KR102125338B1 - Chestnut Inner Shell Fermented Broth and A Cultivating Method of Tomato Plant Using Thereof - Google Patents

Abstract

The present invention relates to a Castanea crenata powder fermented liquor, and a method for cultivating tomatoes using the same. The Castanea crenata powder fermented liquor according to the present invention acts as a fertilizer for tomato cultivation to provide help to improve the functionality of tomatoes. In addition, the Castanea crenata powder itself has no pathogen, etc., thereby having very excellent human body stability, and having excellent effects by containing a lot of excellent functional ingredients itself.

Description

Yulpi fermentation broth and tomato cultivation method using the same {Chestnut Inner Shell Fermented Broth and A Cultivating Method of Tomato Plant Using Thereof}

The present invention relates to a Bacillus subtilus yulpi fermentation broth and a tomato cultivation method using the same.

Tomato (tomato, Lycopersicon esculentum MILL) is a vegetable belonging to the family Asteraceae, and is known to be native to Peru in South America. Tomatoes are perennial in the tropics, but are grown as annual plants in temperate regions. In Korea, premature cultivation, which is usually planted and planted in February to March and transferred to the main field in early May, is common. When the temperature is low, cultivation and semi-cultivation using facilities such as houses and glass greenhouses are also common. In addition, during summer's high-temperature season, the field is cultivated in a cool area of 400 to 800m above sea level. Tomato varieties are classified according to maturity and hyperplasia, and can be roughly divided into four types: mature, immature, pine tomato, and cherry tomato.

These tomatoes have long been widely known as health foods. Tomato contains ingredients such as rich vitamins and minerals, organic acids (containing 0.5 to 1% citric acid), rutin (vitamin P), free amino acids, fiber, and lycopene. Fresh ones are raw It is consumed or eaten as a salad or sandwich, and processed into processed foods such as juice, puree, and ketchup.

In recent years, it has been popular as a well-being food containing a large amount of various components useful for the human body, and accordingly various research and development are in progress.

For example, Korea Patent Publication No. 10-2018-0013887 (tomato plants with improved disease resistance), Korea Patent Registration No. 10-1504368 (tomato plants with high level of resistance to Botrytis), Korea Patent Publication No. 10-2013-0058099 (a method for producing a tomato fruit having a high alkaloid content and food containing the same), or a tomato having improved properties, or a method for manufacturing the same is disclosed.

However, the above prior arts are not cultivated using natural materials, but only have problems that can cause contamination of soil reclaiming tomatoes by cultivating tomatoes through chemical fertilizers or by complex biological methods such as genetic manipulation. In addition, there is a problem that the growth of tomato may be deteriorated, and there is a problem that a large economic cost for genetic manipulation is required.

Under these backgrounds, the present inventors treated the Yulpi fermentation broth, which was cultured with natural ingredients, Yulpi, to tomato plants, thereby significantly increasing the content of the functional ingredients of tomatoes at low cost economically without genetic manipulation or chemical fertilizer treatment. Was prepared to complete the present invention.

An object of the present invention is to provide a fermentation broth fermented with Bacillus subtilis ( Bacillus subtilis ) strain.

The present invention comprises the steps of: (a) culturing Bacillus subtilis strain to prepare a Bacillus subtilus strain culture solution;

(b) preparing a mixed solution by mixing the strain culture with water and yulpi; And

(c) culturing the mixed solution to prepare a yulpi fermentation broth; an object thereof is to provide a method for manufacturing a yulpi fermentation broth.

An object of the present invention is to provide a liquid fertilizer composition for cultivation of tomatoes comprising a fermentation broth fermented with Bacillus subtilis strain.

An object of the present invention is to provide a tomato cultivation method comprising; fertilizing the fermented yulpi fermented with Bacillus subtilis strain.

In order to solve the above problems, the present invention provides an Yulpi fermentation broth fermented with Bacillus subtilis strain as one embodiment.

In the present invention, the Bacillus subtilis strain is usually commercially available or any strain can be used as long as it can be used as a Bacillus subtilis strain or a subspecies thereof.

For example, Bacillus subtilis used in the present invention can be used as long as it is a strain belonging to the same species (species) separable from Cheonggukjang. Bacillus subtilis used in the experiment in the present invention ( Bacillus subtilis ) may be an archaebacteria isolated from traditional Chungkukjang. The separation method of Bacillus subtilis is as follows, for example. First, the traditional Cheonggukjang sample is added to sterile distilled water to suspend it well, then left to stand, and then the supernatant is diluted to 10 ⁴ ~ 10 ⁷ and spread on a prepared agar plate for 24 hours at 37℃ to produce a large amount of viscous material. The strains to be isolated are individually isolated. Then, each of the isolated strains is plated on an agar plate for pure separation. Finally, each isolated strain is cultured in an agar plate for 24 hours to identify it using an Analytical Profile Index (API) kit and 16s rRNA sequencing. Through this method, in the present invention, NCBI Accession No. NR102783.2, No. A strain having a 16s rRNA sequence identified as 027552.1 was secured, and various other Bacillus subtilus strains were identified. Above NCBI Accession No. NR102783.2, No. Strains having a 16s rRNA sequence identified as 027552.1 are each Bacillus subtilis subsp. subtilis strain 168 and Bacillus subtilis strain DSM10 , respectively.

In the present invention, the chestnut inner shell is a material that is mostly discarded during the processing process of chestnuts as the inner skin of chestnuts, which is the fruit of the deciduous tree (Castanea crenata Sieb) belonging to the family Fagaceae. Yulpi contains a large amount of tannic acid, gallic acid, catechin, etc., and the biological components are oxidized by reactive free radicals or free radical peroxide generated inside the body from harmful environments. It can be used as a natural antioxidant that inhibits denaturation. In the present invention, yulpi may be dried and ground chestnut skin, for example, yulpi powder.

Fermentation in the present invention means inoculating yeast such as Bacillus subtilis and culturing under optimal culture conditions thereof.

The Yulpi fermentation broth fermented with the Bacillus subtilis strain according to the present invention itself contains a large amount of polyphenol compounds and tannins, and thus can have excellent antioxidant effects. In addition, when used as a liquid fertilizer composition, it not only enhances the content of functional ingredients in plants, preferably tomatoes, but also helps the plants to grow quickly and healthily.

The present invention comprises the steps of: (a) culturing Bacillus subtilis strain to prepare a Bacillus subtilus strain culture solution;

(b) preparing a mixed solution by mixing the strain culture with water and yulpi; And

(c) culturing the mixed solution to prepare a yulpi fermentation broth; provides a method for preparing a yulpi fermentation broth.

The method for preparing the yulpi fermentation broth according to the present invention includes (a) culturing a Bacillus subtilis strain to prepare a Bacillus subtilus strain culture.

Yulpi fermentation broth in the present invention may be used as a liquid fertilizer composition may require mass production. Accordingly, it is preferable to prepare for mass mixing with Yulpi by mass-culturing the Bacillus subtilis strain itself.

The cultivation of Bacillus subtilis strains in the present invention is a process of inoculating the above strains in a normal liquid medium, followed by cultivation, for example, after inoculating the strains at 26-32°C at 140-180 rpm. It may be cultured in a controlled shaker.

The liquid medium is a medium commonly used in the art. For example, 0.1 to 0.5% by weight of yeast extract, 0.1 to 0.5% by weight of malt extract, 0.1 to 1% by weight of peptone, 0.5 to 2% by weight of dextrose , 96-99.2% by weight of distilled water is mixed and the pH is adjusted to 4~6.5, then sterilized at 115-125°C for 10-20 minutes and cooled to 25-35°C.

In the case of such culture, the culture level can be determined by measuring the absorbance density (OD), which can measure the culture degree of a common Bacillus subtilis strain. For example, the OD value of 0.3, 0.4, 0.5, 0.6 or more can be used by stopping the culture and proceeding to the next step.

The method for preparing the yulpi fermentation broth according to the present invention includes (b) preparing the mixed solution by mixing the strain culture with water and yulpi. The purpose of this step is to provide a mixed solution of conditions suitable for fermentation by mixing the Bacillus subtilus strain culture solution of step (a) with water and Yulpi.

Preferably, the Bacillus subtilus strain culture solution and water are preferably mixed at a volume ratio of 1:50 to 1:150, preferably at a volume ratio of 1:80 to 1:120, more preferably at a volume ratio of about 1:100. .

The mixture of yulpi, water and Bacillus subtilus strain culture is mixed in a weight ratio of 1:6 to 1:15, preferably in a weight ratio of 1:8 to 1:12, more preferably in a weight ratio of 1:9 to 1:11. It is desirable to do.

In the present invention, the Bacillus subtilus strain culture medium is used in an amount of 0.6 to 2.0, preferably 0.8 to 1.25, more preferably about 1.0 part by weight based on 100 parts by weight of water; And Yulpi 6 to 20, preferably 8 to 12.5, more preferably about 10 parts by weight.

According to one embodiment of the present invention, a mixed solution was prepared by mixing 20 L of Bacillus subtilus strain culture, 2000 L of water, and 200 Kg Yulpi.

It is preferable that the mixed solution is well mixed with a mixer or the like, so that the strain culture solution and water can be well mixed.

The method for preparing the yulpi fermentation broth according to the present invention includes a method for preparing the yulpi fermentation broth comprising the step of (c) culturing the mixed solution to prepare the yulpi fermentation broth.

Preparation of yulpi fermentation broth in the present invention includes fermenting the mixed solution under normal fermentation conditions. For example, 20 to 30 ℃, preferably 22 to 28 ℃, more preferably 4 to 14 days at room temperature, preferably 5 to 12 days, more preferably may be to ferment by culturing for 7 to 10 days. For such fermentation conditions, it may be adjusted to an optimal fermentation condition suitable according to the Bacillus subtilus strain used.

By going through this fermentation step, the fermentation broth has the advantage that it can contain more functional components, such as polyphenols and tannins.

The present invention provides a liquid fertilizer composition for cultivation of tomatoes comprising a fermentation broth fermented with Bacillus subtilis strain.

The liquid cultivation composition for tomato cultivation according to the present invention can be processed on tomatoes to significantly increase the content of functional ingredients such as beta-carotene, flavonoids, polyphenols, and tannins of tomatoes. In addition, since it does not use fertilizers based on chemical components, there is no risk of soil contamination, and it is safe from problems caused by plant genetic manipulation, etc. It is advantageous in that it can produce high-functional tomatoes at low cost by using ulpi, which is usually discarded. Have

In the present invention, the liquid fertilizer refers to the fertilizer in a liquid state, and is a fertilizer used in a liquid state, unlike a fertilizer in a solid state, and the original agent may be liquid or powder. Powdered originals can be used by dissolving them in water, and liquid originals can be diluted with water to an appropriate concentration. These liquid fertilizers can be divided into soil, hydroponic, and foliar spraying depending on their use.

In the liquid cultivation composition for tomato cultivation, the Yulpi fermentation broth fermented with the Bacillus subtilis strain may be prepared by the following steps:

(a) culturing a Bacillus subtilis strain to prepare a Bacillus subtilus strain culture solution;

(b) preparing a mixed solution by mixing the strain culture with water and yulpi; And

(c) culturing the mixed solution to prepare a yulpi fermentation broth.

The liquid fertilizer composition according to the present invention may be preferably performed according to the irrigation treatment as a method of spraying a drug by digging a hole in soil or a tree and injecting a chemical liquid.

The present invention provides a tomato cultivation method comprising; fertilizing the fermented yulpi fermentation with Bacillus subtilis strain.

The fermentation broth fermented with the Bacillus subtilis strain comprises: (a) culturing a Bacillus subtilis strain to prepare a Bacillus subtilus strain culture;

(b) preparing a mixed solution by mixing the strain culture with water and yulpi; And

(c) may be prepared by culturing the mixed solution to prepare a yulpi fermentation broth.

In the present invention, the fermentation broth fermented with Bacillus subtilis strain for fertilization may be further diluted with water. For example, the yulpi fermentation broth and water are preferably fertilized by mixing in a volume ratio of 1:50 to 1:150, preferably a volume ratio of 1:80 to 1:120, and more preferably a volume ratio of about 1:100.

In the case of such fertilization, it can be used without limitation as soil treatment, hydroponic treatment, foliar spraying, etc., but it is preferable to treat the tomato through irrigation treatment.

Tomatoes subject to such fertilization can be used without limitation from the initial stage to the late stage depending on the stage of plant development.

In addition, the fertilization can be appropriately applied without limitation once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, or more, preferably once a week to 3 times a week It is preferable to treat it once

By treating the fermentation broth fermented with Bacillus subtilis strain according to the present invention, the content of functional components such as beta-carotene, flavonoids, polyphenols, and tannins can be significantly increased. In addition, since it does not use fertilizers based on chemical components, there is no risk of soil contamination, and it is safe from problems caused by plant genetic manipulation, etc. It is advantageous in that it can produce high-functional tomatoes at low cost by using ulpi, which is usually discarded. Have

Tomato cultivation method according to the present invention more specifically,

(a) culturing a Bacillus subtilis strain to prepare a Bacillus subtilus strain culture solution;

(b) preparing a mixed solution by mixing the strain culture with water and yulpi; And

(c) culturing the mixed solution to prepare a Yulpi fermentation broth;

(d) mixing the yulpi fermentation broth with water to prepare a diluted yulpi fermentation broth; And

(e) fertilizing the diluted yulpi fermentation broth with tomato.

Yulpi powder fermentation broth according to the present invention acts as a fertilizer in tomato cultivation, greatly assisting in improving the functionality of tomatoes, and Yulpi powder fermentation broth itself does not have pathogens, so it has very good human stability and contains many excellent functional ingredients by itself It has an excellent effect in that it does.

Figure 1 shows the experimental results for measuring the number of live bacteria in the fermentation broth of Yulpi powder according to the present invention.
Figure 2 shows the results confirming that there is no pathogenic E. coli ( Escherichia coli O157:H7) in the Yulpi powder fermentation broth according to the present invention.
Figure 3 shows the results confirming that there is no pathogenic Salmonella ( Salmonella spp.) in the Yulpi powder fermentation broth according to the present invention.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

<Example 1> Collection of Bacillus strain

Strains were separately cultured in Cheonggukjang (domestic production). Gene base sequences were obtained from colonies of isolated and cultured strains to analyze 16s rRNA sequences and compare homology with known strains. As a result, all strains separated by Cheonggukjang were identified as Bacillus subtilis strains. Exemplarily isolated Bacillus subtilus strain is NCBI Accession No. 16srRNA sequence. NR102783.2, No. It was confirmed to have the sequence of 027552.1.

<Example 2> Preparation of Yulpi fermentation broth

The Bacillus subtilis strain isolated in Example 1 was cultured under a temperature condition of 30° C. to produce a large amount of Bacillus subtilus strains.

Under normal absorbance density measurement conditions, 20 L of the stomach culture medium was separately isolated after the mass density of the Bacillus subtilus strain culture medium was confirmed to be 0.4 or higher.

Then, the 20 L Bacillus subtilus strain culture solution, water 2000 L, Yulpi powder (source: Made in China) 200 Kg were mixed and cultured at room temperature for 7 to 10 days.

Through the above culture, a fermented broth of yulpi was prepared.

<Example 3> Treatment of yulpi flour fermentation broth in tomato culture

The yulpi powder fermentation broth isolated in Example 2 was treated to a tomato plant to perform tomato culture.

Specifically, the yulpi flour fermentation broth prepared in Example 2 was diluted with 20 L of yulpi flour fermentation broth in 2000 L of water based on 200 pyeong of farmland and irrigated to the root portion. The irrigation treatment was performed twice a week (three times from June in the high temperature period), and the components of the untreated and treated tomatoes were analyzed after the irrigation treatment.

<Example 4> Tomato component analysis

Analysis of the functional components of tomatoes cultured according to the cultivation process of Example 3 was analyzed by requesting an analysis institution Natural Choice.

The analysis was performed on the total polyphenol content, tannin content, and antioxidant scavenging ability {DPPH radical scavenging ability} for the mature tomatoes, and the results are shown in Tables 1 and 2 below.

Table 1 below shows the results of the yulpi powder fermentation broth (10 times a week), and Table 2 below shows the results of the yulpi powder fermentation broth (20 times a week twice).

[Table 1]

[Table 2]

Table 1 shows the results of processing the fermented yulpi powder twice a week from approximately 5 weeks before the ripe tomato comes out, and Table 2 shows the processing of the yulpi powder fermented liquor twice a week 20 times from about 10 weeks before the ripe tomatoes appear. One result is shown.

As can be seen from the results of the experiment, the treatment of Yulpi flour fermentation broth significantly increased the total polyphenol content and tannin content as functional components, and also significantly increased the antioxidant scavenging ability of tomatoes. It was confirmed that these results were larger in the experimental group treated with the long-term fermentation broth.

From the above results, it was confirmed that the fermented yulpi powder fermented with Bacillus subtilus according to the present invention greatly helps to improve the functionality of tomatoes in tomato cultivation.

<Example 5> Analysis of Yulpi powder fermentation broth

(One) Identification of effective microorganisms in Yulpi

The presence of Bacillus subtilus was confirmed once again as an effective microorganism in Yulpi through the Eco-friendly Agricultural Product Stability Center.

First, in order to measure the number of live cells, the number of live cells was measured in three iterations based on the dilution plate method. After determining and counting colonies of the same type as effective microorganism colonies as effective microorganisms, the average value was calculated to measure CFU per 1 ml (g).

The results are shown in FIG. 1. Figure 1 shows the results of performing three measurements of viable cell count, and confirmed that the effective microorganism, Bacillus subtilus, was 5.3 X 10 ⁸ CFU/ml (g).

In addition, similar to Example 1, gene sequence homology search was performed. Specifically, after diluting the Yulpi powder fermentation broth of Example 2 to 1:10 ⁶ , inoculated with 100 μl of DifCo™ LB Agar, Miller (Becton, Dickison and Company) medium, cultured at 30° C. for 24 hours, and dominated microorganisms. Colonies were selected.

Among the selected colonies, colonies of the same type as the effective microorganisms were separated and analyzed by gene sequencing (16s rRNA) to compare homology.

As a result, the Bacillus subtilus strain isolated from the Yulpi powder fermentation broth subjected to fermentation culture was NCBI Accession No. 16s rRNA sequence. NR102783.2, No. It was confirmed to have the same sequence as 027552.1.

(2) Hospital Microbiology Test

Through the Eco-Friendly Agricultural Product Stability Center, the presence of pathogenic microorganisms in Yulpi liquid was confirmed to confirm the stability of the Yulpi powder fermentation broth prepared in the present invention.

Specifically, 25 ml (g) of the yulpi flour fermentation broth was added and added to 225 ml of each growth medium, followed by incubation at a temperature of 35 to 37°C for 24 hours. Then, the pathogenic E. coli ( Escherichia coli O157: H7) and pathogenic Salmonella ( Salmonella spp.) were tested in three replicates.

The results are shown in FIGS. 2 and 3.

2 to 3, colorless colonies were not found in pathogenic E. coli ( Escherichia coli O157:H7), and red colonies having a black ring were not found in pathogenic Salmonella spp.

From the above results, it was confirmed that there was no pathogenic microorganism in the yulpi flour fermentation broth according to the present invention, and thus it was also confirmed that it has stability suitable for treatment with tomatoes.

(3) Functional component analysis of Yulpi powder fermentation broth

Similar to Example 4, Yulpi powder fermentation broth was analyzed for total polyphenol content, tannin content and DPPH radical scavenging ability.

The total polyphenol content was confirmed by the following method. After adding 50 µl of folin-ciocalteu reagent to 0.1 ml of the sample, mixing and reacting for 4 minutes, 1.5 ml of a saturated solution of 20% sodium carbonate anhydrous was added. After the reaction for 2 minutes, the absorbance was measured at 760 nm using a microplate reader (VERSAmax, Molecular Device, CA, USA). The total polyphenol content was calculated from the standard curve prepared using chlorogenic acid.

In addition, the tannin content was confirmed by the following method. After adding 1 ml of 95% ethanol and 5 ml of distilled water to 1 ml of sample, add 1 ml of 5% sodium carbonate anhydrous saturated solution and 0.5 ml of folin-ciocalteu reagent, mix and react for 1 hour. VERSAmax, Molecular Device, CA, USA) was measured at 725 nm. The tannin content was calculated from the standard curve prepared using tannin.

DPPH radical scavenging ability was performed by the following method. Absorbance was measured at 515 nm using a microplate reader (VERSAmax, Molecular Device, CA, USA) by adding 160 µl of DPPH solution dissolved in methanol to a concentration of 0.4 mM and 40 µl of the sample for 30 minutes in the dark. Ascorbic acid was used as a positive control.

The above results are summarized in Table 3 and Table 4.

Yulpi fermentation broth Total polyphenol content (mg/g) 0.95 ± 0.01 Tannin content (mg/g) 0.39 ± 0.01

*Values are mean ± SD

4-fold dilution 2-fold dilution Undiluted 55.80 ± 2.00 74.00 ± 1.18 85.86 ± 0.65

As shown in Table 3, as a result of the analysis, large amounts of polyphenols and tannins were found in the Yulpi powder fermentation broth. In addition, as shown in Table 4, Yulpi powder fermentation broth itself also showed excellent anti-oxidative ability, and in the case of the undiluted solution, it showed better activity than ascorbic acid (25 μg/ml treatment).

From the above results, it was confirmed that yulpi powder fermentation broth according to the present invention can be used as an excellent nutritional component for tomato cultivation. In addition, it was confirmed that the fermentation broth of the stomach Yulpi powder itself does not have pathogens, and thus has excellent human stability, and also contains many excellent functional ingredients.

Claims (5)

(a) culturing a Bacillus subtilis strain to prepare a Bacillus subtilus strain culture solution;
(b) preparing the mixed solution by mixing the Bacillus subtilus strain culture solution with water and Yulpi, using the Bacillus subtilus strain culture solution at 0.6 to 2.0 parts by weight and Yulpi 6 to 20 parts by weight with respect to 100 parts by weight of water. ;
(c) culturing the mixed solution at 20 to 30° C. for 4 to 14 days to prepare a Yulpi fermentation broth;
(d) preparing a diluted yulpi fermentation broth by mixing the yulpi fermentation broth and water in a volume ratio of 1:50 to 1:150; And
(e) fertilizing the diluted Yulpi fermentation broth with tomato; tomato cultivation method comprising a. delete delete The method according to claim 1, wherein the fertilization is performed once to three times a week. delete

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