JP2006348270A - Method for preserving xanthophyll in algal body - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preserving xanthophyll in a xanthophyll-containing algal body. <P>SOLUTION: The method for preserving xanthophyll in an algal body comprises the steps of culturing an algal body containing xanthophyll and then collecting the algal body, and drying the collected algal body to a state that it has a moisture content of 3% by mass or less. Preferably, drying is performed using a spray drying method under drying conditions wherein the temperature of the air discharged from a spray drier is 125°C or higher. In such a dry algal body, xanthophyll can be preserved stably for a long term even at room temperature in air. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for preserving xanthophyll in algae containing xanthophylls, particularly astaxanthin. Furthermore, this invention relates to the method of improving the storage stability of the xanthophyll in a xanthophyll containing dry alga body.

  Xanthophyll (a type of carotenoid, such as astaxanthin, canthaxanthin, zeaxanthin, adonilpine, adonixanthin, cryptoxanthin) is used for various applications. For example, astaxanthin is a kind of red carotenoid and is known to have a strong antioxidant effect. Therefore, it is used as a feed for coloring fish, coloring materials for foodstuffs, cosmetics, health foods, and the like. As a feed containing astaxanthin, for example, a feed obtained by treating Phaffia yeast with NaOH (Patent Document 1), a feed obtained by crushing alga bodies, lyophilized and powdered (Patent Document 2) ).

  Astaxanthin is chemically synthesized or extracted from natural products such as shrimp such as krill and flax shrimp, faffia yeast, and algae. When extracting astaxanthin from crustaceans such as krill, lobster, crab, or Phaffia yeast, the yield is very low due to their low astaxanthin content. Therefore, it is generally extracted from algae with a high astaxanthin content, for example, green algae belonging to the genus Haematococcus.

  Astaxanthin is unstable to heat, oxygen, light, etc., and the culture solution of algal cells cannot be stored as it is. Therefore, extraction of astaxanthin is often performed immediately after culturing algal cells (see, for example, Patent Documents 3 and 4). In this case, it is necessary to provide the culture device and the extraction device in the same place. When a large amount of algal cells are cultured at once, it is necessary to store the culture solution or extract once. To prevent oxidation and decomposition of astaxanthin, block light in a nitrogen atmosphere and store it at a low temperature. Must. There is also a need to prevent spoilage caused by bacterial growth. Therefore, further storage facilities are required, leading to an increase in the cost of the extraction process.

  In order to solve the above problem, there is a method of drying an algal body containing astaxanthin. Patent Document 5 describes a method of drying algal bodies containing carotenoids and crushing them in edible vegetable oil. Patent Document 6 also describes drying alga bodies containing astaxanthin. However, these Patent Documents 5 and 6 do not discuss the stability of astaxanthin itself against heat, oxygen, light and the like during drying. For example, according to the study of the present inventor, it has been clarified that astaxanthin in the alga body is decomposed and the astaxanthin content is lowered when it is simply dried using the drying method described in Patent Document 6.

  Furthermore, a method for improving the long-term storage stability of astaxanthin has been developed by mixing a crushed alga and various additives into a powder composition (Patent Document 7). However, this method requires an additive such as a surfactant and an antioxidant, which increases the cost. Alternatively, since the surfactant is mixed in the extract, it is necessary to remove the surfactant thereafter. Therefore, this method of using an additive is not preferable.

Thus, the method of preserving xanthophylls in alga bodies for a long time and stably by a simple method is not yet known.
JP-A-6-105657 JP-A-3-83577 Japanese Patent Laid-Open No. 9-111139 JP 53-38653 A Japanese Patent Laid-Open No. 7-8212 JP 2004-129504 A International Publication No. 2002/077105 Pamphlet Atsuko Sato et al., Japanese Journal of Food Hygiene, 39, 6, 368-374, 1998

  An object of the present invention is to provide a method for preserving xanthophyll in a xanthophyll-containing alga body, thereby providing a method for enhancing the storage stability of xanthophyll, particularly astaxanthin, in the xanthophyll-containing dry alga body.

The present invention provides a method for preserving xanthophyll in algal cells, the method comprising:
A step of culturing algal bodies containing xanthophyll and then recovering; and a step of drying the recovered algal bodies so that the water content is 3% by mass or less;
including.

  The present invention also provides a method for enhancing the storage stability of xanthophyll in a dry alga body containing xanthophyll, the method comprising drying the alga body so that the water content in the alga body is 3% by mass or less. including.

  In certain embodiments, in any of the above methods, the xanthophyll is astaxanthin.

  In a further embodiment, in any of the above methods, the algal body is a green alga belonging to the genus Haematococcus.

  In a still further embodiment, in any of the above methods, the green algae is Haematococcus pluvialis.

  In another embodiment, in any of the above methods, the drying is performed by a spray drying method.

  In a further embodiment, in any of the above methods, the spray drying method is performed using a spray drying device, and the exhaust air temperature is adjusted to 125 ° C. or higher in the spray drying device.

  The present invention further provides a method for producing xanthophyll, which method comprises enhancing the storage stability of any of the above xanthophylls.

  According to the method of the present invention, xanthophyll in xanthophyll-containing dry algae can be stably maintained at room temperature (that is, without being frozen or cooled) for a long period of time. The xanthophyll-containing dry alga obtained by the method of the present invention can be stored for a long time at room temperature, and can be appropriately used for extraction and purification of xanthophyll, particularly astaxanthin. Therefore, for example, an astaxanthin-containing alga body culture step and an astaxanthin extraction / purification step can be separated, and astaxanthin can be produced efficiently and at low cost. Moreover, according to the method of the present invention, a high concentration of astaxanthin can be easily stored, so that extraction and recovery of astaxanthin can be performed efficiently. Furthermore, when the astaxanthin-containing dry alga obtained by the method of the present invention is used as a feed for fish, the astaxanthin in the feed is stably stored for a long period of time, so that the fish color-raising effect is improved.

(Algae)
The algae used in the present invention is not particularly limited as long as it is an algae that produces xanthophyll. Typically, green algae such as unicellular algae belonging to the genus Haematococcus are preferably used. Algae of the genus Haematococcus include Haematococcus prubialis, H. lacustris, H. capensis, Hematococcus droebakensi, Hematococcus・ Zimbabwiensis (H. zimbabwiensis) and the like.

  As H. pluvialis, NIES 144 strain deposited at the National Institute for Environmental Studies, UTEX 2505 strain deposited at the University of Texas Algae Conservation Facility, Scandinavian Culture Center for Copenhagen University of Denmark, Denmark Examples include the K0084 strain preserved in Algae and Protozoa, Botanical Institute.

  As H. lacustris, ATCC 30402 strain and 30453 strain deposited with ATCC, IAM C-392 strain and C-393 strain deposited with University of Tokyo Institute for Molecular Cell Biology, C-394 and C-339, or UTEX 16 and 294.

  Examples of Hematococcus capensis (H. capensis) include UTEX LB1023 strain.

  Examples of Haematococcus droebakensi (H. droebakensi) include UTEX 55 strain.

  Examples of Hematococcus zimbabwiensis (H. zimbabwiensis) include UTEX LB1758 strain.

  Among these, Haematococcus prubiaris is preferably used.

(Culture of alga bodies containing xanthophyll)
An algal body containing xanthophyll is obtained by culturing the algal body under conditions capable of producing xanthophyll. There is no restriction | limiting in particular in the production conditions of a xanthophyll. For example, after growing vegetative cells of algal cells in a medium containing a nitrogen source and trace metals under light conditions with aeration of carbon dioxide, the grown algal cells are subjected to physical stress such as light irradiation, Examples thereof include a method of inducing vegetative cells to cyst formation by applying biological stress such as nutrient starvation or stress such as chemical stress caused by addition of hydrogen peroxide and / or iron compounds, alone or in combination. Moreover, acetic acid etc. may be added as a carbon source, and dark culture may be performed to vegetatively grow algal bodies, followed by cystization (see, for example, Patent Documents 2 and 6).

  The obtained algal bodies are collected, for example, by centrifugation, washed with water as necessary, and then adjusted to a solid content concentration of about 5 to 40%, preferably 10 to 30%. It attaches | subjects to the drying process explained in full detail.

  In the present invention, xanthophyll is a generic name for carotenoids containing oxygen-containing functional groups, and does not mean a specific compound. In the present invention, it refers to xanthophylls produced mainly by algae, and typical examples of such xanthophylls include astaxanthin, canthaxanthin, zeaxanthin, adonilpine, adonixanthin, cryptoxanthin, and fatty acid esters thereof.

(Algae drying)
The algal bodies containing xanthophyll are dried so that the moisture content in the dried algal bodies is 3% by mass or less. If the moisture content in the dried alga body exceeds 3% by mass, the decomposition of xanthophyll in the dried alga body proceeds during storage, and the storage stability of xanthophyll deteriorates.

  There is no particular limitation on the drying method. Drum drying (for example, refer to the pamphlet of International Publication No. 2000/057724), hot air drying method (for example, see JP-A No. 2002-119252), freeze drying method (for example, US Pat. No. 6,163,379) And the spray drying method. From the viewpoint of preventing thermal decomposition of xanthophyll, low-temperature drying such as a freeze drying method is preferred, but there are problems such as time-consuming drying and cost of drying equipment. In the case of the drum drying method, the hot air drying method, or the spray drying method, since xanthophyll may be decomposed by heating, attention should be paid to the heating temperature and the heating time.

  The drying temperature is preferably as low as possible so that xanthophylls (particularly esters with fatty acids) in the alga are not decomposed, and the temperature in the alga is preferably not more than 100 ° C. In the drum drying method and the hot air drying method, if the heating temperature is 100 ° C. or higher, the temperature in the algal cells may rise to 100 ° C. or higher. In spray drying, since a slurry of algal bodies is sprayed, the sprayed algal bodies hardly form a lump. Therefore, the algal bodies can be dried uniformly in a short time, and thermal decomposition of xanthophyll in the algal bodies can be suppressed. Therefore, in the present invention, the spray drying method is preferably employed.

  The spray drying method can be performed using any spray drying apparatus. A typical spray drying apparatus is schematically shown in FIG. The alga body drying process using such a spray drying apparatus will be described with reference to FIG.

  A slurry containing algal bodies is supplied from a slurry tank 2 to the main body 1 of the spray drying apparatus. The slurry is sprayed into the main body 1 from the tip of the atomizer 4 together with the compressed air from the compressor 3. Air heated by the heater 6 in the heater chamber 5 is supplied into the main body 1 and the sprayed alga bodies are dried. The algal bodies fall and accumulate on the slope portion 7 of the main body 1. The hammer 8 provided on the slope portion 7 gives an impact to the slope portion 7 and drops the accumulated algal bodies to the lower part of the main body 1. A fan 10 is provided at the rear end of the flow path 9. By rotating the fan 10, air heated by the heater 6 is introduced into the main body 1 from the heater chamber 5 and dried. Is introduced into a cyclone (collector) 11. In addition, the thermometer 12 provided in the flow path 9 measures the temperature of the air containing the dried alga bodies that are guided from the main body 1 to the cyclone (collector) 11. In the cyclone 11, dry algal bodies accumulate on the lower part, so that the algal bodies can be recovered in the container 14 by opening the valve 13.

  In such a spray-drying apparatus, the temperature of hot air introduced from the heater chamber 6 into the main body 1 is preferably 200 to 400 ° C, more preferably 250 to 350 ° C, and even more preferably around 300 ° C. In the spray drying apparatus, the exhaust air temperature in the flow path 9 is preferably higher than 120 ° C, more preferably 125 ° C or higher, and further preferably 130 ° C or higher. However, if the exhaust air temperature exceeds 150 ° C., the temperature in the alga body becomes too high and the xanthophyll may be thermally decomposed. Therefore, the exhaust air temperature is preferably less than 150 ° C., more preferably 140 ° C. or less. By performing drying by such a spray drying method, the moisture content of the dried alga body becomes 3% by mass or less in a relatively short time. In particular, when the xanthophyll is astaxanthin, it is preferable to keep the exhaust air temperature at 150 ° C. or lower. Furthermore, the spray drying method is preferable because the moisture content in the dried alga can be adjusted almost independently of the concentration of the dried alga by adjusting the exhaust air temperature.

  By setting the water content to 3% by mass or less by the above process, more preferably, the dry air alga body is dried by adjusting the exhaust air temperature to a temperature of 125 ° C. or higher by using a spray drying method. The water content in it becomes 3% or less. Even when the obtained dried alga body is placed in a glass bottle or the like in air of 30 to 40% humidity, sealed and shielded from light, and stored at room temperature, the decomposition of xanthophyll hardly occurs. In an embodiment, when alga bodies containing astaxanthin and having a water content of 3% or less are stored in air at 30 to 40% humidity at room temperature for 50 days, the degradation rate of astaxanthin is less than 10%. . On the other hand, when the moisture content in the dried algae exceeds 3%, the astaxanthin degradation rate reaches 40% when stored for 50 days under the same conditions. Thus, according to the method of the present invention, the storage stability of xanthophyll in algal cells is enhanced, and therefore xanthophyll can be stored in algal cells for a long period of time. In order to prevent further decomposition of xanthophyll after drying, it is preferable to store the dried alga body in a low humidity and low oxygen atmosphere, for example, in a vacuum aluminum pack so as not to absorb moisture.

In addition, the moisture content of the dried alga body is determined by the following method. Place the glass petri dish with a lid in a low-temperature dryer adjusted to a predetermined temperature, heat for 1 hour, and transfer to a desiccator. When the temperature of the glass petri dish reaches room temperature after standing to cool, it is immediately weighed to the nearest 0.1 mg. The operation of heating, cooling, and weighing is repeated again to obtain a constant weight (W ₀ g). Next, a 1-2 g sample is quickly collected, spread flat on a glass petri dish, capped, and accurately weighed (W ₁ g). Slide the lid into the low temperature dryer. After the low-temperature dryer reaches 105 ° C., after drying for 3 hours, the glass petri dish is quickly covered in the dryer, transferred to a desiccator and allowed to cool. Weigh immediately after reaching room temperature (W ₂ g). The moisture content in the sample can be obtained by the following formula.
Moisture in sample (%) = (W ₁ −W ₂ ) / (W ₁ −W ₀ ) × 100

(Extraction, recovery and purification of xanthophyll)
Xanthophyll can be extracted and recovered from dried algal bodies having a water content of 3% by mass or less. There is no restriction | limiting in particular in the extraction and collection | recovery method of xanthophyll, The method normally used by those skilled in the art is used. For example, a chemical extraction method using an organic solvent such as chloroform, hexane, acetone, methanol, or ethanol, an edible fat or the like, or a physical extraction method such as pressing is used. Or you may extract and collect | recover using a supercritical extraction method. After extraction, it can be purified by methods such as concentration / crystallization, fractionation with a synthetic resin (for example, styrene-divinylbenzene copolymer).

  According to the present invention, since xanthophyll is stored at a high concentration in an alga body whose water content is adjusted to 3% by mass or less, the amount of astaxanthin extracted and recovered from the alga body does not decrease. Therefore, xanthophyll can be efficiently recovered and produced from algae cultured in large quantities by using the storage method or the method for enhancing storage stability of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to examples using an algal body of the genus Haematococcus, but it is needless to say that the present invention is not limited to these examples.

  First, a method for measuring xanthophylls, a method for measuring astaxanthin isomers, and a method for measuring the dry mass of algal cells will be described.

(Measurement of xanthophyll)
A certain amount of sample is taken into a micro tube dedicated to the bead beader. Add zirconia beads to the tube, add acetone, and crush the sample with a bead beader. After disruption, the sample is separated into a supernatant and a precipitate by centrifugation, and the supernatant (ie, acetone fraction) is collected. Acetone is added again to the precipitate, and the same operation as described above is repeated until the color of the precipitate becomes almost completely white. The collected acetone fractions are combined, diluted 100-fold with dimethyl sulfoxide (DMSO), and the absorbance at ₄₉₂ nm (A ₄₉₂ ) and the absorbance at 750 nm (A ₇₅₀ ) are measured. The concentration of xanthophyll (free body conversion) in the recovered acetone fraction (sample) can be obtained by the following equation.

Xanthophyll concentration in sample (μg / mL) = 4.5 × 100 × (A ₄₉₂ −A ₇₅₀ )

  The xanthophylls produced by Haematococcus pluvialis used in the following examples are mostly astaxanthin. The above xanthophyll measurement method is also applied to the measurement of astaxanthin.

(Measurement method of astaxanthin isomer)
The sample prepared for the measurement of xanthophyll (recovered acetone fraction) is dissolved in 0.15M phosphate buffer (pH 7.0), and cholesterol esterase is added to hydrolyze the astaxanthin ester. The hydrolyzate is analyzed by HPLC with a C18 column. A UV detector or a photodiode array detector is used as the detector. HPLC analysis conditions are the same as those described in Non-Patent Document 1. Under these conditions, trans-astaxanthin (retention time of about 7.9 minutes), 9-cis-astaxanthin (retention time of about 8.9 minutes), and 13-cis-astaxanthin (retention time of about 9.3 minutes) were eluted in this order. To do. Assuming that these compounds have the same molar extinction coefficient, the ratio of each compound is determined by comparing the area values.

(Measurement method of alga body concentration)
The algal concentration is measured as follows. A predetermined amount of the culture solution is collected and suction filtered on a GC50 glass fiber filter paper (manufactured by TOYO ADVANTEC). In order to dissolve inorganic salts, the filter paper is washed twice with 5 mL of aqueous hydrochloric acid at pH 4. Next, the filter paper is dried with a constant temperature dryer at 105 ° C. for 3 hours, cooled to room temperature in a vacuum desiccator for 1 hour, and the dry weight of the filter paper is measured. The GC50 glass fiber filter paper is dried in advance at 105 ° C. for 1 hour with the above-mentioned constant temperature dryer, and its mass is measured. The alga mass concentration per unit volume or per unit mass is measured by subtracting the dry mass of the filter paper measured in advance from the mass of the dry filter paper adsorbed with algal bodies to obtain the dry mass of the algal bodies. In addition, the algal body density | concentration in the dispersion liquid of a dry algal body is measured by the method similar to the above except not wash | cleaning with hydrochloric acid aqueous solution.

(Preparation Example: Culture of alga bodies containing astaxanthin)
A Haematococcus pluvialis K0084 strain (hereinafter simply referred to as K0084 strain) that produces astaxanthin was used. Place 1 L of MBG-11 medium containing the components shown in Table 1 below into a 1.5 L light path 25 mm closed flat culture bottle and inoculate K0084 strain so that the initial concentration is 0.6 g / L. did.

While the strain K0084 was aerated with air containing 3% by volume of CO ₂ at a rate of 600 mL / min (ie, 0.6 vvm), the culture temperature was adjusted between 25 ° C. and pH between 6-8, The cells were cultured for 5 days under the light irradiation conditions shown.

For light irradiation, a white fluorescent lamp (manufactured by Matsushita Electric Industrial Co., Ltd., FL40SSW / 37) was used as a light source. The intensity of the light irradiation was adjusted so that the PPFD in the culture tank light receiving direction measured using a LICOR-190SA planar photon sensor was 100 μmol-p / m ² s.

  The K0084 strain after the culture changed from green to brown to brown, and it was confirmed that it was cysted.

  Next, the same medium (MBG-11 medium) is put in the same flat culture bottle as described above, and the K0084 strain cystized so that the initial concentration is 0.6 g / L is inoculated. Incubate for hours. By this culture, algal bodies containing 4.2% by mass of astaxanthin were obtained.

(Example: Preparation of dried alga bodies)
The algal bodies obtained by the above preparation examples were collected by centrifugation, washed with water, adjusted so that the amount of algal bodies was approximately 18% by mass, and subjected to drying by a spray drying method. Using a spray dryer (Anhydro APV PSD52), set the hot air temperature on the inlet side to 300 ° C and the exhaust air temperature on the outlet side to 105 ° C, 120 ° C, 130 ° C, or 140 ° C. Got. For each of the dry alga bodies A to D, the water content, the astaxanthin concentration, and the concentration of the astaxanthin isomer were measured. The results are shown in Table 2.

  As can be seen from this table, the water content in the dried alga body decreased as the exhaust air temperature increased, and was 1.9% when the exhaust air temperature was 140 ° C. Moreover, most of the astaxanthin in the algal cells is originally a trans form in which the double bonds at the 9th and 13th positions are both. However, as the exhaust air temperature increased, the isomers 9-cis (9-cis) and 13-cis (13-cis) tended to increase. This indicates that isomerization of astaxanthin proceeds due to heat.

  Next, each obtained dry alga body AD was put into a glass bottle so that it might become 30 volume%. This was sealed and stored at room temperature under a humidity of 30 to 40%, and the concentration of each astaxanthin was measured over time and used as an index of storage stability. The results are shown in FIG.

  As can be seen from FIG. 2, in the dry algal bodies C and D having a water content of 3% by mass or less, the decomposition of astaxanthin was extremely gradual, despite being stored in an air atmosphere at room temperature in a glass bottle. . In particular, in dry algal bodies D having a water content of 2% by mass or less, astaxanthin hardly decomposed even after 80 days or more. Moreover, in the dry algae C, the degradation rate was 10% or less even on the 50th day. On the other hand, in the dry alga bodies A and B having a water content of 3% by mass or more, astaxanthin decomposition progressed, and on the 50th day, 30 to 40% of astaxanthin was decomposed. Considering these facts, it has been clarified that the residual moisture in the dried alga body has a greater influence on the decomposition of astaxanthin than the heat when the alga body is dried. From these facts, it was shown that astaxanthin can be stably stored by controlling the water content in the dried algal bodies to 3% by mass or less. Moreover, when the dry algal bodies C and D obtained by the method of the present invention were compared with the dry algal bodies A and B corresponding to the comparative examples, a large difference was found in the amount of residual astaxanthin after the 20th day from the start of storage. Therefore, the method of the present invention is particularly useful when storing for 20 days or more.

  From the above, when using the spray drying method, it was found that even if some astaxanthin thermal decomposition was allowed, the moisture content of 3% by mass or less was finally effective for stable storage of astaxanthin. It was.

  According to the method of the present invention, xanthophyll (astaxanthin) in dry algae containing xanthophylls, particularly astaxanthin that is easily decomposed by heat, oxygen, light, etc., can be maintained stably at room temperature and in air for a long period of time. Can do. Therefore, it is useful for the stabilization and production of xanthophyll, particularly astaxanthin. Moreover, according to the method of the present invention, when the astaxanthin-containing dry alga is used as a feed for fish, the astaxanthin in the feed is stably stored for a long period of time, so that the fish color-raising effect is improved.

It is a schematic diagram of the spray-dry apparatus which can be used for the method of this invention. It is a graph which shows a time-dependent change of the astaxanthin content in each dry alga body.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Main body 2 Slurry tank 3 Compressor 4 Atomizer 5 Heater chamber 6 Heater 7 Slope part 8 Hammer 9 Flow path 10 Fan 11 Cyclone (collector)
12 Thermometer 13 Valve 14 Container

Claims (13)

A method for preserving xanthophyll in algal cells,
A step of culturing algal bodies containing xanthophyll and then recovering; and a step of drying the recovered algal bodies so that the water content is 3% by mass or less;
Including a method.   The method of claim 1, wherein the xanthophyll is astaxanthin.   The method according to claim 1, wherein the algal body is a green alga belonging to the genus Haematococcus.   The method according to claim 3, wherein the green algae is Haematococcus pluvialis.   The method according to claim 1, wherein the drying is performed by a spray drying method.   The method according to claim 5, wherein the spray drying method is performed using a spray drying device, and the exhaust air temperature is adjusted to be 125 ° C. or higher in the spray drying device.   A method for enhancing the storage stability of xanthophyll in a dried xanthophyllium-containing alga body, the method comprising the step of drying the alga body so that the water content in the alga body is 3% by mass or less.   8. The method of claim 7, wherein the xanthophyll is astaxanthin.   The method according to claim 7 or 8, wherein the drying is performed by a spray drying method.   The method according to claim 9, wherein the spray drying method is performed using a spray drying device, and the exhaust air temperature is adjusted to be 125 ° C. or higher in the spray drying device.   The method according to any one of claims 7 to 10, wherein the algal body is a green alga belonging to the genus Haematococcus.   The method according to claim 11, wherein the green algae is Haematococcus pluvialis.   A method for producing xanthophyll comprising the method according to any one of claims 7 to 12.

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