butter, cream, cheese, etc. Examples of dairy drinks include, but are not limited to, cafe au lait, cafe latte, fruit au lait, nutritionally enhanced milk, etc.

[0023] The liquid raw material may contain raw materials other than milk and / or dairy products, so long as they do not interfere with the growth of the microorganisms described below, the production of amino acids and organic acids, and the growth of crops. Specific examples of the other raw materials include juices such as fruit juice and vegetable juice, and tea-based beverages such as coffee, green tea, and black tea. The juice may contain fruit juice or vegetable juice, and the amount of fruit juice or vegetable juice is not limited. The coffee and tea-based beverages may be unsweetened or sweetened.

[0024] These raw materials may be used in combination of one or more kinds. In addition, even if a raw material is in a solid state, it can be used if it can be made into a liquid state as a whole by mixing it with another raw material in a liquid state.

[0025] It is desirable to use industrial waste from dairy factories and other sources as raw materials for these purposes in terms of reducing food waste and waste disposal costs. In addition, it is desirable to use only food-derived raw materials as liquid raw materials in terms of the safety of crops and workers.

[0026] [Microbial group] The microbial community used in this embodiment is composed of multiple types of microorganisms capable of growing in the liquid raw material and producing amino acids and / or organic acids. From the viewpoint of the safety of crops and workers, it is desirable that the microbial community is composed only of microorganisms contained in the fermented food.

[0027] Specifically, the microorganism group may include lactic acid bacteria and yeast. Lactic acid bacteria may include, but are not limited to, lactobacillus lactic acid bacteria. Lactobacillus lactic acid bacteria may include, but are not limited to, L. rhamnosus, L. harbinensis, L. farraginis, L. buchneri, L. rapi, L. casei, L. composti, L. kimchicus, etc. Yeast may include, but are not limited to, Pichia yeast. Pichia yeast may include, but are not limited to, P. membranifaciens, P. deserticola, etc.

[0028] The microorganism group may contain other microorganisms than Lactobacillus lactic acid bacteria and Pichia yeast, so long as they do not interfere with the production of amino acids and organic acids by the above-mentioned microorganisms and the growth of crops. Examples of other microorganisms include, but are not limited to, any lactic acid bacteria, yeast, acetic acid bacteria, etc. Examples of any lactic acid bacteria other than Lactobacillus include, but are not limited to, Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, L. chungangensis, L. garvieae, L. piscium, L. plantarum, L. raffinolactis, etc. Examples of any yeast other than Pichia include, but are not limited to, Saccharomyces yeast, etc. Examples of any acetic acid bacteria include, but are not limited to, Acetobacter acetic acid bacteria. Examples of Acetobacter acetic acid bacteria include, but are not limited to, A. fabarum, A. lovaniensis, etc.

[0029] [Fermentation process] 1 is a schematic diagram showing the mechanism of fermentation in this embodiment. Under anaerobic conditions, yeast (e.g., Pichia yeast) decomposes proteins into peptides and amino acids, and the peptides are decomposed into amino acids by lactic acid bacteria (e.g., Lactobacillus lactic acid bacteria). Meanwhile, lactic acid bacteria also perform lactic acid fermentation using sugars as a raw material. In addition, acetic acid bacteria (e.g., Acetobacter acetic acid bacteria) perform acetic acid fermentation under aerobic conditions using alcohol produced by yeast in alcohol fermentation using sugars as a raw material. In this way, amino acids and organic acids such as lactic acid and acetic acid are produced by the microorganisms in the fermentation process in this embodiment.

[0030] The fermentation step in this embodiment is a step of producing a fermentation liquid by inoculating the liquid raw material with the microorganisms and fermenting the liquid raw material at room temperature for 24 hours or more.

[0031] The inoculation of the microbial group can usually be carried out by adding a part of the fermentation liquid (bulk starter) obtained by the main fermentation process to the liquid raw material. Alternatively, a preculture liquid of the microbial group can be added to the liquid raw material. The amount of the bulk starter or preculture liquid added is not particularly limited, but can usually be 1 to 50% (v / v) of the liquid raw material. The medium used for preculture is not particularly limited as long as the microbial group can grow therein, and for example, a medium containing milk and / or dairy products or a synthetic medium can be used. Examples of synthetic media include, but are not limited to, MRS medium.

[0032] Fermentation can be carried out substantially at room temperature (for example, about 20 to 35°C). In this specification, "substantially room temperature" means that even if the temperature temporarily deviates from room temperature, the total time during one day during which the temperature deviates from room temperature is within about 6 hours. During fermentation, stirring may be performed, or static fermentation may be performed. The rotation speed when stirring is not particularly limited, but may be, for example, 500 to 1000 rpm. The fermentation time can usually be 24 hours or more, preferably 30 hours or more, and more preferably 36 hours or more.

[0033] [Aging process] In this embodiment, a maturation step can be provided after the fermentation step. This step is carried out by leaving the fermentation liquid at room temperature. By carrying out this step, fermentation can be further advanced and more amino acids and organic acids can be produced. The maturation time is not particularly limited, but can be, for example, 24 hours or more, preferably 48 hours or more, and more preferably 72 hours or more.

[0034] In the fermentation process and optional maturation process, the microorganisms assimilate the milk proteins and sugars contained in the liquid raw material to produce amino acids and organic acids, so that as the fermentation progresses, the sugars in the liquid raw material decrease and the organic acids increase. Therefore, the practitioner can manage the fermentation by measuring the sugar content and / or pH of the fermentation liquid.

[0035] Specifically, fermentation is desirably continued until the pH of the fermentation liquid is 5 or less, preferably 4.8 or less, more preferably 4.6 or less, even more preferably 4.4 or less, particularly preferably 4.2 or less, and especially preferably 4.0 or less, and / or until the sugar content (Brix) is 7% or less, preferably 6.8% or less, more preferably 6.6% or less, even more preferably 6.5% or less, particularly preferably 6.4% or less, and especially preferably 6.3% or less.

[0036] [Biostimulants] The method produces a fermentation liquid containing the microorganisms, amino acids, and organic acids. This fermentation liquid is rich in free amino acids that can be absorbed by plants, and is particularly rich in proline, which has functions such as regulating the water level of plants and promoting sugar synthesis, and glutamic acid, which has functions such as promoting plant growth and improving the quality of pollen and fruits. In addition, the lactic acid bacteria, lactic acid bacteria metabolites, and lactic acid contained in the fermentation liquid not only promote plant growth, but also activate soil microorganisms and are said to be effective in preventing pests and diseases, and also have an effect of preventing the fermentation liquid from spoiling.

[0037] Therefore, the fermentation broth produced by the method can be used as an agricultural material, in particular as a biostimulant, soil conditioner or fertilizer that stimulates the physiological processes of crops and reduces abiotic stress.

[0038] That is, according to the present embodiment, a biostimulant is provided that contains a microorganism group including lactic acid bacteria and yeast, and an amino acid and an organic acid. Here, the pH of the biostimulant (fermentation liquid) may be 5 or less, preferably 4.8 or less, more preferably 4.6 or less, even more preferably 4.4 or less, particularly preferably 4.2 or less, and especially preferably 4.0 or less. The sugar content (Brix) of the biostimulant (fermentation liquid) may be 7% or less, preferably 6.8% or less, more preferably 6.6% or less, even more preferably 6.5% or less, particularly preferably 6.4% or less, and especially preferably 6.3% or less.

[0039] It is presumed that amino acids are present in the fermentation broth as both free amino acids and peptides, and the total content of free amino acids is desirably 40 mg or more, preferably 50 mg or more, more preferably 60 mg or more per 100 g of the fermentation broth. Examples of the types of free amino acids in the fermentation broth include proline, threonine, leucine, glutamic acid, valine, lysine, histidine, serine, and glycine.

[0040] In addition, the organic acids are presumed to be mainly composed of lactic acid and acetic acid, and it is desirable that the total content of the organic acids is 0.5 mass% or more, preferably 0.7 mass% or more, and more preferably 1.0 mass% or more, relative to the fermentation liquid.

[0041] By applying the biostimulant of this embodiment to plants or soil, as described in detail in the examples, the effects of promoting plant growth and improving taste are achieved. This is thought to be because the plant absorbs free amino acids, and the energy consumed to synthesize amino acids from nitrate in the plant body can be directed to growth. In addition, the plant growth promotion effect of lactic acid and lactic acid bacteria metabolites, and the effect of increasing the sugar content of plants by acetic acid are also thought to have an effect.

[0042] [Method of cultivating plants] The plant cultivation method of the present embodiment is characterized in that the biostimulant is applied to the plant or soil. The means for applying the biostimulant to the plant or soil is not particularly limited, but examples thereof include foliar spraying and irrigation. In particular, foliar spraying is preferable because amino acids and the like can be absorbed directly from the leaves.

[0043] The amount and concentration of the biostimulant to be used per time can be appropriately determined by those skilled in the art depending on the type and growth stage of the plant, the cultivation environment, etc. Usually, the fermentation liquid is diluted with water, or the undiluted liquid is applied to the plant or soil. When the fermentation liquid is used after dilution, the dilution ratio is not particularly limited, but can be, for example, 10 to 100,000 times, preferably 100 to 10,000 times.

[0044] The interval for using the biostimulant is not particularly limited, but may be, for example, 24 hours or more, 3 days or more, 5 days or more, 7 days or more, 9 days or more, or 11 days or more, or 2 weeks or less, or may be 1 to 7 days. In addition to the use of the biostimulant, it is desirable to perform watering appropriately according to the type of plant and the cultivation environment. In this embodiment, except for the above points, the plant can be cultivated in the same manner as the conventionally known cultivation technique.

[0045] The cultivation method of the present embodiment can be applied to any plant, such as vegetables, fruit trees, flowers, trees, foliage plants, etc. Specific examples of plants include plants belonging to the Rosaceae family, Brassicaceae family, Poaceae family, Cucurbitaceae family, Asteraceae family, Solanaceae family, Amaranthaceae family, Nymphaeaceae family, etc. EXAMPLES

[0046] The present disclosure will be specifically described below with reference to examples and comparative examples. In the following, "%" means mass % (w / w) unless otherwise specified.

[0047] (Example 1) Subculture of microbial communities using food waste as a raw material As described below, microbial communities were subcultured using liquid raw materials such as discarded milk, and the bacterial flora was analyzed.

[0048] (1) Subculture method The seed culture was provided by Zeus Industry Co., Ltd. The liquid raw materials were made by mixing milk, dairy beverages, fruit juice, or vegetable juice, and coffee (unsweetened, slightly sweetened, or sweetened), which were discarded after daily quality inspections at dairy factories, in the ratios shown in Table 1. The sugar content of the entire liquid raw materials was about 3 to 7%. The seed culture was added to the liquid raw materials in the ratios shown in Table 1, and fermentation was carried out with stirring at room temperature (approximately 22°C to 26°C). The total liquid volume was 1 to 2 L. Subculture was carried out by using the fermentation liquid after fermentation for 6 hours to 7 days as a bulk starter and adding the bulk starter to new liquid raw materials in the ratios shown in Table 1.

[0049] [Table 1]

[0050] (2) Analysis of bacterial flora Using the method described above, metagenomic analysis was performed a total of four times during subcultivation to investigate the microbial composition in the fermentation liquid. DNA extraction to metagenomic analysis was outsourced to Techno Suruga Lab. Analysis of fungi was performed using the DNA sequence of the ITS2 region, with an analysis model: MiSeq (registered trademark) (Illumina, Inc.) and database: RDP. Analysis of bacteria was performed using the 16S rDNA sequence, with an analysis model: MiSeq (registered trademark) (Illumina, Inc.) and database: RDP + Techno Suruga Lab "Microorganism Identification Database." The number of microorganisms in the fermentation broth was measured by the plate spread method.

[0051] (3) Results and Discussion The results of the metagenomic analysis are shown in Figure 2, Tables 2 and 3. Figure 2 is a diagram showing the composition ratio of microorganisms involved in fermentation, based on a synthesis of the results of the metagenomic analysis. Tables 2 and 3 show the results of each round of metagenomic analysis for fungi and bacteria, respectively. In the tables, the numbers "passage (1)" to "passage (3)" are used to distinguish between three times of subculture performed from the same seed bacteria. In addition, "passage (1) 10th" indicates that the number of passages in passage (1) is the 10th. The ratio of bacteria whose species could not be identified is not included in the tables below.

[0052] [Table 2] *In the table, the numbers indicate the composition ratio (unit: %).

[0053] [Table 3] *In the table, the numbers indicate the composition ratio (unit: %).

[0054] Among fungi, Pichia yeasts accounted for more than 30%. Excluding passage (2), Pichia yeasts (Pichia membranifaciens) accounted for more than 92% (Table 2). Among bacteria, Lactobacillus lactic acid bacteria accounted for 29-92%. Among the Lactobacillus lactic acid bacteria, Lactobacillus rhamnosus accounted for 25-74%, Lactobacillus harbinensis for 2-40%, and Lactobacillus farraginis for 0.1-32%. Acetobacter fabarum was not detected in passage (3), but was present in 23-39% of the other passages (Table 3).

[0055] These results indicate that the microorganisms involved in the fermentation in this embodiment are mainly Lactobacillus lactic acid bacteria (particularly Lactobacillus rhamnosus) and Pichia yeast (particularly Pichia membranifaciens). In some cases, Acetobacter acetic acid bacteria (particularly Acetobacter fabarum) are also involved.

[0056] (Example 2) Amino acid analysis of fermentation liquid using food waste as raw material Fermentation tests were carried out using liquid raw materials such as waste milk as follows, and amino acid analysis was carried out.

[0057] The fermentation liquid subcultured by the method of Example 1 was used as a bulk starter and added to the liquid raw material at the blending ratio shown in Table 4. The total liquid volume was set to 2 L, and fermentation was carried out for 120 hours with stirring at room temperature (approximately 22°C to 26°C). The pH and sugar content (Brix) were measured during and after fermentation. The measurement results are shown in Table 5. It was shown that the pH and sugar content decreased as fermentation progressed (Table 5).

[0058] [Table 4]

[0059] [Table 5]

[0060] The fermented liquid after 72 hours of fermentation was stored at room temperature (approximately 22°C to 26°C) for 8 weeks. The pH, sugar content (Brix), and free amino acid content were measured during and after storage. The free amino acid content was measured by HPLC under the conditions shown in Table 6. The results are shown in Tables 7 and 8 and in FIG. 3.

[0061] [Table 6]

[0062] Table 7 shows the changes in pH and sugar content during storage. Table 8 and Figure 3 show the changes in free amino acid content during storage. In Figure 3, the bar graphs show the free amino acid concentration (unit: mg / 100 g), and for each amino acid, from left to right, the bar graphs show the storage periods of 0 days, 2 weeks, 6 weeks, and 8 weeks.

[0063] [Table 7]

[0064] [Table 8] *In the table, the numbers indicate free amino acid concentration (unit: mg / 100 g).

[0065] Even when the stirring was stopped and the mixture was stored, the pH and sugar content decreased over time (Table 7), and the free amino acid content increased over time (Table 8, Figure 3).

[0066] These results suggest that in this embodiment, the action of lactic acid bacteria and yeast produces free amino acids and organic acids from milk proteins and sugars contained in the liquid raw material, resulting in a decrease in the pH and sugar content of the fermentation liquid and an increase in the free amino acid content. It was also shown that fermentation continues even when stirring of the fermentation liquid is stopped. It was shown that the decrease in pH and sugar content stopped after the storage period exceeded 6 weeks (Table 7), but the increase in the free amino acid content continued (Table 8, Figure 3). This was thought to be due to the decrease in pH of the fermentation liquid, which fell outside the optimal pH range for growth of organic acid-producing bacteria such as lactic acid bacteria and acetic bacteria, causing the organic acid fermentation using sugars as a raw material to stop.

[0067] (Example 3) Measurement of organic acid content in fermentation liquid using food waste as raw material Fermentation was carried out in the same manner as in Example 2, and the changes in the organic acid content (the total content of lactic acid and acetic acid) and pH during fermentation were examined. The organic acid content was measured by neutralization titration.

[0068] The measurement results are shown in Table 9. It was shown that as the fermentation proceeded, the pH decreased and the organic acid content increased (Table 9). This indicated that lactic acid and acetic acid were produced in the fermentation of this embodiment. It was also shown that the decrease in pH in the fermentation liquid was associated with an increase in the lactic acid and acetic acid contents.

[0069] [Table 9]

[0070] (Example 4) Plant Damage Test (Komatsuna) Using the fermented liquid produced in Example 2 above, a plant damage test was carried out on Komatsuna (Brassicaceae). The fermented liquid was fermented for 72 hours using the method described in Example 2, and then left to stand for 4 weeks. The fermented liquid was diluted 10-fold (pH 4.3), 50-fold (pH 4.2), or 100-fold (pH 4.1) with water (diluted liquid) and used as a sample. The test was carried out under the test conditions shown in Table 10 in accordance with "Method of Cultivation Test for Harm to Plants" (Notice of Nosan No. 1943, 1984).

[0071] [Table 10]

[0072] That is, Neubauer pots were filled with black soil or gray lowland soil, and 20 Komatsuna seeds were sown per pot (3 pots for each group). After that, the diluted solution of each concentration was sprayed or irrigated once per week, a total of three times, to replenish the lost moisture, and the plants were cultivated for three weeks. As a control group, the plants were cultivated in the same manner as above, except that water was used instead of the diluted solution, and compared. The items shown in Table 11 were investigated.

[0073] [Table 11]

[0074] The results are shown in Figures 4 to 6 and Tables 12 to 15. Figure 4 is a photographic image showing the growth of Komatsuna in Andosol and Figure 5 is a photographic image showing the growth of Komatsuna in gray lowland soil. In Figures 4 and 5, (a) shows the aboveground part 7 days after sowing, (b) shows the aboveground part 21 days after sowing (at the time of the final survey), and (c) shows the root part 21 days after sowing (at the time of the final survey). Also, Figure 6 is a photographic image showing the growth of Komatsuna (root part) in gray lowland soil. In Figure 6, the dilution rates of 10x, 50x, and 100x are shown from the left in each column, and the control, spray, and irrigation groups are shown from the top in each row. Tables 12 and 13 show the measurement results in Andosol, and Tables 14 and 15 show the measurement results in gray lowland soil. In the tables, there was a significant difference between the treatment sections that did not share a common alphabet (Tukey-Kramer test, P<0.05).

[0075] [Table 12]

[0076] [Table 13]

[0077] [Table 14]

[0078] [Table 15]

[0079] In the black soil, no abnormal discoloration of leaves or root rot was observed as a result of spraying or irrigation of the diluted solution. Furthermore, no significant differences were observed in plant vigor and the extent of root expansion compared to the control (untreated) (Fig. 4, Tables 12 and 13). In the gray lowland soil, improved root expansion and development of fine roots (root hairs) were observed in the diluted solution-treated plots compared to the control plots (Fig. 6). Furthermore, in the gray lowland soil, no adverse effects on growth (discoloration of leaves, root rot, etc.) were observed as a result of spraying or irrigation of the diluted solution, and plant vigor was not significantly different from the control plot (Fig. 5, Tables 14 and 15).

[0080] The fresh weight in the diluted solution-treated plots was 89-107% (fresh weight index) of the control plot (criteria for growth impairment: 80% or less) (Tables 13 and 15). In addition, when the soil pH was measured before and after the test, no significant difference was observed before and after the test.

[0081] Thus, no significant growth impairment was observed due to the treatment with the diluted solution. Furthermore, in the gray lowland soil, root establishment was improved compared to the control area, and the development of fine roots (root hairs) was observed, suggesting the possibility that the diluted solution may have an effect of improving the root system in poor physicochemical soil. Furthermore, although the diluted solution is weakly acidic (pH 4.1 to 4.3), its effect on soil pH is small, suggesting that there is no problem with soil acidification.

[0082] (Example 5) Rice cultivation test A cultivation test of rice (Poaceae) was carried out using the fermented liquid produced in Example 2 above. The fermented liquid was fermented for 72 hours using the method described in Example 2, and then left to stand for 4 weeks. The fermented liquid was diluted 500-fold with water (diluted liquid) and used as a sample. Rice seedlings were planted at one plant per pot, and cultivation was started by spraying or irrigating with the diluted liquid (4 pots per group). Thereafter, cultivation was continued for 5 months by spraying or irrigating with the diluted liquid every 2 weeks. As a control, cultivation was carried out in the same manner as above except that water was given instead of the diluted liquid, and comparison was made.

[0083] The results are shown in Figure 7. Figure 7 is a graph comparing brown rice weight, with the vertical axis representing the brown rice weight per plant (unit: g / plant) and the horizontal axis representing each test plot, and there was a significant difference between treatment plots that did not share a common alphabet (Tukey-Kramer test, P<0.05).

[0084] The weight of brown rice in the sprayed area increased significantly compared to the control area (P<0.05), but no significant difference was observed in the irrigated area (Figure 7). In addition, no growth disorders or abnormal symptoms were observed in the treated area compared to the control area. These results indicate that the treatment with the diluted solution does not cause growth disorders, abnormal symptoms, or adverse effects on the quality of the harvest (Figure 7).

[0085] (Example 6) Strawberry cultivation test A strawberry (Rosaceae) cultivation test was carried out using the fermented liquid produced in Example 2 above. The fermented liquid was fermented for 72 hours using the method described in Example 2, and then left to stand for 4 weeks. The fermented liquid was diluted 100 times with water (diluted liquid) and used as a sample. In December, strawberry (variety "Benihoppe") seedlings were planted in a cultivation bed in a greenhouse, and the diluted liquid was sprayed on the leaves or irrigated (sprayed on the soil) to start cultivation (5 plants per group x 3 replicates). After that, the diluted liquid was sprayed or irrigated every 2 weeks and cultivated for 4 months. Irrigation was performed appropriately during the cultivation period. As a control group, cultivation was carried out in the same manner as above except that water was given instead of the diluted liquid, and comparison was made.

[0086] Harvesting was performed every 3-4 days, and on each harvest day, fruit weights were measured, and physicochemical and taste evaluations were performed. In the physicochemical tests, the sugar and acidity of the fruit were measured using a saccharometer. At the end of the test, the strawberries were removed with the roots and separated into above-ground and below-ground parts, and the weights of each were measured.

[0087] Taste evaluation was carried out by 2 to 7 inspectors who tasted one strawberry from each treatment group. Each inspector ranked (1st to 3rd place) each evaluation item shown in Table 16, and the average ranking value was calculated for each harvest date. The average ranking values ​​for each harvest date (29 days in total) were added up to form the evaluation value for each item, and the sum of these was used as the overall evaluation value, which are shown in Table 16. It was shown that treatment with the diluted solution resulted in strawberries that were sweeter, less sour, firmer, and juicier. The foliar spray group in particular received a higher evaluation, but there were no significant differences in any of the items (Table 16).

[0088] [Table 16] *The smaller the number, the higher the rating.

[0089] The results of the physicochemical tests are shown in Table 17. In the table, the sugar-acid ratio is the value obtained by dividing the sugar content (Brix (%)) by the acidity (citric acidity (%)). Table 17 shows that treatment with the diluted solution tended to result in a higher sugar content compared to the control, and in particular, the sugar content of the foliar spray group was significantly higher than that of the control (Tukey-Kramer test, P<0.05). This result can be said to correlate with the results of the taste evaluation (Table 16). However, there were only slight differences between the groups in citric acid acidity and sugar-acid ratio.

[0090] [Table 17] *Significant difference compared to the control group (Tukey-Kramer test, P<0.05).

[0091] The results of the yield measurements are shown in Figure 8. Figure 8(A) is a graph showing the progress of the cumulative yield, with the solid line representing the irrigation group, the dashed line representing the spray group, and the dotted line representing the control group. The vertical axis represents the yield (g / plant) and the horizontal axis represents the harvest date. Figure 8(B) is a graph showing the average yield, with the vertical axis representing the yield (g / plant) and the horizontal axis representing the test group, and the values ​​represent the average value ± standard deviation. Figure 9 is a scatter plot showing the relationship between the weight of each fruit and the number of fruits harvested per harvest. In Figure 9, (A) represents the control group, (B) represents the foliar spray group, and (C) represents the irrigation group. In Figure 9, the vertical axis represents the weight of each fruit (g / fruit) and the horizontal axis represents the number of fruits harvested per harvest.

[0092] The yield improved in all test plots from mid-February onwards (Figure 8(A)). Although there was no statistically significant difference in the yields of the test plots, the yields were 3.4% higher in the irrigation plot and 11% higher in the foliar spray plot compared to the control plot (Figure 8(B)). Thus, there was a tendency for the irrigation or foliar spray treatments to improve yields compared to the control plot (Figure 8). Furthermore, as shown in Figure 9(A), there was a tendency for the weight per fruit to decrease as the number of harvested fruits increased in the control plot, but no such tendency was observed in the treatment plots (Figures 9(B) and (C)). This suggests that the diluted solution may have the effect of alleviating the so-called "ripening fatigue (plant fatigue)" (Figure 9).

[0093] Next, the results of measuring the plant weight at the end of the test are shown in Figure 10. In Figure 10, the vertical axis shows the plant weight (g / plant fresh weight) and the horizontal axis shows the treatment groups. In the bar graph, the diagonal line shows the weight of the above-ground parts and the white line shows the weight of the underground parts, and the values ​​show the average value ± standard deviation. As can be seen from Figure 10, growth of both above-ground and underground parts was slightly more vigorous in the foliar spray group, but no significant difference was observed (Tukey-Kramer test).

[0094] These results indicate that treatment with the diluted solution improves strawberry taste and yield, and tends to alleviate the so-called "fruit fatigue." This tendency was particularly strong in the case of foliar spray, where the sugar content of the fruit was significantly higher than in the control area (Table 17, P<0.05). This is thought to be because the diluted solution was efficiently absorbed by the plant body when sprayed directly on the leaves. It is also suggested that absorbing the amino acids in the diluted solution reduces the energy consumption required to synthesize amino acids from nitrate nitrogen within the plant body, leading to the accumulation of more sugar in the fruit and improved plant growth.

[0095] Example 7: Petri dish test A petri dish test was carried out on cucumber (Cucurbitaceae) using the fermented liquid produced in Example 2 above. The fermented liquid was fermented for 72 hours using the method described in Example 2, and then left to stand for 4 weeks. The fermented liquid was diluted 100 times with water (diluted liquid) and used as a sample. In addition, a commercially available soil improvement material "EM No. 1" (manufactured by EM Research Institute) was diluted 1000 times with water and used as a positive control.

[0096] Filter paper was placed in a 10 cm diameter petri dish, and 5 mL of sample was added per dish. 10 cucumber seeds per dish were placed on the filter paper and cultured for 7 days (one dish per group). Culture conditions were a constant temperature of 25°C. After 7 days of culture, the above-ground length (stem length) and underground length (root length) of the top 5 individuals with the best growth were measured and the average values ​​were calculated. Culture was performed in the same manner as above, except that distilled water was used instead of the sample as a negative control (NC), and comparisons were made.

[0097] Figure 11 is a graph comparing (A) stem length and (B) root length of cucumber. In Figure 11, the vertical axis indicates stem length or root length (unit: cm), the values ​​indicate the average value ± standard deviation, the horizontal axis indicates each test area, and NC indicates the negative control (distilled water). There was a significant difference between treatment areas that did not share a common alphabet (Tukey-Kramer test, P<0.05).

[0098] Stem length was significantly longer than any of the control plots (Figure 11(A)) (Tukey-Kramer test, P<0.05). On the other hand, root length was the longest in NC (distilled water), but the difference was small and a significant difference was observed compared to EM1 (Figure 11(B)) (Tukey-Kramer test, P<0.05). These results suggest that the fermentation liquid of this embodiment has a growth-promoting effect.

[0099] Although the embodiments and examples of the present invention have been described in detail above with reference to the drawings, the specific configurations are not limited to these, and design changes that do not deviate from the gist of the present invention are included in the present invention.

[0100] For example, in the above examples, milk, fruit juice, vegetable juice, coffee, etc. were used as liquid raw materials, but any liquid raw material that can be fermented with lactic acid bacteria or yeast to produce amino acids or organic acids can be used as the liquid raw material in the present disclosure. More specifically, milk with various pasteurization histories such as LL milk and HTST milk, as well as unpasteurized raw milk, can be used as milk. In addition, animal milk other than cow's milk, skim milk, whey, and other liquid raw materials containing milk components can also be used.

[0101] Furthermore, for example, in the above examples, a microbial group was used that was subcultured from a commercially available seed culture, but this is not limited thereto, and any lactic acid bacteria and any yeast that can be fermented in food waste such as milk to produce amino acids and organic acids can be used as the microbial group in the present disclosure.

Claims

1. A method for producing a biostimulant, comprising inoculating a liquid raw material containing milk and / or dairy products with a group of microorganisms including lactic acid bacteria and yeast, and fermenting the liquid raw material at room temperature for 24 hours or more.

2. The method for producing a biostimulant according to claim 1, wherein the microbial community further comprises acetic acid bacteria.

3. The method for producing a biostimulant according to claim 1 or 2, wherein the liquid raw material further contains fruit juice and / or vegetable juice.

4. The method for producing a biostimulant according to claim 1 or 2, wherein the liquid raw material further contains coffee.

5. A biostimulant characterized by containing a microorganism group including lactic acid bacteria and yeast, 40 mg or more of amino acids per 100 g of biostimulant, and lactic acid, and having a pH of 5 or less and a Brix value of 7% or less.

6. The biostimulant according to claim 5, further comprising acetic acid bacteria and acetic acid, and wherein the total content of the lactic acid and the acetic acid is 0.5 mass% or more.

7. A method for cultivating plants, comprising producing a biostimulant by the method according to claim 1 or 2, and applying the obtained biostimulant, or the biostimulant according to claim 5 or 6, to plants or soil.

8. 8. The method for cultivating a plant according to claim 7, wherein the plant is one or more species selected from the family Rosaceae, Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Solanaceae, Amaranthaceae, and Nymphaeaceae.

9. A method for circulating waste milk, characterized in that it includes a step of producing a biostimulant from a liquid raw material containing waste milk by the method described in claim 1 or 2, and cultivating crops using the obtained biostimulant.

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