JP2000152778A - Production of lycopene-containing alga and lycopene- containing alga thus produced - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain lycopene-contg. algae useful as a health food, food colorant, food material, or the like without inhibiting its proliferation by heterotrophic culture of algae proliferable by heterotrophic culture in a medium containing a lycopene cyclase inhibitor. SOLUTION: The lycopene-contg. algae useful as a health food, food colorant, food material, or the like is efficiently obtained without inhibiting its proliferation by heterotrophic culture of algae proliferable by heterotrophic culture (e.g. chlorella) in a medium containing a lycopene cyclase inhibitor consisting of at least one kind selected from nicotine, N,N-dimethyl-2-phenylziridinium (DPA), 2-(4-chlorophenylthio)-triethylamine (CPTA), 2-(4-methylphenyl)- triethylamine (MPTA), azasqualene, dodecyltrimethylammonium chloride, and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing lycopene-containing algae useful for health foods, food colorings and food materials, and to lycopene-containing algae obtained by the production method.

[0002]

2. Description of the Related Art Chlorella, Senedesmus, Spirulina,
Algae such as Synechococcus and Donariella inhabit tropical and temperate lakes and marshes and have been used as food for local people since ancient times. At present, the dried algal bodies of the algae are used as dietary supplements and health foods, and have already formed a large market. The nutrient component of the dried algal body contains 60% or more of protein, and also contains dietary fiber, various vitamins, minerals such as iron and potassium, essential fatty acids, chlorophyll of photosynthetic pigment, and carotenoid. However, carotenoids produced by the algae include α-carotene, β-carotene, lutein, zeaxanthin, anteraxanthin,
It is limited to violaxanthin, neoxanthine and the like, and lycopene, whose physiological activity has attracted attention in recent years, does not accumulate.

[0003] Lycopene is a C40 linear carotene and has a ruby-like red color, and is abundantly contained in tomatoes, watermelons and the like. In recent years, it has been revealed that lycopene has excellent carcinogenesis inhibitory and antioxidant activities (Monthly Food Chemical
1995-3, pp. 38-43), and its physiological activity has attracted attention.
For example, regarding the carcinogenesis inhibitory effect, epidemiological studies have reported that the higher the level of lycopene in human blood, the lower the incidence of pancreatic cancer (Am.J.Clin.Nutr. 53,
260s, 1991). Lycopene has also been reported to exhibit a growth inhibitory effect on human lung cells and breast cancer cells (Nu
tr, Cancer, 24 (3), 1995).

As for the antioxidant activity, it has been reported that lycopene has an ability to scavenge active oxygen more than twice that of β-carotene (Arch. Biochem. Biophys., 1988).
274, 532, 1989). Thus, as the biological activity of lycopene becomes apparent, the demand for lycopene-containing foods is increasing, but the foods are limited to tomatoes, watermelons, etc., and their lycopene content is extremely low. Was. Under such circumstances, in the health food market, a supplement food that can easily supplement lycopene has been desired.

On the other hand, nicotine, N, N-dimethyl
1-2-phenylziridinium (DP
A), 2- (4-chlorophenylthio)
-Triethylamine (CPTA), 2- (4
-Methyl-phenyl) -triethyla
triethylamines such as mine (MPTA) and azasqualene, dodecyltrimethylam
Lycopene analogs such as monium chloride have been known in the literature to inhibit lycopene cyclase in vitro (Arch. Biochem. Biophys. 346, 5).
3, 1997). For this reason, several attempts have been made to accumulate lycopene in algae. For example, Dunariella bardaw, a type of algae
il) has been confirmed to accumulate lycopene in the presence of nicotine (Plant Cell Physiol. 31 (5): 689-69).
6, 1990).

[0006] Synechococcus s
p. PCC7942) also reports that nicotine inhibits lycopene cyclase (FEBS LETTERS Vol 328,
1, 2, 130-138, 1993). However, since the former is a study aimed at elucidating the carotenoid metabolic pathway, it does not mention growth of lycopene-containing algae. On the other hand, in the latter, Synechococcus is killed at a nicotine concentration of 2 micromol / liter due to low nicotine resistance.
Japanese Patent Application No. 9-313167 discloses a technique for accumulating lycopene in spirulina by culturing spirulina in a medium containing nicotine at 200 to 500 micromol / liter. However, in this production method, when the nicotine concentration exceeds 500 micromol / liter, the sequential growth of spirulina is inhibited, and
When the amount is more than micromol / liter, the growth is remarkably suppressed. The upper limit of the nicotine concentration is 50.
Even lycopene accumulated at 0 micromol / liter was less than 20 percent of total carotenoids.

[0007] As described above, although attempts have been made to accumulate lycopene by causing nicotine to act on algae, the growth of the algae is inhibited in that case. Was impossible to supply. Therefore, it is desired to establish a production method for producing algae containing lycopene at a high concentration without significantly inhibiting the growth of algae.

[0008]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for efficiently producing lycopene-containing algae without significantly inhibiting the growth of algae and a high concentration of lycopene obtained by the production method. It is to provide a lycopene-containing algae.

[0009]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems. The present inventors have found that even a medium containing a lycopene cyclase inhibitor such as nicotine does not cause significant growth inhibition and can actively biosynthesize a photosynthetic dye, thus completing the present invention.

As the algae used for producing the lycopene-containing algal body of the present invention, any algae can be suitably used as long as they proliferate heterotrophically and biosynthesize a photosynthetic pigment. At present, there are many unclear points about the classification and physiological actions of algae,
Normally, algae such as Donariella, Synechococcus, and Spirulina perform photosynthesis and grow autotrophically. For this reason,
When lycopene cyclase is inhibited using nicotine or the like, a photosynthetic system required for autotrophic culture is not constructed, and the growth of algal cells is also inhibited. However, if algae that can proliferate heterotrophically and actively biosynthesize photosynthetic pigments are used, even if they inhibit carotenoid biosynthesis, which is important for the photosynthetic system, the algae will grow heterotrophically. In addition, it is possible to efficiently produce an alga body containing lycopene without being inhibited by growth. Algae that can be heterotrophically cultivated include those capable of biosynthesizing photosynthetic pigments even when heterotrophic culture is performed under dark conditions, such as Chlorella spp., Synedesmus spp., And Chlamydomonas spp., Hematococcus spp. There are some that do not biosynthesize photosynthetic pigments without light.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION As specific algae, the genus Chlorella, the genus Senedesmus, the genus Gorenkinia, the genus Selenastrum, the genus Protosiphon, the genus Chlorococcum and the like are mentioned as examples of the former, and the examples of the latter are Chlamydomonas,
Examples include the genus Haematococcus, the genus Euglena, the genus Potriococcus, and a small portion of the genus Spirulina. As the algae used in the present invention, an algae such as a genus Chlorella and a genus Senedesmus that can biosynthesize a photosynthetic pigment even when subjected to heterotrophic culture under dark conditions is preferable. When using algae that do not biosynthesize photosynthetic pigments without light, such as Chlamydomonas, when the concentration of the lycopene cyclase inhibitor is high, the growth of algae may be significantly suppressed by light irradiation, and Because it is necessary to perform light irradiation to accumulate a large amount of lycopene (mixed nutrition culture),
This is because the cost of facilities and the like, the complexity of workability, and the power consumption increase.

Further, as these algae, mutants which have been subjected to a mutation treatment or the like and which are imparted with a trait of actively biosynthesizing a photosynthetic pigment under dark heterotrophic nutrition may be suitably used. For algae culture, add a lycopene cyclase inhibitor to the medium,
Perform heterotrophically. Light irradiation may be performed as appropriate according to the algae used at this time, but chlorella, scenedesmus, and the like are not particularly required since sufficient growth and photosynthetic pigment content can be obtained by culturing in a dark place. Further, the light irradiation condition may be determined according to the type of algae.

The medium to be used may be determined according to the type of algae, and the culture method may be appropriately selected from static culture, stirring culture, shaking culture, aerobic culture, anaerobic culture and the like. Furthermore, various culturing conditions such as culturing temperature and medium pH may be set to optimal conditions according to the type of algae.

The lycopene cyclase inhibitors include nicotine, N, N-dimethyl-2-phe
nylziridinium (DPA), 2- (4-c
chlorophenylthio) -triethyl
amine (CPTA), 2- (4-methyl-p
henyl) -triethylamine (MPT
A), Azasquare, dodecyltrimeth
For example, nicotine and DPA are preferable for culturing algae used as a material for foods such as chlorella, from the viewpoint of safety, availability, easy removal from algal cells, and the like. As with the various culture conditions described above, the addition concentration of the inhibitor is adjusted according to the algae used,
It may be determined in consideration of cytotoxicity, osmotic pressure, ionic strength and the like.

As an embodiment of the present invention, lycopene accumulation in chlorella will be described. Mutation treatment is applied to chlorella regularis isolated from freshwater to impart a trait of actively biosynthesizing photosynthetic pigment under dark heterotrophic nutrition. The resulting mutant strain, Chlorella regularis Y-21 strain, is used (hereinafter simply referred to as "Y-21 strain"). The Y-21 strain is a high pigment-containing mutant derived from the M-1 strain, which is a wild strain of Chlorella regularis, by NTG mutation treatment. Incidentally, the Y-21 strain has a chlorophyll content of 30 under dark heterotrophic nutrition.
Milligram / gram or more, total carotenoid content is 5.0
It has a high pigment-containing trait of milligrams / gram or more (for the Y-21 strain, Japanese Patent Application No. 9-25 / 1990).
0352).

The carbon source used for heterotrophic culture of Chlorella includes glucose, acetic acid, sucrose, fructose, galactose and the like, and glucose and acetic acid are preferable from the viewpoint of growth. As the nitrogen source, ammonium sulfate, potassium nitrate, urea and the like are preferable from the viewpoint of food additives and ease of handling, and urea is particularly preferable. The medium used for culturing Chlorella is not particularly limited, but an A medium commonly used in culturing algae is preferable. The culture pH is preferably from 6.0 to 8.0, particularly preferably 6.5. The cultivation temperature is preferably from 20 to 35C, particularly preferably 30C. The shaking method is preferably a rotary method, and the speed is preferably 100 to 200 rpm, particularly 180 rpm.
rpm is preferred. When heterotrophic culture of Chlorella is performed, it is not necessary to perform light irradiation, and the culture may be performed in a dark place.

In order to accumulate lycopene in Chlorella,
The concentration of the lycopene cyclase inhibitor to be added to the medium varies depending on the difference in inhibitor sensitivity among chlorella strains. For example, nicotine has a concentration of 3.0 to 10 mmol / mol.
Liter is preferred, and especially 5.0 to 8.0 mmol / liter. When the nicotine concentration is less than 5.0 mmol / l, lycopene accumulation in chlorella decreases, and when the nicotine concentration is 10 mmol / l or more, remarkable growth inhibition is exhibited and the productivity of algal cells is reduced.

Arch. Biochem. Biophys. 346,5
According to 3,1997, the I ₅₀ of each inhibitor of lycopene cyclase (inhibitor concentration when inhibiting 50% of the enzyme activity) is 40 μmol / liter for nicotine and 15 μmol / DPA for nicotine. Liters, CPT
A10 micromol / liter, MPTA 12 micromol / liter, Azaxarene 1 micromol / liter, dodecyltrimethylammoni
um chloride 10 micromol / liter. Therefore, the concentration of each inhibitor to be added may be set by comparing I ₅₀ of the inhibitor with I ₅₀ of nicotine.

As described above, it is possible to industrially produce lycopene-containing chlorella by heterotrophically culturing chlorella in a medium supplemented with a lycopene cyclase inhibitor. In the production method of the present invention, as described later, the growth of chlorella is suppressed to some extent, but this can be dealt with by extending the culture time or increasing the initial chlorella concentration. It is also possible to select chlorella strains that are less susceptible to growth inhibition. On the other hand, the proportion of lycopene in the total carotenoids in chlorella is close to 90%, and an incomparable lycopene content is obtained.

The chlorella accumulating lycopene as described above may be used as feed after removing nicotine by a method such as washing with water. Also, heat and dry,
Powders can also be provided in the form of tablets or the like. Furthermore, dyes and the like in the cells can be extracted with an organic solvent or supercritical carbon dioxide and used.
As for the use, it can be suitably used for food materials, cosmetics, pharmaceutical raw materials, and the like, in addition to use as health foods, feeds, and pigments. Algae obtained by the present invention, such as chlorella, have reduced chlorophyll and increased lycopene content. For this reason, it can take a new use form as a food material, a pigment, a cosmetic raw material, or the like.

[0021]

EXAMPLES Hereinafter, an example in which the Y-21 strain is used as an algae will be specifically described. Chlorella regularis (chlo
rella regularis) Y-21 strain in medium A (Table 1) at 180 rpm at 30 ° C under dark conditions until stationary phase is reached.
Was shake-cultured.

[0022]

[Table 1]

Since nicotine, a lycopene cyclase inhibitor, used in this example is a basic compound, it is necessary to adjust the pH after the addition of nicotine. Since nicotine is a compound that is relatively resistant to heat, it can be sterilized by adjusting the pH after addition.

The above-mentioned strain Y-21 was subjected to photoautotrophic culture in an A medium (excluding glucose) supplemented with 5 mmol / liter of nicotine. As a result, as shown in Table 2, the strain was significantly inhibited by nicotine,
It was not possible to produce algal cells with lycopene accumulated in the cells. As shown in Table 2, growth inhibition did not occur in the same A medium to which nicotine was not added (0 mM).

[0025]

[Table 2]

Next, the above Y-21 strain was transformed into 1, 2, 5, 10
Heterotrophic cultivation was performed in A medium (excluding glucose) supplemented with mmol / liter nicotine. As a result, as shown in Table 3, although the growth of the strain was inhibited by nicotine, the degree of inhibition was significantly reduced as compared with the case of photoautotrophic culture. Under these culture conditions, sufficient growth of the strain was observed, and it was possible to produce alga bodies in which lycopene had accumulated in a considerable amount. In addition,
As shown in Table 3, no nicotine was added (0 mM)
No growth inhibition occurred in the same A medium.

[0027]

[Table 3]

As a whole, A with various nicotine concentrations
When heterotrophic culture was performed in the medium, the nicotine concentration was 1
No effect was observed on the growth of chlorella in the range of ２2 mmol / l, and when the nicotine concentration was 10 mmol / l or more, the growth of chlorella was inhibited, and the growth rate was 1/100 of that without nicotine. 10 or less.

After heterotrophic culture of the Y-21 strain, the pigment was quantified. As a means of quantification, the chlorophyll and total carotenoid contents were measured for absorbance after acetone extraction (vegetable pigments: Kozo Hayashi, Yokendo). That is, chlorella alga bodies were precipitated from 50 ml of the chlorella culture solution by centrifugation (3000 rpm, 10 minutes), and then washed twice with distilled water. The washed chlorella alga was resuspended in 10 ml of distilled water, 0.1 ml of the chlorella suspension was suction-filtered with a glass fiber filter (Advantech GA55), and the chlorella alga was trapped in the glass fiber filter. A glass fiber filter trapped with Chlorella algae was placed in a mortar, 1 ml of distilled water was added, and the chlorella algae together with the glass fiber filter were ground with a pestle until a porridge was formed. Then, 9 ml of acetone was added, and the mixture was transferred to a graduated centrifuge tube as if the mortar was washed. After making up the solution volume to 10 ml with acetone, centrifugation (3.
000 rpm, 10 minutes) to precipitate the algal cells and the ground matter of the glass fiber filter, and absorbance of the supernatant (480
nm, 630 nm, 645 nm, 663 nm, 750 n
m) was measured, and chlorophyll and total carotenoid concentrations were calculated from the following formula. The dried algal body weight was measured after drying at 105 ° C. overnight.

[0030]

## EQU1 ## Chlorophyll a (μg / ml) = 11.64 (A
663-A750) -2.16 (A645-A750) +
0.1 (A630-A750)

[0031]

Chlorophyll b (μg / ml) = 3.94 (A6
63-A750) -20.97 (A645-A750) +
3.66 (A630-A750)

[0032]

## EQU3 ## Carotenoid (μg / ml) = 4.0 (A480
-3.0A750)

The pigment content of the chlorella algae at the stationary stage cultured in the examples was determined by the above method. As a result, FIGS.
As shown in Fig. 5, it was confirmed that when the nicotine concentration was 5 mM or more, chlorella hardly biosynthesized chlorophyll, but the carotenoids were biosynthesized similarly when nicotine was not added.

Chlorella algae in the stationary phase after culturing were freeze-dried, and carotenoids were extracted from a 100 milligram sample. 10 ml of a 6N potassium hydroxide / methanol solution and a small amount of anhydrous sodium sulfate were added to the sample, the cells were crushed with a glass homogenizer, centrifuged (3000 rpm, 10 minutes), and the supernatant was collected as an alkali methanol extract. Then, 10 ml of dichloromethane was added to the residue containing the cell lysate and resuspended.
After centrifugation (3000 rpm, 10 minutes), the supernatant was collected as a dichloromethane extract.

The above operation was repeated until the residue became colorless. The finally obtained alkali methanol extract and dichloromethane extract were mixed, and the solvent was evaporated as much as possible using an evaporator. Thereafter, 50% of 5% saline solution and methanol were added thereto, and the mixture was transferred to a separating funnel, and 100 ml of ethyl ether was added. The mixture was separated into two layers, a water tank and an ether tank, and an ether layer was collected. The extraction operation was repeated with ethyl ether until no dye was extracted in the ether layer. The collected ether extracts were combined, transferred to a separating funnel, and distilled water was added to wash the ether layer. Anhydrous sodium sulfate was added to the washed ether layer, and the mixture was kept refrigerated for 24 hours under light shielding to dehydrate. The dehydrated ether solution was evaporated to dryness by an evaporator and redissolved in 1 ml of dichloromethane to obtain a sample for high-performance liquid chromatography described later. The sample was stored in a light-shielded frozen state after replacing the head space with nitrogen.

The analysis of carotenoids was performed using high performance liquid chromatography. The analysis conditions are as follows (Table 4).

[0037]

[Table 4]

From the analysis of high performance liquid chromatography,
α-carotene, β-carotene, lutein and lycopene were detected. In addition to these carotenoids, two unknown carotenoids were detected. At nicotine concentrations below 2 mmol / l, chlorella did not accumulate lycopene. On the other hand, when the nicotine concentration reached 5 mmol / L or more, lycopene accumulated at 1.0 mg / gram or more (Table 5).

[0039]

[Table 5]

[0040]

According to the present invention, alga bodies containing lycopene can be obtained efficiently and economically. In particular, by setting the nicotine concentration to 5 mmol / L or more, chlorella containing lycopene at 1.0 mg / gram or more can be obtained.

[Brief description of the drawings]

FIG. 1 is a correlation diagram of nicotine concentration and chlorophyll content showing the results of the production method according to Example 1.

FIG. 2 is a correlation diagram of carotenoid content with respect to nicotine concentration, showing the results of the production method.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/00 A61K 31/00 603 4C088 35/00 635 A61K 31/47 31/47 35/80 35/80 A C07D 401/04 207 C07D 401/04 207 C12P 5/02 C12P 5/02 (C12N 1/12 C12R 1:89) (C12N 1/12 C12R 1:89) (C12P 5/02 C12R 1:89) F Terms (Reference) 4B018 MA01 MD89 ME06 ME08 4B064 AH01 CA08 CC03 CC04 CD12 DA05 DA10 4B065 AA84X AC09 AC14 BB12 BC50 CA03 CA41 CA44 CA52 4C063 AA01 BB01 CC12 DD03 EE01 4C086 BC17 GA07 GA08 4C088 AA16B07 Z

Claims (6)

[Claims] 1. A method for producing lycopene-containing algae, which comprises heterotrophically cultivating an algae that can be propagated by heterotrophic culture in a medium containing a lycopene cyclase inhibitor. 2. The method for producing lycopene-containing algae according to claim 1, wherein the algae are alga that can be proliferated by heterotrophic culture in a dark place. 3. The lycopene cyclase inhibitor is nicotine,
N, N-dimethyl-2-phenylziri
dinium (DPA), 2- (4-chloroph)
anyylthio) -triethylamine (C
PTA), 2- (4-methyl-phenyl)-
triethylamine (MPTA), azasqualene, dodecyltrimethylammoni
The method for producing lycopene-containing algae according to claim 1 or 2, wherein the lycopene-containing algae is at least one selected from um chloride. 4. Lycopene-containing algae produced by the production method according to any one of claims 1 to 3. 5. The method for producing lycopene-containing chlorella according to any one of claims 1 to 3, wherein the algae is chlorella. 6. A chlorella containing lycopene.