KR20140000790A - Method for cultivating tomato containing high content of lycopene - Google Patents

Abstract

The present invention relates to a method for cultivating tomatoes with high content lycopene and, more specifically, to a method for cultivating tomatoes containing high content lycopene, which is an active ingredient of the tomatoes and which has anti-oxidative and anti-cancer activities. A feature of the method for cultivating tomatoes with high content lycopene is that Flavobacterium sp. or Chryseobacterium indologenes is treated to the soil at a planting step. According to the present invention, the lycopene content of the tomatoes is increased by treating Flavobacterium sp. or Chryseobacterium indologenes to the soil at the planting step. Therefore, the cultivating method has an advantage of mass producing the lycopene having functions in controlling or inactivating free oxygen radicals, which damages a human body.

Description

How to grow lycopene-containing tomatoes {Method for Cultivating Tomato Containing High Content of Lycopene}

The present invention relates to a method for culturing lycopene-containing tomatoes, and more particularly, to a method for culturing lycopene-containing tomatoes, which are useful components of tomatoes having antioxidant and anticancer activity.

Tomatoes grow well at relatively high temperatures and love strong rays. Generally, it is divided into finite growth tomato and infinite growth tomato. Finite growth tomato is mainly used for processing, and the tomato grown and eaten mainly in Korea is infinite growth tomato.

Tomatoes contain citric acid, malic acid, succinic acid, amino acids, rutin, protein sugars, calcium, iron, phosphorus, vitamins A, B1, B2, C, and dietary fiber. For vitamin C, one tomato contains about half of the recommended daily intake. Tomatoes contain antioxidants such as lycopene and beta-carotene, and the color of the tomatoes is due to this carotenoid.

Lycopene is usually about 0.02g from a kilogram of fresh, ripe tomatoes. Lycopene binds to and releases free radicals from the body's metabolism that cause aging and damage DNA. Lycopene has also been reported to significantly reduce the risk of various cancers, including prostate cancer, and slow the aging of the arteries. Tomato's anti-cancer effect is better than its anti-cancer drug, beta-carotene, and in fact, at the University of Illinois, USA in 1999, when a patient with prostate cancer ate a plate of tomato sauce per day, the damage of oxidized DNA in white blood cells was reduced. Reported.

Currently, various lycopene-related health supplements are sold at home and abroad. These supplements usually contain up to 15 mg of lycopene in one capsule.

However, as described above, since only about 0.02 g of lycopene can be recovered from 1 kg of fresh and ripe tomatoes, development of a technique for increasing the content of lycopene in the tomato is urgently needed.

In Korean Patent No. 10-0506796, in order to increase the content of lycopene in tomatoes, fermentation of natural tomato fermentation and fermentation for 3 months or more at room temperature after mixing low-commercial low tomato and sugar in a ratio of 5: 1 to 1: 1 Fertilizer was mixed and fertilized at a ratio of 5 to 50% by weight of the entire fertilizer, but because a large amount of tomatoes are required to produce fermented fertilizer, there was a problem of low productivity.

Accordingly, the present inventors have made diligent efforts to solve the above problems, when the tomato formulation step , when treated with Flavobacterium sp. Or Chryseobacterium indologenes strain to the soil, It was confirmed that the lycopene synthesis related gene of tomato is highly expressed by the strain, thereby increasing the content of lycopene, thereby completing the present invention.

An object of the present invention is to provide a method for culturing lycopene-containing tomatoes.

In order to achieve the above object, the present invention, in the formal stage of tomato, Flavobacterium sp. ( Cryseobacterium indologenes ) or Chryseobacterium indologenes Provided is a cultivation method of lycopene-containing tomatoes, characterized in that the strain is treated in the soil.

In the present invention, the lycopene-containing tomato cultivation method is 0.5 × 10 ⁸ ~ 1.5 × 10 ⁸ bacterial cell / ml Flavobacterium sp. Or Chryseobacterium indologenes ( Chryseobacterium indologenes ) 300 to 500 ml of strain is characterized in that the treatment for each pot of 20 ~ 30cm in diameter.

In the present invention, the Flavobacterium sp. ( Flavobacterium sp. ) The strain is Flavobacterium sp. GSE09, and the Chryseobacterium indologenes strain is Chryseobacterium indologenes ISE14.

In the present invention, the Flavobacterium sp. Or Chryseobacterium indologenes The strain is characterized by increasing the expression of genes ( PSY1, PSY2 ) encoding tomato phytone synthase.

According to the invention, in the formal stages of tomato, Flavobacterium sp. Or Chryseobacterium indologenes By treating the soil with soil, the lycopene content of tomatoes can be increased. Therefore, it is possible to mass-produce lycopene, a natural antioxidant having a function of inhibiting or inactivating free radicals that damage cells of the human body.

1 is a pathway for lycopene synthesis in higher plants.
Figure 2 is a photograph according to the degree of ripening of tomatoes at the time of tomato harvest (1: unripe tomatoes, 2: tomatoes at the start of ripening, 3: ripe tomatoes).
Figure 3 is a real-time PCR results to confirm the expression of lycopene synthesis related gene in tomato according to an embodiment of the present invention (C: untreated tomato, GSE09: Flavobacterium sp. ) GSE09-treated tomatoes, ISE14: Chryseobacterium indologenes Tomatoes treated with ISE14, MgSO ₄ : Tomatoes treated with MgSO ₄ , number of ripening tomatoes (1: unripe tomatoes, 2: Half ripe tomatoes, 3: ripe tomatoes)).
4 is an HPLC analysis graph of standard lycopene.
5 is a result of measuring the lycopene content in mature tomatoes grown according to an embodiment of the present invention.

In the present invention, when treated with Flavobacterium sp. Or Chryseobacterium indologenes strains in the soil during the tomato formulation step , Flavobacterium sp. Or Chryseobacterium indologenes The strain was intended to confirm that the lycopene synthesis gene of tomato is highly expressed and the lycopene content is increased.

In the present invention, Flavobacterium sp. ( Flavobacterium sp. ) GSE09 and Chryseobacterium indologenes Each strain of ISE14 was treated at the formal stage of tomato, and harvested after dividing into three stages according to the degree of ripening from 4 weeks after the treatment, and then the lycopene content of the tomato was measured. As a result, Flavobacterium sp. GSE09 or Chryseobacterium indologenes In the case of the tomato treated with ISE14, it was confirmed that the content of lycopene increased.

Accordingly, the present invention, in one aspect, in the formal stages of tomato, Flavobacterium sp. Or Chryseobacterium indologenes It relates to a method for growing a lycopene-containing tomato, characterized in that the strain is treated in the soil.

In the present invention, the Flavobacterium sp. Or Chryseobacterium indologenes Strains contain 0.5 × 10 ⁸ to 1.5 × 10 ⁸ bacterial cells / ml for each pollen 20 to 30 cm in diameter. It is preferable to treat 300 to 500 ml of Flavobacterium sp. Or Chryseobacterium indologenes strains. If the throughput of the Flavobacterium sp. Or Chryseobacterium indologenes strain is outside the above range, it is unlikely to increase the lycopene content of the tomato or inhibit the growth of the tomato. have.

The number of treatments of the Flavobacterium sp. Or Chryseobacterium indologenes strains is not particularly limited, but only one treatment is necessary because the strain is alive in the soil. Suffice.

In the present invention, the Flavobacterium sp. Or Chryseobacterium indologenes The strain can be used without limitation as long as it increases the expression of genes ( PSY1, PSY2 ) encoding tomato phytone synthase. Flavobacterium sp. The strain is Flavobacterium sp. GSE09 (KACC 91368P), and the Chryseobacterium indologenes strain is Chryseobacterium indologenes ISE14 (KACC 91370P), etc. can be illustrated.

In the present invention, the Flavobacterium sp. Or Chryseobacterium indologenes The strain is characterized by increasing the expression of genes ( PSY1, PSY2 ) encoding tomato phytone synthase.

The gene in tomato is known to overexpress or reduce a specific gene by a defense mechanism against the influx of external stimulants, Flavobacterium sp. Or Chryseobacterium indologenes strain Is thought to increase tomato lycopene content by overexpressing phytone synthase, a gene involved in lycopene synthesis.

1 is a pathway for lycopene synthesis in higher plants. As shown in Figure 1, the biosynthesis process of lycopene in higher plants is 2 × Geranylgeranyl diphosphate is synthesized as Phytone by Phytone synthase, changed to ζ-catotene by Phytone desaturase, finally lycopene by ζ-catotene desaturase It is known to be synthesized. Synthesized lycopene is synthesized into α-carotene and β-carotene by Lycopene cyclase.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Cultivation of Lycopene-Containing Tomato

Example 1-1 Sowing and Cultivation of Tomatoes

Tomato seeds were purchased from commercially available Do Tarang Dia varieties (Takiage, Japan), and germinated at 24 ° C. in Petri dishes under 16 hours light condition and 8 hours dark condition. After 1-2 weeks, the germinated seeds were transplanted into soil mixed with Bioplug No. 2 (heungjong seed) and vermiculite 2: 1. After 3-4 weeks, they were planted in pots of 20-30cm in diameter and grown in greenhouses.

Example 1-2 Strain Culture and Treatment

Flavobacterium sp. GSE09 (KACC 91368P) and Chryseobacterium indologenes After culturing ISE14 (KACC 91370P) strain in a solid medium NA (nutrient agar, Difco, Detroit, USA) for 48 hours at 28 ℃, single colony (Single colony) in 5 ml of NB (nutrient broth, Difco, Detroit, USA) Inoculated and incubated in a shaking incubator for 24 hours at a speed of 160rpm at 28 ℃. Pre-cultured Flavobacterium sp. GSE09 (KACC 91368P) and Chryseobacterium indologenes ISE14 (KACC 91370P) strain was placed in the same liquid medium NB 500ml and incubated in a shaking incubator for 48 hours at a speed of 160rpm at 28 ℃. The cultured strains were divided into 250ml tubes and centrifuged at 5000g for 20 minutes at 20 ° C. Centrifuged strains were mixed in 10mM MgSO ₄ to 1 × 10 ⁸ bacteria cell / ml (OD ₆₀₀ = 0.5), and in the tomato planting step, 400 ml of each pollen was treated. At this time, as a control, 10mM MgSO ₄ was also treated together.

Example 1-3 Harvesting Tomatoes

The strain was treated in the formal stage of Example 1-2, and after 4 weeks, the tomato seeds were harvested by dividing it into three stages according to maturity (Fig. 2). Tomato is a ripening crop, but in the present invention, the harvest is waited until ripening.

Example 2: Confirmation of the gene related to lycopene synthesis of tomato

Phytone synthase ( PSY1, PSY2 ), Phytone desaturase ( PDS ), ζ-carotene desaturase ( ZDS ), Lycopene β-cyclase ( Lcy- ), genes involved in lycopene synthesis in tomatoes harvested in Example 1 (see FIG. 1) b ), Lycopene ε-cyclase ( Lcy-e ) and elongation factor 1 alpha ( EF-1a ), which are house keeping genes, were prepared to confirm the expression level of genes using real-time PCR (Table 1). One).

SEQ ID NO: primer name Base sequence One EF-1α-F 5 '-TCC TGA AGT GGA AGA CGC AAG GGT T-3' 2 EF-1α-R 5 '-CCC TGA CAA GAT CCC ATT TGT GCC G-3' 3 PSY1-F 5 '-TGC TGG TAC GGT TGG GTT GAT GAG T-3' 4 PSY1-R 5 '-TGC GAT CCC CAG AGC CAA AGC A-3' 5 PSY2-F 5 '-AGC ACA GGC AGG GCT CTC CGA T-3' 6 PSY2-R 5 '-ACG ATG CCA ACA CCG GCC ATC T-3' 7 PDS-F 5 '-CGT ACT CCC CAT GCC ACG ACC A-3' 8 PDS-R 5 '-GGG CGC GGA GAA GCA CGG AA-3' 9 ZDS-F 5 '-GCT GGG CTT GCA GGC ATG TCG-3' 10 ZDS-R 5 '-GCG CCC ACC TTT TTC AAC AGA CGG A-3' 11 LCYB-F 5 '-CGT AGC TCG TCC TGG CTT GCG T-3' 12 LCYB-R 5 '-ACC ATG CCA GCT GTA CCA CCG A-3' 13 LCYE-F 5 '-TGA ACG TGG GGC TCG TTG GGC-3' 14 LCYE-R 5 '-CCC GCC AAA CAT GTT CAA TGC AGG C-3'

The harvested tomatoes were separated by total RNA using a Plant RNA Purification Reagent (Ivitrongen, Carlsbad, CA) by maturity, and cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA).

 Expression of each gene using the synthesized cDNA as a template was confirmed using SYBR Premix EX Taq (TaKaRa, Japan), and the results are shown in FIG. 4.

As shown in Figure 4, it was confirmed that the expression level of these genes increased in the tomato (C-2), the second degree of ripening in the tomato untreated strain.

Tomatoes treated with MgSO ₄ also showed similar results as untreated tomatoes (MgSO ₄ -1 ~ 3).

In contrast, Flavobacterium sp. GSE09 (KACC 91368P) or Chryseobacterium indologenes Tomatoes treated with ISE14 (KACC 91370P) did not show significant differences in other genes, but the expression of phytone synthase ( PSY1, PSY2 ) increased.

And it was confirmed that the expression pattern is different depending on the type of strain treated. Chryseobacterium indologenes In tomato treated with ISE14 (KACC 91370P), the expression level was increased in tomato (ISE14-2), the second tomato ripening, but Flavobacterium sp. In the tomato treated with GSE09 (KACC 91368P), it was confirmed that the expression level was increased in the third ripened tomato (GSE09-3).

From these results, Flavobacterium sp. GSE09 (KACC 91368P) or Chryseobacterium indologenes The strain ISE14 (KACC 91370P) was found to affect the increased expression of genes ( PSY1, PSY2 ) encoding phytone synthase among genes involved in lycopene synthesis of tomatoes.

Example 3: Determination of Lycopene Content in Tomato

Lycopene content of tomatoes is described in Lidia et al., Spectrophotometric Analysis of Lycopene in Tomatoes and Watermelons: A Practical Class, Chem. Educator , 13, 11-13, (2008)] and Mario et al. [Analysis of lycopene geometrical isomers in biological microsamples by liquid chromatography with coulometric array detection, Journal of Chromatography B , 760, 289-299, (2001)]. It was measured by deformation.

0.1 g of the tomato dried material harvested in Example 1, which was shaded with aluminum foil and freeze-dried, was placed in a 1.5 ml conical tube, and 1 ml of an extraction solvent (hexane: acetone = 1: 1, v / v) was added thereto and extracted at room temperature for 20 minutes. . The supernatant obtained by centrifuging the extract (13,000 rpm, 1 min) was filtered through a 0.45 mm membrane filter, and the filtrate was measured by HPLC. At this time, all experiments were performed while shading in order to minimize the decomposition of lycopene by light. HPLC analysis conditions used in the analysis are shown in Table 2 below.

Insrtument: Gilson HPLC system (Gilson Inc. USA) Column: YMC Carotenoid (YMC Co. JAPAN) Column diameter: 4.6 mm Particle size: 5 mm Column length: 250 mm Column Temp. : 30 ℃ Flow rate: 1ml / min Sample injection volume: 20 ml Detector: Vis (503 nm) Mobile phase A; MeOH: MTBE: H ₂ O = 85: 12: 3
Mobile phase B; MeOH: MTBE: H ₂ O = 8: 90: 2 Gradient: 0-3 min 100% A; 3-18 min 80% A linear; 18 ~ 35 min 45% A linear, 35 ~ 45 min 5% A linear, 45 ~ 50 min 5% A

The calibration curve was prepared from the peak area ratio of lycopene obtained by lycopene standards (Sigma Co., Ltd.) at 0.05, 0.1 0.2 0.4 mg / ml, 20 ml each and analyzed by HPLC (FIG. 4).

In general, since tomatoes consume ripe tomatoes, lycopene content was measured in ripe tomatoes. As a result, as shown in Figure 5, the tomato was not treated in the tomato formulation stage (control) and tomato treated with MgSO ₄ contains 1.2mg / g of lycopene, Chryseobacterium indologenes ( Chryseobacterium indologenes ) ISE14 (KACC 91370P) and Flavobacterium sp. In ripened tomatoes treated with GSE09 (KACC 91368P), the content of lycopene increased by about 3 to 5 times to 7.4 mg / g and 4.2 mg / g, respectively.

In conclusion, the real-time PCR results of Example 2 and the results of measuring the lycopene content of Example 3, the expression level of the gene encoding the phytone synthase involved in the lycopene synthesis is about half ripe than in ripe tomatoes Chryseobacterium indologenes with increased expression in tomato Increased lycopene content in tomatoes treated with ISE14 (KACC 91370P) suggests that increased gene expression in less ripe tomatoes is more involved in lycopene content than in mature tomatoes. .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Dong-A University Research Foundation for Industry-Academy Cooperation <120> Method for Cultivating Tomato Containing High Content of Lycopene <160> 14 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> EF-1a-F primer <400> 1 tcctgaagtg gaagacgcaa gggtt 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> EF-1a-R primer <400> 2 ccctgacaag atcccatttg tgccg 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PSY1-F primer <400> 3 tgctggtacg gttgggttga tgagt 25 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PSY1-R primer <400> 4 tgcgatcccc agagccaaag ca 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> P2232-F primer <400> 5 agcacaggca gggctctccg at 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PSY2-R primer <400> 6 acgatgccaa caccggccat ct 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PDS-F <400> 7 cgtactcccc atgccacgac ca 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDS-R <400> 8 gggcgcggag aagcacggaa 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ZDS-F <400> 9 gctgggcttg caggcatgtc g 21 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ZDS-R <400> 10 gcgcccacct ttttcaacag acgga 25 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LCYB-F <400> 11 cgtagctcgt cctggcttgc gt 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LCYB-R <400> 12 accatgccag ctgtaccacc ga 22 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCYE-F <400> 13 tgaacgtggg gctcgttggg c 21 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LCYE-R <400> 14 cccgccaaac atgttcaatg caggc 25

Claims (4)

In the formal stages of tomato, Flavobacterium sp. Or Chryseobacterium indologenes Cultivation method of lycopene-containing tomatoes, characterized in that the strain is treated in the soil.
According to claim 1 , The lycopene-containing tomato cultivation method is Flavobacterium sp. Or Chryseobacterium indologenes of 0.5 × 10 ⁸ ~ 1.5 × 10 ⁸ bacterial cell / ml ) Cultivation method of lycopene-containing tomatoes, characterized in that the strain is treated 300-500ml per pollen having a diameter of 20 ~ 30cm.
According to claim 1, The Flavobacterium sp. ( Flavobacterium sp. ) The strain is Flavobacterium sp. GSE09, and the Chryseobacterium indologenes strain is Chryseobacterium indologenes Cultivation method of lycopene-containing tomatoes, characterized in that ISE14.
According to claim 1, Flavobacterium sp. Or Chryseobacterium indologenes The strain is a method for culturing lycopene-containing tomatoes, characterized in that to increase the expression of the gene ( PSY1, PSY2 ) encoding a phytone synthase of tomatoes.

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