JPH04108374A - New alga of chlorella - Google Patents

Abstract

PURPOSE:To use a new alga of Chlorella widely in a food industry and a cosmetic industry by using a specific yellowish green or yellow alga of the genus Chlorella having <=0.1% total chlorophyll content and <=220ppm total PPB amount. CONSTITUTION:A mother stock of algae of the genus Chlorella belonging to Chlorella pyrenoidosa is varied by irradiation with ultraviolet light to give variants MK01 (ATCC n. 40,867) and MK02 (ATCC No.40,838), which are cultured, recovered and dried by a general method to give a dried alga. The alga of the genus Chlorella has <=0.1% extremely low chlorophyll content and <=220ppm total amount of PPB formed by decomposition of chlorophyll and a light color tone of yellowish green or yellow. Then the alga or a treated material thereof is added to a food and a drink such as breads or juice in a proper ratio to produce a Chlorella-containing food and drink. Consequently, the alga of the genus Chlorella having a color tone of yellowish green or yellow can be used for a food and a drink, a health food and prevention of stain.

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Utilization and Decomposition The present invention relates to a novel microalga belonging to the genus Chlorella and its utilization. The algae of the present invention are useful as materials in the food industry and cosmetics industry.

BACKGROUND OF THE INVENTION Algae belonging to the genus Chlorella are a type of unicellular algae, and their bodies contain 50 to 60% protein and have a high composition of essential amino acids, so they are used as a vegetable source of protein. Chlorella algae has long been in strong demand as a health food because it contains vitamins, minerals, fiber, chlorella gross factor (CGF), and the like.

There are no known techniques for improving the cultivation method of Chlorella algae to obtain algae with a high content of photosynthetic pigments.
(Tokuko Showa 5'nee 18', 92), but the glue in the algae was 0.
Mutant strains with reduced chlorophyll content and pheophorbite (PPB) content due to chlorophyll degradation are known.

Problems to be Solved by the Invention Algae belonging to the genus Chlorella exhibit a green color, and their use is extremely limited because the color fades or turns brown when exposed to light or heat. If Chlorella algae with a light green tone or a different tone can be obtained, it is expected that its use in the food and cosmetics industries will expand.

Means for Solving the Problems The present inventors have discovered that a mutant strain obtained by subjecting Chlorella algae to mutation treatment has an extremely low chlorophyll content and a bright yellow-green or yellow tone. These mutant strains do not cause any discomfort in color tone even when used in combination with various food materials, and they do not contain pheophorbite, which is known as a decomposition product of chlorophyll and is said to cause dermatitis.
Bide, PPB), it can be expected to be used as a food additive.

The present invention: Chlorella pyrenotdosa has an extremely low content of chlorophyll and a bright yellow-green or yellow tone.
Species:: Provides a novel algae belonging to the genus Chlorella.

The novel Chlorella algae of the present invention produces 1100 ppm or less of the existing PPB by decomposing chlorophyll in the algal body, and produces 220 ppm or less of the total PPB.

Specific examples of Chlorella R algae of the present invention include those named MKOI and MKo2.

This algae is American Type Cu1tu
re Co11ection (ATCC) on July 6, 1990, ATCC No. 40867.
and No. 40838, respectively.

The above-mentioned algae of the genus Chlorella are obtained by subjecting a parent strain belonging to the species Chlorella virenodosa to mutation treatment by irradiation with ultraviolet rays.

The table below shows an example of algal body component analysis of MKOI strain, MKO2 strain, and parent strain.

yellow-green Book 12. To the standard color chart (Japan Color Research Institute) MKO1! The MK02 strain differs from the parent strain in color tone, chlorophyll content, and amount of PPB produced by decomposition of chlorophyll, as described above, but no differences from the parent strain are observed in other components and properties.

Strains MKo 1 and MK02 have been transplanted and preserved for two years after isolation, and are stable mutant strains without any reversion. The methods for obtaining both strains are shown in Example 1.

The present invention provides algae of a novel Chlorella genus algae belonging to the species Chlorella pyrenoidosa, which has an extremely low content of chlorophyll and a bright yellow green or vivid yellow (v+v+d yellow) color tone, or the algae of the algae. Provide food and drinks containing processed products. Examples of foods and drinks include breads such as white bread and butter rolls, noodles such as fresh Chinese noodles, juices from vegetables and fruits, and agar.

Examples of processed products of algae include extracts obtained by extracting algae with organic solvents such as ethyl alcohol and methyl alcohol, or hot water, or (freeze) dried products thereof.

The chlorella-containing food or drink of the present invention can be produced by adding these algae or processed products of algae to raw materials for the food or drink or during processing.

The mutant algae have lower chlorophyll content and PP compared to the parent strain.
Since the B content is extremely low and there is no difference in other ingredients and properties; -), we use powder as a health food, like the parent strain, and we use fine powder made by dry cell crushing of the powder, and tablets of Soreru. Available in solid or granule form.

Moreover, it can be used in foods that dislike green color, and its range of use is significantly wider than that of the parent strain.

The content of algae and processed algae in food and drink is 0.1 to 2.
For example, bread 0.5-2.0%, noodles 0.5-1.5%, juice 0.1-0.3%, agar 0.1-0.3%. Just add it.

The cultivation, collection and extraction treatment of algae of the present invention involves
The methods used in ships are used.

For example, as the carbon source, saccharides such as glucose, fructose, and sucrose, and organic acids such as acetic acid, citric acid, malic acid, fumaric acid, and succinic acid are used.

Ammonium salts, nitrates, urea, etc. are used as nitrogen sources.

In addition, potassium salts, magnesium salts, phosphates, trace metal salts such as iron salts, cobalt salts, manganese salts, boric acid or borates, zinc salts, copper salts, molybdates, calcium salts, and chelating agents such as EDTA and A medium containing citric acid or the like is used.

The culture temperature is 25-35°C, pH 6.5-7.5, and the aeration and stirring conditions are different for flask culture and jar culture.
For flask culture, aerobic conditions are used; for jar culture, the aeration rate is 1/4 to 1/2 vvm, and the number of stirring is 250 to 400.
rpm, and the pH during the culture with NaOH solution or N
Adjust it to within the range of 6.5 to 7.5 with an alkaline solution such as H, OH water.

The culture time is also different between flask culture and jar culture, with flask culture being cultured for 3 to 4 days, and jar culture being cultured for 2 to 3 days. A centrifugal separator or a p-filter is used to collect the algae after culturing and to wash the algae. Chlorophylase (PPB production), which causes photosensitivity, can be removed by adjusting the pH of the washed algal suspension to 9 with an alkaline solution such as NaOH, NH, or OH.
．． Adjust to a range of 5 to 11.0, and 5 to 1 at 95 to 97°C.
It can be deactivated by heating for 0 minutes.

For drying the algal bodies, a freeze vacuum dryer, a warm air dryer (a spray dryer is particularly suitable), etc. are used. Further, by heating and drying this dried algae at 100 to 105°C for 30 to 60 minutes, dried algae with good aroma and flavor can be obtained.

Pigments can be extracted from the Chlorella algae of the present invention and used as colorants for foods and the like. Furthermore, the hot water extract of Chlorella algae of the present invention has an effect on preventing rough skin, an effect on preventing stains, and can be expected to be used as a cosmetic product.

Examples of the present invention are shown below.

Example I Obtaining MKOI strain and MKO2 strain Two parent strains (Ch! orella pyrenoidosa) were diluted with sterile water to form a suspension with a cell count of approximately 10,000 cells/ml.

Transfer 0.5 ml of this suspension + 30 rIJ of this suspension to a Petri dish agar medium.
1 (glucose 1.5%, monopotassium phosphate 0.122
%, magnesium sulfate 0.246%, ferrous sulfate 0.0
03%, potassium nitrate 0.125%, sodium citrate 0.1%, A-5 solution 0.1%, agar 1.5%, pII
In the medium with composition 7.3, solution A-5 contains 2.9 g of boric acid, 2.0 g of manganese chloride, and 2.2 g of zinc sulfate in deionized water of 11.
, copper sulfate 0.8 g, sodium molybdate 0.5
g, a solution prepared by adding one drop of concentrated sulfuric acid. ) and spread it over the entire surface of the agar using a glass rod. The top lid of the petri dish was removed, and the agar plane was placed 50 cm directly under a germicidal lamp (4-shiba GL-15), and UV irradiation was performed.

The irradiation time was set to be the time (100 seconds) at which the survival rate of algal cells after irradiation was 5% or less. After UV irradiation, the top of the dish was covered and cultured for 8 days at 28°C. A strain with extremely low chlorophyll was isolated from the resulting colonies and named strain MKOI.

Furthermore, the MKOI strain was treated with UV irradiation in the same manner as above.
Perform static culture and isolate yellow colonies, which are MK
It was named strain 02.

Example 2 Production of MKOI strain and MK02 strain algal bodies: Each of the MKOI strain and MK02 strain was cultured as described below to produce algal bodies.

1 platinum loop of algae from the slant was placed in a 200 ml Erlenmeyer flask with a culture medium (glucose 1.0%, potassium phosphate 0,
07%, dipotassium phosphate 0.03%, magnesium sulfate 0.03%, ferrous sulfate 0.0015%, EDTAo
, 005%, A-5 liquid 0.1%, pH 7.0) and cultured with shaking at 28° C. and 20 Orpm for 4 days. 50 ml of this culture was placed in an 11-volume Erlenmeyer flask as a medium (glucose 1.0%, monopotassium phosphate 0.07%, dipotassium phosphate 0.03%, magnesium sulfate 0.03%, ferrous sulfate 0.0%). 0015%, EDT
The medium was inoculated into a medium (Ao, 005%, A-5 liquid 0.1%, pH 7.0) and cultured with shaking at 28° C. and 20 Orpm for 4 days. 1.5 l of this culture (5 flasks) was placed in a 301 volume culture tank (3.5 g of glucose).
3%, monopotassium phosphate 0.066%, dipotassium phosphate 0.029%, magnesium sulfate 0.055%, ferrous sulfate 0.0029%, EDTAo, 0088%, urea 0.497%, A- 5 solution 0.11%, pH 7.0), inoculated into 14.5 Il, 30°C, 350 rp
m.

The cells were cultured with aeration and agitation for 60 hours at 1/4 VVM (41/m1n). During culture, the pH of the medium was adjusted to 5N NHaO.
It was adjusted to about 7.0 at H. After the culture is completed, the culture (16
A) Continuous centrifugation (10,00 Orpm, 60 mf
+/min> and collected algal bodies (wet weight 1,530 g).

Suspend the washed algae in distilled water (PCV 60-70%),
After adjusting the pH to 9.5 to 9.7 with NaOH, the mixture was heated in a water bath (97°C) for 10 minutes and cooled with cold water. The cooled treatment liquid was dried with a small spray dryer (manufactured by Tokyo Rika) to obtain 250 g of dried algal bodies (chlorella powder).

Example 3 A loaf of bread was manufactured using materials having the following composition and following the steps ① to ②.

Ingredients for 1 loaf of chlorella bread Wheat flour (strong flour) 280g Sugar
16g butter 30g salt 2
g Dry yeast 5g Chlorella powder (yellow) (MKO2 strain) 4g Milk 120ml Step ■ Add 5g of dry yeast to 40°C hot water (60°C) containing 2g of sugar and perform preliminary fermentation of the yeast.

■ 280g of strong flour, 4g of chlorella powder, and 2g of salt.

Mix 120ml of milk, 1.14g of sand, 30g of butter and 1 pre-fermented yeast liquid and knead well.

■ Place the bread dough in a double plastic bag, remove the air from the bag, tie the bag tightly, and let it ferment for 1 hour at 40℃.

■ Degas the fermented bread dough and pour it over the wet mixture 5
Let rest for ~10 minutes.

■ Put the bread dough into a mold, wrap it in a plastic bag, and bake for 30~4
Ferment at 0°C for 30 minutes.

■ Bake at 180-200℃ for 30 minutes.

The bread made with MK02 strain chlorella powder had a moderate castella-like color, especially the inside.

Instead of MK02 strain (vivid yellow),
Bread made using the parent strain's chlorella powder (dark green) has a green color on the inside and a dark green color on the surface (the skin).
It had a darker brown color and looked like overbaked bread, and was not appetizing.

Example 4 A butter roll was manufactured using materials having the following composition and following steps 5) ① to 3).

Ingredients for butter rolls with chlorella (18 pieces) Flour (strong flour) 40.0g Sand a
33g butter 80g salt
2g dry yeast 10g whole egg 50g egg yolk 120g chlorella powder (yellow> (MKO2 strain) 8g
Milk 80ml Process■ Add 10g of dry yeast to 60ml of 40℃ hot water and 113g of sand to pre-ferment the yeast.

■ Mix 400 g of strong flour, 8 g of chlorella powder, 2 g of salt, 1.30 g of milk 80-1 sand, 80 g of butter, 50 g of whole eggs, 120 g of egg yolk, and pre-fermented yeast liquid and knead well.

■ Place the bread dough in a double plastic bag, remove the air from the bag, tie the bag tightly, and let it ferment for 1 hour at 40℃.

■ After degassing the fermented bread dough and shaping it,
Secondary fermentation is performed at 0-40°C for 30 minutes.

[F] Bake at 200℃ for 10 minutes.

A comparison similar to that made with white bread revealed that when the parent strain of chlorella was used, the surface had a strong dark brown color, resembling overbaked bread. In the case of the MKO2 strain, the color tone was the same as that of white bread.

Example 5 Raw Chinese noodles were manufactured using materials having the following composition and following the steps (1) to (2).

Ingredients for raw Chinese noodles with chlorella: 350g wheat flour (strong flour) (
(soft flour) 150g Chlorella powder (yellow) (MK02 strain> 5g potato starch (used for flouring) Egg yolk 120g
Process■ Strong flour 350g, soft flour 150g, chlorella powder 5
Mix g and pass through a sieve.

■ Add 120g of egg yolk and mix. Pour water containing 2g of salt and 2g of herbs and knead.

■ Sprinkle with potato starch and repeatedly press and stretch the dough. Wrap it in a damp cloth and let it sit in the refrigerator (0-5℃) for 3 hours.

■ Sprinkle with potato starch and roll out thinly, then cut with a knife.

When powder from the MK02 strain was used, the color of the noodles became darker and the color tone was better than that of noodles containing egg yolk, making them more similar to Chinese noodles. When chlorella powder from the parent strain was used, the noodles were green, which was not desirable for Chinese noodles.

Example 6 A juice was produced using materials having the following composition and following the steps below.

Ingredients for chlorella juice: Sugar 10g
Maltose 5g citric acid
0.2g Chlorella powder (yellow> (MKO2 strain) 0.5g water
Sugar (Log) and citric acid (0,
Add 2g> to bring out the sweetness and sourness. Add maltose (5g) to eliminate the astringent taste of citric acid.

Add chlorella powder (0.5g) for coloring.

When the MK02 strain (vivid yellow) is used, the color becomes yellow with a juice-like feel, and the MK02 strain is suitable for use as a beverage. Parent stock (dark green)
When the chlorella powder (n) was used for juice, the green color became dark and it was not suitable for drinking.

In addition, the parent plant could not be used for juice at all because it browned within a few days.

Example 7 (Three-color agar for Mitsume beans) Three-color agar for Mitsume beans was produced using materials with the following composition and according to the following steps.

Ingredients for tricolor agar with chlorella Agar 3.
0g chlorella powder 0.5g parent stock ■ MKOI strain ■ MK02 strain Add chlorella powder (0.5g) to process water (200ml),
Add agar (3.0g) and heat to dissolve, cool and mash into molds.

Agar was made using three types of chlorella powder: the parent strain, the MKOI strain, and the MK02 strain. Pale green, white, and pale yellow agar are produced, which are diced and mixed to create a colorful and appetizing product.Example 8 Extraction of Pigment Fruit 4 Duck Chlorella mutant strain obtained according to the method of Example 2~ 1
After adding a small amount of water to the KO2 algae powder Log to moisten it, 500 ml of methyl alcohol was added and extracted for 1 hour at room temperature. Extract No. 131 + Togami (Toyo P Paper)
After removing algal bodies and removing methyl alcohol using a vacuum concentrator, the mixture was dried at 80° C. for 7 hours. In this way 1.16 g of oily pigment was obtained.

Example 9 (Skin irritation prevention test) The following was conducted on a total of 805 female subjects: 101 women aged 25-30, 150 aged 30-35, 221 aged 3540, 180 aged 40-45, and 153 aged 45 and over. A skin irritation prevention test was conducted. After washing the facial skin, the main pores were opened with ozone and steam, and the hot water extract of chlorella according to the present invention (10 g of dried chlorella was mixed with 100 g of water, 100°C, 2
After treatment for 0 minutes and centrifugation at 2,000×g, the supernatant was injected to allow penetration into the skin. After one treatment, 65% of those with rough skin improved, 21% of those with slight improvement, 10% of those with no change, and 4% of those with worsened skin, m = <, this Chlorella. It was found that the extract is effective for treating rough skin and is also suitable for preventing fine wrinkles.

Example 10 (Test for preventive effect on stains) The backs of mice were irradiated with ultraviolet rays for 60 minutes to produce erythema, and the chlorella hot water extract used in Example 9 was impregnated into a collagen nonwoven fabric, and the erythema was applied to the erythema. pasted. Rapid disappearance of erythema was observed in treated mice compared to untreated mice. Therefore, the chlorella extract of the present invention can be expected to have a preventive effect on human sunburn and the stains that are exacerbated by it.

Effects of the Invention According to the present invention, the content of chlorophyll is extremely low;
A novel Lorella algae having a bright yellow-green or yellow tone is provided.

Hokkaido Sugar Co., Ltd. Representative Sasaki good

Claims (6)

[Claims] (1) The content of total chlorophyll is 0.1% or less, and the amount of total PPB generated by decomposition of chlorophyll is 22%.
Chlorella pyrenoidosa (Chlorella pyrenoidosa) characterized by having a content of 0 ppm or less
) A new Chlorella algae belonging to the species. (2) The algae of the genus Chlorella according to claim 1, which has a bright yellow-green color tone. (3) Chlorella pyrenoidosa MK01ATCCNo
．． The Chlorella algae according to claim 2, which is 40867. (4) The Chlorella algae according to claim 1, which has a yellow color. (5) Chlorella pyrenoidosa MK02ATCCNo
．． 5. The Chlorella algae according to claim 4, which is 40838. (6) The content of total chlorophyll is 0.1% or less, and the amount of total PPB produced by decomposition of chlorophyll is 22%.
A food or drink containing Chlorella pyrenoidosa algae or a processed product of the algae with a concentration of 0 ppm or less.