JPS6114119B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for cultivating vegetable vegetables with enhanced flavor, and particularly to a method for cultivating vegetable vegetables characterized by obtaining vegetable vegetables with enhanced amino acid content or lycopene content. Recently, as dietary habits have improved, there has been a high demand for vegetables that are well-colored, visually appealing, and have a good flavor.The same applies to vegetables that can be used as raw materials for processing, such as juices, ketchups, and piures made from tomatoes. In order to improve the quality of tomatoes, there is a demand for improved tomato quality, especially tomatoes for processing that are rich in components such as amino acids and lycopene. The biggest influence on the flavor of these vegetables is the level of soil moisture during the ripening stage.
Also, the temperature is high or low, and the amount of sunshine hours. Therefore,
Conventionally, methods used to improve the quality of vegetables, such as improved agricultural techniques through cultivating methods and the use of fertilizers, pesticides, etc., are difficult to expect to have a certain effect depending on the climate and climate conditions. On the other hand, there are currently limits to the breeding of vegetables themselves, so improving the quality of vegetables, especially stably increasing the amount of amino acids and lycopene, is a technical challenge in the industry. Therefore, as a result of various studies, the present inventors found that the genus Microbacterium, Aslobacter, Corynebacterium, and Brevibacterium belong to the genus Microbacterium, Aslobacter, and Brevibacterium.
Cyclic-3′,5′-adenylic acid (hereinafter referred to as
By cultivating bacteria with the production ability (called CAMP) in a medium and adding the bacterial cell content to the soil and cultivating vegetables in the soil, the quality is significantly improved, and the amount of amino acids and lycopene is unprecedented. The inventors have discovered that it is possible to obtain vegetable vegetables with enhanced flavor and enhanced flavor, and have completed the present invention based on this knowledge. The present invention will be explained in detail below. First, the microorganisms used in the present invention are those belonging to the genus Microbacterium, Aslobacter, Corynebacterium, and Brevibacterium, and the microorganisms include carbon sources, nitrogen sources, inorganic salts, or CAMP as necessary. Any bacteria that produces CAMP when cultured in a medium containing an appropriate precursor can be used. Specific examples of these bacteria include Microbacterium No. 205 (FERM-P No.
106, ATCC21376), Microbacterium No.MT
-3 (FERM-P No.787, ATCC21981), Microbacterium No.205-AgAzMsTh-2 (FERM-
PNo.3237), Microbacterium No.205-M-32
(FERM-PNo.1559, ATCC31001), Microbacterium No.205-MP-197 (FERM-PNo.2499)
As bacteria belonging to the genus Aslobacter, for example, Aslobacter 11 (FERM-P No. 207,
ATCC21375), Aslobacter 11-211 (FERM
- P No. 1556, ATCC 21978), and other bacteria belonging to the genus Corynebacterium, such as Corynebacterium muriseputecum No. 7 (FERM-P No. 206,
ATCC21374), Corynebacterium muriceptecum No.7-10 (FERM-P No.1555,
Examples of bacteria belonging to the genus Brevibacterium include Brevibacterium liquefaciens (ATCC 14929). Next, a bacterium capable of producing CAMP is inoculated into a CAMP production medium that can be used by the bacterium.
Culture at ~40°C for 10-80 hours. Carbon sources for the culture medium include, for example, carbohydrate compounds such as glucose, starch hydrolysates, and glycerin, and nitrogen sources include, for example, ammonium sulfate, ammonium chloride, urea, various amino acids, amino polymer hydrolysates, and corn staple liquor. and biological extracts such as yeast extract. As the inorganic phosphate, for example, monopotassium phosphate or soda, dipotassium phosphate or soda, ammonium phosphate, etc. are used. Inorganic salts other than inorganic phosphates that may be added as necessary include:
Examples include magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, zinc sulfate, cobalt sulfate, manganese sulfate, manganese chloride, and the like. Depending on the CAMP production mode of the strain used, CAMP precursors such as adenine, hypoxanthine, succinyladenine, 5-amino-4-imidazolecarboxamide, 7-amino-pyrazolo-(4,
3-d)-pyrimidine, pyrazolo-(4,3-d)
-pyrimidine, 4-amino-pyrrolo-(2,3-
d)-pyrimidine, pyrrolo-(2,3-d)-pyrimidine, or riboside, ribonucleotide, deoxyriboside, deoxyribonucleotide, etc. using these as a base are used. Culture can be carried out using an appropriate culture method such as shaking culture, agitation culture, or aeration culture.
When the production of CAMP reaches the maximum, the culture is stopped and a culture containing bacterial cells is obtained. The bacterial cell-containing material used in the method of the present invention is the above-mentioned
A cell-containing culture of CAMP-producing bacteria, an isolated cell culture obtained by centrifuging, filtration, etc. of the culture using a conventional method, or a suitable drying method such as a heating drying method (hot air drying method, rotating drum drying method, etc.) (drying method, etc.), spray drying method, etc.Also, the bacterial cells can be crushed and used as appropriate, but in addition, excipients,
It can be mixed with fertilizers, etc., and sometimes used with herbicides and nematicides. Next, vegetables used in the method of the present invention include tomatoes, strawberries, cucumbers, watermelons, eggplants, melons,
Examples include gourd. The amount of the bacterial cell-containing material used in the method of the present invention is preferably 1 to 1000 kg/10 are as dry bacterial cell material, and the optimum amount to be used is 50 to 500 kg/10 are. The period of use is generally 2 to 3 months before planting and after planting, depending on the form of use, for example, if it is in a fixed form, it is mixed into the vegetable cultivation soil or applied to the soil surface, and if it is in a solution form, it is removed from the soil. do. The vegetable cultivation soil is not particularly limited, and for example, volcanic ash soil can be used as long as it does not hinder the growth of vegetables. In this way, the amino acid content and lycopene content of vegetables grown by the method of the present invention are extremely high compared to conventional vegetables, and fruits with high commercial value and good flavor can be obtained. When used in this way, very high quality products can be obtained, which has great industrial merits. Next, the present invention will be specifically explained with reference to Examples. Example 1 Glucose 2%, polypeptone 1%, yeast extract 0.5%, NaCl 0.3%, soybean oil 0.2%, silicone
KM70 (antifoaming agent) (product name: Shin-Etsu Chemical) 0.14%
Dispense 30 ml of seed medium (adjusted to pH 7.0 with 3N NaOH before sterilization) into 500 ml Sakaguchi flasks, heat sterilize at 115°C for 15 minutes, and then microbacterium No. 205 on the bouillon slant. (FERM−
PNo. 106, ATCC 21376) was inoculated into the medium and cultured with shaking at 140 rpm for 16 hours at 30°C.
Next, ZnSO ₄ 7H ₂ O 0.01%, KH ₂ PO ₄ 2%,
MgSO ₄ 7H ₂ O 1%, FeSO ₄ 7H ₂ O 0.01%, polypeptone 1%, yeast extract 0.5%, 5'-inosinic acid 0.7%, glucose 10%, soybean oil 0.2%, silicone KM70 (product name: Of the fermentation medium consisting of 0.14% (manufactured by Shin-Etsu Chemical Co., Ltd.) (adjusted to pH 7.5 with KOH before sterilization), glucose and MgSO ₄ 7H ₂ O were sterilized separately from other components at 120°C for 15 minutes. In addition to the other ingredients that were sterilized for 15 minutes at 120°C in a 30-volume jar fermentor (manufactured by Marubishi), 0.8 of the above seed culture solution was inoculated and cultured at 28°C for 72 hours.
After the cultivation was completed, the culture solution containing the bacterial cells was centrifuged using a shear press type centrifuge, and the CAMP in the supernatant was measured and found to be 7.8 mg/ml. Next, the obtained isolated bacterial cells were dried with hot air at 70°C to obtain 3.8 kg of dried bacterial cells with a moisture content of 13%, N8.2%, P2.1%, and CAMP of 3 mg/g (dried bacterial cells). A total of 3,800 kg of bacterial cells were obtained by culturing twice. The dried bacterial cells were first added to the soil before planting tomato seedlings.
Use the amounts shown in the table with fertilizer, and apply so that the total amount is 9.6 Kg/10 are of nitrogen, 27 Kg/10 are of phosphoric acid (as P ₂ O ₅ ), and 8.4 Kg/10 are of potassium (as K ₂ O). did. For comparison,
Soil (a) in which only fertilizer was applied to the above composition without using dried Microbacterium No. 205 cells, and the CAMP non-producing bacterium Brevibacterium protoformiae.
Soil (b) in which 300 kg of dried bacterial cells obtained by culturing Protophormiae (IFO12128) in the same manner as Microbacterium No. 205 were used together with fertilizer to have the above composition.
was used. Seeds of tomato (variety K705) were sown on April 5th, and the resulting seedlings were planted on May 26th in each of the above cultivation soils. Tomato fruits were collected from each control plot and treatment plot on the days shown in Table 1, and the lycopene content and amino acid content of the collected tomato fruits were measured. The amount of lycopene was measured using the method of Miki et al. (Miki, Akatsu: Journal of the Japan Food Industry Association, Vol. 17, No. 5,
175-177, 1970), as follows. After crushing 1 kg of tomato fruit with a mixer, puree it with a sieve, thoroughly deaerate the air in the tomato pulp using a satsuka, take exactly 1 g of tomato pulp into a 50 c.c. beaker, add about 15 ml of methanol, and soak for 10 minutes. rear,
Filter through a 3-G-3 glass filter, and thoroughly wash the residue with methanol until the color of the immersion liquid disappears. Next, lycopene in the methanol washing residue was extracted with benzene, and the benzene extract was made up to 50 ml with benzene, and the color was compared at 480 nm, and the amount of lycopene was calculated using the following formula. Alternatively, the amount of amino acids can be determined by crushing 1 kg of the collected tomatoes with a mixer, centrifuging at 10,000 x g for 30 minutes,
The supernatant was filtered through No. 5C filter paper, and the resulting filtrate was subjected to amino acid measurements. Amino acid analysis is performed using an amino acid analyzer [Hitachi KLA-
Type 5, resin: Durrum DC-6A, butter: pico buffer (manufactured by Durrm Chemical Corporation, US
A.) (Reiland, J., JRBenson, Durrum Resin
Report No. 7, 1976), coloration: ninhydrin method]
Each amino acid was analyzed using the method, the total of each amino acid was taken as the amino acid amount, and the average value of the amino acid amount in the tomatoes collected on August 19th and August 28th was calculated as the amino acid amount in the tomatoes collected in soil (a). The results are shown in Table 1, expressed as % of the average amount. Below, the amount of lycopene and the amount of amino acids were measured in the same manner.

[Table] Example 2 Corynebacterium muriceptecum No.7
(FERM-P No. 206, ATCC 21374) and Aslobacter 11 (FERM-P No. 207, ATCC 21375) were cultured in the same manner as in Example 1, and the CAMP in the supernatant was measured to be 4.8 mg/ml and 3.9 mg, respectively. /ml. Next, the obtained isolated bacterial cells were dried with hot air at 50°C to obtain dried bacterial cells having the compositions shown in Table 2. The dried bacterial cells
300 kg/10 are was used together with fertilizer on the soil before planting tomato seedlings, and the soil was fertilized so that the overall amount of nitrogen, phosphoric acid, and potassium was the same as in Example 1. The control was the same as in Example 1. Tomato seedlings (varieties) were planted in each cultivation soil on May 26th.
K705) was planted, mature tomato fruits were collected from each control plot and treated plot, and the amount of amino acids was measured.The results were expressed as % of the amount of amino acids in tomatoes in soil a, and are shown in Table 2.

[Table] Example 3 The strains shown in Table 3 were cultured as in Example 1, and dried cells were obtained in the same manner as in Example 1. 200Kg/10A of each dry bacterial cell was used together with fertilizer in the soil before planting strawberry seedlings, and the total content was 7.5Kg/10A of nitrogen, 20Kg/10A of phosphoric acid (as P ₂ O ₅ ), and 8.5Kg/10A of potassium. (as K ₂ O). As a control, soil to which only fertilizer was applied and 200 kg of dried bacterial cells of Brevibacterium protoformiae (IFO12128)
Soil was used in which 10 are was used together with fertilizer to have the above composition. Meanwhile, strawberry seedlings (variety: Shure crop) were grown on October 1st.
Strawberry fruits were planted in the above cultivation soil on June 1st, and strawberry fruits were collected twice on June 1st and June 7th as they reached full ripeness the following year. Crush 500g of collected strawberries with a mixer,
Centrifuge at 10,000×g for 30 minutes and remove the supernatant No.5C.
Amino acid measurements were performed on the filtrate obtained by filtration through a filter paper. The average value of the amino acid content of the two times was expressed as a percentage of the average value of the amino acid content in strawberries grown in soil to which only fertilizer was applied, and is shown in Table 3.

[Table] Example 4 Glucose 2%, polypeptone 1%, yeast extract 0.5%, FeSO ₄ 5H ₂ O 0.05%, NaCl 0.3%, soybean oil 0.2%, silicone KM70 (antifoaming agent) (product name: Manufactured by Shin-Etsu Chemical Co., Ltd.) 0.14% (PH with 3N KOH before sterilization)
7.0) was sterilized in the same manner as in Example 1, one platinum loop of bacterial cells of each strain shown in Table 4 on a bouillon slant was inoculated into the medium, and the culture was incubated at 30°C for 16
Cultured with shaking for hours. Next, from the medium composition of Example 1
The fermentation medium except for 0.7% of 5'-inosinic acid was sterilized in the same manner as in Example 1, and then 0.8% of each of the above seed cultures of the strains shown in Table 4 was inoculated and cultured at 28°C for 72 hours. After culturing, the culture solution containing each bacterial cell is centrifuged using a shear press centrifuge, and the CAMP in the supernatant is
When measured, the results were as shown in Table 4. Next, each isolated bacterial cell obtained (300 dried bacterial cells)
Kg) was suspended in 5,000 ml of water and applied to 10 are of cultivation soil before planting tomato seedlings, resulting in a total nitrogen content of 9.6
Kg/10 are phosphoric acid 27 Kg/10 are (as P ₂ O ₅ ) and potassium 8.4 Kg/10 are (as K ₂ O) were fertilized. Tomato seedlings (variety K705) were planted in each cultivation soil, ripe tomatoes were collected, and the amount of amino acids was measured in the same manner as in Example 1. The results were calculated as Kl for amino acids in tomatoes grown in soil to which only fertilizer was applied. and shown in Table 4. As a control, soil to which only fertilizer was applied and isolated bacterial cells of Brevibacterium protoformiae (IFO12128) (300 kg in terms of dry bacterial cells)
Amino acids were measured in the same manner using tomato fruits grown in soil fertilized to have the above-mentioned composition as a whole. The dry bacterial cell components of each isolated bacterial cell are also shown in Table 4.

[Table] Example 5 Meat extract 1%, polypeptone 1%, yeast extract
0.5%, NaCl0.3%, _{MnCl2.4H2O0.0005} % _,
FeSO ₄ 5H ₂ O 0.01%, soybean oil 0.2%, silicone
KM70 (product name: Shin-Etsu Chemical) 0.14% (before sterilization)
After sterilizing a seed medium (adjusted to pH 7.0 with 3N NaOH) in the same manner as in Example 1, one platinum loopful of each strain shown in Table 5 was inoculated onto the bouillon slant and incubated at 30°C for 16 hours. , and cultured with shaking at 140 rpm.
Next, ZnSO ₄ 7H ₂ O 0.01%, KH ₂ PO ₄ 2%,
_MgSO4・_7H2O1 %, _FeSO4・_7H2O0.025 %, _Fe2
(SO ₄ ) ₃・XH ₂ O0.025%, MnCl ₂・4H ₂ O0.0001
%, glucose 10%, soybean oil 0.2%, silicone
KM70 (antifoaming agent) (product name: Shin-Etsu Chemical) 0.14%
After sterilizing the fermentation medium (adjusted to pH 7.5 with 3N KOH before sterilization) in the same manner as in Example 1, 0.8% of each of the above seed cultures of the strains shown in Table 5 was inoculated.
Cultured at 28°C for 72 hours. After the cultivation was completed, the culture solution containing each bacterial cell was centrifuged using a shear press type centrifuge, and the isolated bacterial cells obtained were dried with hot air at 70°C to obtain dried bacterial cells. 200 kg/10 are of the dried bacterial cells are used together with fertilizer in the soil before planting tomato seedlings, and the total nitrogen content is
9.6Kg/10ares, phosphoric acid 27Kg/10ares (P ₂ O ₅
) and potassium at 8.4 kg/10 are (as K ₂ O), and tomato seedlings (variety K705) were planted in the cultivation soil. As a control, 200 kg/10 are of dried bacterial cells obtained by culturing Brevibacterium protoformiae (IFO12128) in the same manner were applied together with fertilizer. Collect mature tomato fruits from each treatment plot and control plot, measure the amount of amino acids, and display the results as a percentage of the amount of amino acids in tomatoes in the control plot,
It is shown in Table 5.

[Table] Example 6 Glucol 2%, polypeptone 1%, yeast extract 0.5%, FeSO _{4.5H 2} O 0.01%, Fe ₂ (SO ₄ ) ₃ .
_XH2O0.01 %, soybean oil 0.2%, silicone KM70
(Product name: Shin-Etsu Chemical) 0.14% (3N before sterilization)
After sterilizing the seed medium consisting of Brevibacterium liquefaciens (adjusted to pH 7.0 with NaOH) in the same manner as in Example 1,
Iiquefaciens) (ATCC14929) was inoculated with one platinum loop and cultured at 30°C for 16 hours with shaking at 140 rpm.
Then KH ₂ PO ₄ 1%, K ₂ HPO ₄ 1%, (NH ₄ ) ₂ SO ₄ 2%,
_MgSO4・_7H2O0.05 %, _FeSO4・_5H2O0.005 %,
Glucose 10%, Soybean oil 0.2%, Silicone KM70
A fermentation medium consisting of 0.14% (trade name: manufactured by Shin-Etsu Chemical) and tap water (adjusted to pH 7.5 with 3N KOH before sterilization) was sterilized in the same manner as in Example 1, and a 200 volume jar fermenter (manufactured by Marubishi) was sterilized. ) was inoculated with 0.8% seed culture solution and cultured in the same manner as in Example 1.
The culture solution was subjected to shear press centrifugation, and CAMP in the supernatant was measured and found to be 0.8 mg/ml. Next, the isolated bacterial cells obtained were dried with hot air at 50°C.
1000 culture solutions yielded 30Kg of dried bacterial cells. 250 kg of the dried bacterial cells were used together with fertilizer on the soil before planting tomato seedlings, and applied so that the overall amount of nitrogen, phosphoric acid, and potassium was the same as in Example 1, and the tomato seedlings (variety K705) were planted with the above tomato seedlings. It was planted in cultivation soil.
As a control, 250 kg of dried bacterial cells obtained by culturing Brevibacterium protoformiae (IFO12128) in the same manner as above was applied together with fertilizer. When mature tomato fruits were collected from each treatment plot and the control plot and the amount of amino acids was measured, it was found that the amount of amino acids in tomatoes obtained from the cultivation soil using Brevibacterium liquefaciens (ATCC14929) was higher than that of tomatoes in the control plot. It was 125% of the amount of amino acids in it.

Claims (1)

[Scope of Claims] 1. A method for cultivating vegetables, which comprises applying to soil a substance containing cells of cyclic-3',5'-adenylic acid producing bacteria. 2. The method for cultivating vegetables according to claim 1, wherein the cyclic-3',5'-adenylic acid-producing bacteria belong to the genus Microbacterium, Aslobacter, Corynebacterium, and Brevibacterium. . 3. The method for cultivating vegetables according to claim 1, wherein the bacterial cell-containing material is bacterial cells obtained by culturing in a cyclic-3',5'-adenylic acid production medium.