Method for preparing high-purity carotenoids in high yield

Abstract

The present invention relates to a method for preparing high-purity carotenoids in high yield by using a carotenoid-producing strain. According to the present invention, carotenoids vulnerable to oxidation can be stably produced, and high-purity carotenoids can be produced in high yield.

Description

High-yield production method of high-purity carotenoids

[0001] The present invention relates to a method for producing carotenoids, and more particularly, to a method for producing high-purity carotenoids at high yield from a carotenoid-producing strain.

[0002]

[0003] Carotenoids are isoprenoid compounds with 40 carbon atoms and possess antioxidant activity, and are widely distributed throughout nature. To date, over 600 carotenoids with different chemical structures have been reported. Carotenoids are a group of pigments that exhibit vibrant colors, such as yellow, orange, and red, depending on their chemical structure. Representative carotenoids include lycopene, beta-carotene, lutein, astaxanthin, and zeaxanthin, and are primarily used as nutritional supplements, pharmaceuticals, food coloring, and animal feed additives. Among these, carotenoids that have the characteristic of being colorless include phytoene and phytofluene. Phytoene, which has three conjugated double bonds, is known to have excellent short-wavelength ultraviolet ray absorption in the UVB range (280–320 nm), and phytofluene, which has five conjugated double bonds, is known to have excellent long-wavelength ultraviolet ray absorption in the UVA range (320–400 nm) (Antonio J. Melendez-Martinezet et al., Arch. Biochem. Biophys., 72:188-200, 2015). In addition, phytoene and phytofluene have been found to have excellent skin whitening effects, and have recently been attracting attention for use in cosmetics and health functional foods (Korean Patent No. 10-1448317).

[0004] Phytoene and phytofluene can be produced through various methods, including direct extraction from natural products, organic chemical synthesis, and microbial fermentation. However, because phytoene and phytofluene are known to exist in small quantities in plants and some microorganisms, production and extraction using highly phytoene- and phytofluene-producing recombinant microorganisms is an economical method.

[0005] However, when extracting phytoene and phytofluene from cultures of highly phytoene- and phytofluene-producing recombinant microorganisms, there were problems such as low extraction yields, low phytoene and phytofluene contents in the final extract, and dark color of the extract. Adding a purification process to the extract could increase the purity of phytoene and phytofluene and improve color. However, disadvantages such as increased unit cost and environmental pollution due to the additional extraction and purification processes became clear.

[0006] Phytoene and phytofluene are chemically highly nonpolar and accumulate by forming granules inside the cell, which is a hydrophilic environment, rather than being released outside the cell. Therefore, phytoene and phytofluene present inside the cell must be extracted using a solvent with low polarity. Since phytoene and phytofluene have high polarity due to the high moisture content of the microbial cells and culture medium after microbial fermentation, extraction is required using microbial disruption and an organic solvent with an appropriate polarity. Hexane, which has a high nonpolarity, can dissolve carotenoids best, but the difference in polarity between water and hexane in the culture medium makes extraction impossible. In addition, fermentation alcohol also dissolves various substances of the microorganism, such as phospholipids, making it difficult to use for solvent extraction. Ethyl acetate was determined to be a relatively usable solvent for the phytoene and phytofluene extraction process.

[0007] When the inventors of the present invention use ethyl acetate as a solvent in the existing method, by changing the ratio of culture medium to ethyl acetate, RPM, and extraction time, phytoene and phytofluene are extracted by about 30%, and when the extraction time and stirring speed are increased, the extraction rate of phytoene and phytofluene increases to 40%, but there was still a problem with solvent recovery. After extraction, excluding the solid content, the microorganisms are partially dissolved in ethyl acetate, and the lipids that are generated cause three solvent layers: an aqueous solution layer, an ethyl acetate and lipid layer, and an ethyl acetate layer. Since the ethyl acetate in which phytoene and phytofluene are extracted in the ethyl acetate and lipid layer accounts for about 40% of the initial input, the ethyl acetate recovery rate decreases. In summary, about 20% of phytoene and phytofluene were extracted from the culture medium, resulting in a low extraction rate, low phytoene and phytofluene content in the final extract, and a dark color. The recovery rate of ethyl acetate can be increased by increasing the initial input amount of ethyl acetate or by adding an additional process, and the purity of phytoene and phytofluene can be increased and the color can be improved by adding a purification process to the extract. However, there are disadvantages such as increased unit price due to the additional extraction and purification process and environmental pollution issues.

[0008] To overcome this, if the cultured microorganisms are freeze-dried to remove moisture, they can be extracted in an environmentally friendly manner using supercritical carbon dioxide. However, the extraction efficiency of the supercritical carbon dioxide extraction after freeze-drying the microorganisms was less than 20%, and the purity of the extracted phytoene / phytofluene oil was also low at less than 40%. That is, the content of phytoene and phytofluene decreased rapidly during the drying process of the cells, and the content of phytoene and phytofluene in the supercritical extract was low, and the color of the extract was dark oil. To confirm whether the problem was due to the supercritical extraction, extraction was performed with solvents such as hexane, ethyl acetate, and fermentation alcohol on freeze-dried E. coli, but the extraction efficiency of less than 20%, similar to that of supercritical extraction, was observed. Subsequently, the drying method was changed to high-temperature spray drying to dry the microorganisms, but the phytoene and phytofluene contents of the dried cells decreased rapidly to less than 1%.

[0009] In addition, phytoene and phytofluene are reduced by UVA and UVB regions, and when exposed to light, the content decreased by about 15% as of 7 days compared to the unexposed experimental group. The content decreased by 40% as of 7 days when stored at room temperature of 25℃ rather than at low temperature storage below 4℃, and under high temperature conditions above 60℃, isomerization occurred in addition to the decrease in the content of phytoene and phytofluene (data omitted).

[0010] In summary, when phytoene and phytofluene-producing recombinant microorganisms are freeze-dried and supercritical extraction is performed, environmentally friendly extraction is possible. However, phytoene and phytofluene have the characteristic of being vulnerable to oxidation, which is a characteristic of antioxidants, and it was confirmed that they are easily oxidized and decomposed by oxygen, light, temperature, etc. in the environment (Alan A. Woodallet et al., Arch. Biochem. Biophys., 1336:33-42, 1997; HD Martin et al., Pure Appl. Chem., 71:2253-2262, 1999).

[0011] Accordingly, the inventors of the present invention have made diligent efforts to solve the above problems and have confirmed that when a recombinant microbial cell producing high levels of phytoene and phytofluene is freeze-dried by adding polyol, stored while blocking oxygen, light, and heat, and then extracted, high-purity phytoene and phytofluene can be obtained with high efficiency, thereby completing the present invention.

[0012]

[0013] Summary of the invention

[0014] The purpose of the present invention is to provide a method for producing high-purity carotenoids at high yield from a carotenoid-producing strain.

[0015] To achieve the above object, the present invention provides a method for producing carotenoids using a carotenoid-producing strain, comprising the following steps:

[0016] (a) A step of culturing a carotenoid-producing strain to obtain a strain in which carotenoids are accumulated within the strain;

[0017] (b) a step of treating the cell body in which the carotenoid has accumulated with a polyol stabilizer and drying it to obtain a dried cell body;

[0018] (c) a step of storing the dried fungi; and

[0019] (d) A step of extracting carotenoids from the stored dried fungi to obtain a carotenoid extract.

[0020]

[0021] Figure 1 is a schematic diagram of a process for producing high-purity, high-efficiency phytoene and phytofluene oil from highly carotenoid-producing microorganisms.

[0022] Figure 2 shows the results of confirming the one-day stability of phytoene and phytofluene in a control group not treated with a polyol stabilizer, an experimental group treated with polyols arbutin, trehalose, and glycerol, respectively, and an experimental group not treated with polyol but with an oxygen absorber added.

[0023] Figure 3 shows the results of confirming the 7-day stability of phytoene and phytofluene in room temperature vacuum storage of freeze-dried products obtained by air saturation and nitrogen saturation, respectively, at the saturation stage at the end of freeze-drying.

[0024] Figure 4 shows the results of confirming the 5-day stability of phytoene and phytofluene under untreated storage, nitrogen-filled storage, and oxygen absorber addition conditions in dry room temperature storage.

[0025] Figure 5 shows the properties of the initial supercritical carbon dioxide extract of phytoene and phytofluene, the ethyl acetate solvent extract, and the supercritical carbon dioxide extract obtained by applying the process of the present invention.

[0026] Figure 6 shows the results of confirming the 68-day stability of the untreated group, tocopheryl acetate, and Tinoguard TT treated group of 10% diluted phytoene and phytofluene products.

[0027]

[0028] Detailed description of the invention and preferred embodiments

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, unless otherwise defined herein. Generally, the nomenclature used herein is well known and commonly used in the art.

[0030] The present invention aimed to develop a stable method for producing carotenoids, which have high antioxidant activity but are themselves vulnerable to oxidation, and developed a high-purity, high-efficiency method for producing representative carotenoids, phytoene and phytofluene. Specifically, a process was developed for culturing a recombinant microorganism (Korean Patent No. 10-2229379) that simultaneously produces phytoene and phytofluene, and obtaining an extract containing highly pure phytoene and phytofluene in high yield from the cultured cells.

[0031] In the present invention, it was confirmed that when a recombinant microbial cell producing high levels of phytoene and phytofluene is freeze-dried by adding polyol and then blocking oxygen, light, and heat to increase storage stability, high-purity phytoene and phytofluene can be extracted with high efficiency.

[0032]

[0033] Accordingly, the present invention relates to a method for producing carotenoids using a carotenoid-producing strain comprising the following steps:

[0034] (a) A step of culturing a carotenoid-producing strain to obtain a strain in which carotenoids are accumulated within the strain;

[0035] (b) a step of treating the cell body in which the carotenoid has accumulated with a polyol stabilizer and drying it to obtain a dried cell body;

[0036] (c) a step of storing the dried fungi; and

[0037] (d) A step of extracting carotenoids from the stored dried fungi to obtain a carotenoid extract.

[0038] In the present invention, the polyol stabilizer may include, but is not limited to, glycerol, trehalose, arbutin, etc.

[0039] In the present invention, the polyol helps maintain the microbial membrane structure, thereby increasing the storage stability of carotenoids within the cells.

[0040] In the present invention, the drying in step (b) is preferably performed in a vacuum at room temperature or in a frozen state to minimize oxygen contact, and the storage in step (c) is preferably performed by blocking oxygen, light, and heat.

[0041] In addition, it is preferable to store the dried fungi using at least one method selected from the group consisting of vacuum packaging, nitrogen substitution packaging, oxygen absorber treatment, low-temperature storage, and sealed storage.

[0042] In the present invention, the storage in step (c) is preferably performed for 0 to 7 days, more preferably for 0 to 2 days, and even more preferably, it is performed immediately without storage.

[0043] In the present invention, the extraction in step (d) may be characterized by extraction using supercritical carbon dioxide.

[0044] In the present invention, (e) it may be characterized by further including a step of formulating by mixing an antioxidant into the carotenoid extract, and the antioxidant may be tocopheryl acetate or Tinoguard TT, but is not limited thereto.

[0045] In the present invention, the carotenoid may be characterized as being phytoene or phytofluene.

[0046] In the present invention, the carotenoid-producing strain can be used without limitation as long as it is a strain capable of producing carotenoids and accumulating them within the cell body, and preferably, a recombinant microorganism having high carotenoid productivity can be used.

[0047] In one embodiment of the present invention, a recombinant microorganism PTFsynS-MGLB that simultaneously produces phytoene and phytofluene disclosed in Korean Patent No. 10-2229379 was used, but the present invention is not limited thereto. The recombinant microorganism that simultaneously produces phytoene and phytofluene is a recombinant microorganism having the ability to produce phytofluene, in which a gene encoding an enzyme that polymerizes farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP), a gene encoding an enzyme that polymerizes geranylgeranyl pyrophosphate (GGPP) into phytoene, and a gene derived from Synechocystis sp. encoding an enzyme that converts phytoene into phytofluene have been introduced into the microorganism that inherently has a farnesyl pyrophosphate (FPP) biosynthetic pathway (Table 1).

[0048]

[0049] In one embodiment of the present invention, after the fermentation culture of a microorganism that simultaneously produces phytoene and phytofluene is completed, a polyol such as arbutin, trehalose, or glycerol is added. When the polyol is added, the polyol is located on the cell membrane of the microorganism, thereby minimizing changes in the cell membrane structure that occur during the drying step and reducing oxygen penetration into the membrane. The microorganism is dried while maintaining a vacuum state, and moisture inside the culture medium and the microorganism is removed through room temperature vacuum drying or freeze vacuum drying. When the vacuum is released at the end of the drying step, nitrogen gas is used to saturate the space inside the dryer with nitrogen and complete the drying process. This minimizes oxygen contact by filling the internal micro spaces between the dried cells with nitrogen.

[0050] For storage of dried phytoene and phytofluene-producing microorganisms, the storage containers are filled with nitrogen or vacuumed and oxygen absorbers are added in low-temperature conditions, blocking light, to minimize oxygen contact, light exposure, and temperature changes.

[0051] Highly productive microorganisms producing phytoene and phytofluene in storage can recover high-purity phytoene and phytofluene oils with a purity of 90% or more through an extraction process using supercritical carbon dioxide.

[0052] Extracted phytoene and phytofluene oils are stored in a sealed, low-temperature, oxygen-free environment. When diluting oils, the microscopic air spaces within the oil are replaced with nitrogen. Additionally, a non-polar antioxidant, such as tocopheryl acetate or Tinoguard TT, is added to the product, which is then stored in a sealed, low-temperature, oxygen-free environment.

[0053]

[0054] [Example]

[0055] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0056]

[0057] Example 1: Cultivation of carotenoid-producing strains

[0058] A recombinant E. coli strain PTFsynS-MGLB (Korean Patent No. 10-2229379), which can simultaneously produce phytoene and phytofluene as carotenoids, was inoculated into Terrific Broth (Difco) and cultured at 30°C and 200 rpm for 48 hours.

[0059] The above recombinant strain PTFsynS-MGLB is a recombinant microorganism having the ability to produce phytofluene, which has been introduced into a microorganism endogenously possessing a farnesyl pyrophosphate (FPP) biosynthetic pathway a gene encoding an enzyme that polymerizes farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP), a gene encoding an enzyme that polymerizes geranylgeranyl pyrophosphate (GGPP) into phytoene, and a gene derived from Synechocystis sp. encoding an enzyme that converts phytoene into phytofluene.

[0060]

[0061] Example 2: Preparation of strains treated with polyol protectants

[0062] In Example 1, after the culture of the high-carotenoid producing strain was completed, in order to increase the stability of the cells, a polyol protective agent that helps maintain the microbial membrane structure was added to the culture solution as shown in Table 2, stirred for 1 hour, and then the culture solution was frozen at -80°C.

[0063]

[0064] Vacuum freeze-drying was performed for 3 days, and the dried microorganisms were stored in a sealed container at room temperature for 1 day, after which the contents of phytoene and phytofluene were measured.

[0065] As a result, as shown in Table 2 and Figure 2, in all groups containing the polyols arbutin, trehalose, and glycerol, carotenoid stability increased, and the maximum storage stability on the first day was 97% for phytoene and 90% for phytofluene.

[0066]

[0067] Example 3: Nitrogen saturation and vacuum storage of dried microorganisms at the end of vacuum drying.

[0068] At the end of the vacuum drying process, the vacuum was released using nitrogen gas instead of atmospheric air. The dried cells were then vacuum-packed to minimize the amount of air inside the storage container and stored at room temperature for 7 days. When nitrogen saturation was performed at the end of the vacuum drying process, the storage stability of phytoene increased by 19% and that of phytofluene increased by 32% compared to the air saturation experimental group (Fig. 3).

[0069]

[0070] Example 4: Method for preserving dried microorganisms

[0071] In addition to the vacuum packaging of Example 3, when the packaging container was filled with nitrogen to replace the residual air with nitrogen and minimize oxygen contact as a storage method for dried carotenoid-producing microorganisms, the storage stability of phytoene and phytofluene increased by 80% compared to the untreated group based on 5 days of storage at room temperature (Fig. 4).

[0072] In addition, when an oxygen absorber was added inside the sealed container, storage stability of carotenoids of more than 99% could be achieved through chemical removal of oxygen molecules (Fig. 4).

[0073]

[0074] Example 5: Extraction of carotenoids from dried microorganisms using supercritical carbon dioxide

[0075] A dried cell with high carotenoid productivity was produced by applying the methods of Examples 2 to 4, and a control dried cell was produced without applying the methods of Examples 2 to 4, and supercritical carbon dioxide extraction was performed on each.

[0076] The culture solution prepared by the method of Example 1 was directly extracted with ethyl acetate solvent, and the properties of the extracts from which the ethyl acetate solvent was removed using a rotary still were compared. The color of the supercritical carbon dioxide extract without applying the method of Examples 2 to 4 was the darkest, and the color of the supercritical carbon dioxide extract to which the method of Examples 2 to 4 was applied was the lightest (Fig. 5). The lighter the color of each extract, the greater the phytoene and phytofluene content, and the supercritical carbon dioxide extract to which the process was applied showed a purity of approximately 97% (Table 4).

[0077]

[0078] In Table 4, the extraction rate is the ratio of the total amount of phytoene and phytofluene obtained by extraction to the total phytoene and phytofluene of the initial input sample, and the residual rate is the ratio of the total amount of phytoene and phytofluene obtained by re-extracting the remaining sample after the extraction process. By applying the carotenoid stability enhancement process, high-purity, high-extraction efficiency phytoene and phytofluene oils could be obtained.

[0079]

[0080] Example 6: Formulation of phytoene and phytofluene oils

[0081] To enhance the stability of phytoene and phytofluene formulations, the diluted products were saturated with nitrogen gas to replace the microscopic air spaces with nitrogen, and tocopheryl acetate or Tinoguard TT, a hydrophobic antioxidant, was added. In the case of Tinoguard TT, stability was secured for up to 68 days at room temperature (Fig. 6).

[0082]

[0083] According to the present invention, stable production of carotenoids having a property of being vulnerable to oxidation is possible, and high-purity carotenoids can be produced at a high yield.

[0084]

[0085] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for producing carotenoids using a carotenoid-producing strain comprising the following steps: (a) A step of culturing a carotenoid-producing strain to obtain a strain in which carotenoids are accumulated within the strain; (b) a step of treating the cell body in which the carotenoid has accumulated with a polyol stabilizer and drying it to obtain a dried cell body; (c) a step of storing the dried fungi; and (d) A step of extracting carotenoids from the stored dried fungi to obtain a carotenoid extract.

2. A method according to claim 1, characterized in that the polyol stabilizer is selected from the group consisting of glycerol, trehalose, and arbutin.

3. In the first paragraph, the drying in step (b) is characterized in that it is dried in a vacuum at room temperature or in a frozen state to minimize oxygen contact.

4. A method characterized in that, in the first paragraph, oxygen, light and heat are blocked during storage in step (c).

5. A method according to claim 1, characterized in that the storage in step (c) is performed for 0 to 7 days.

6. A method according to claim 1, characterized in that the extraction in step (d) is performed using supercritical carbon dioxide.

7. A method characterized in that, in the first paragraph, the storage in step (c) uses at least one method selected from the group consisting of vacuum packaging, nitrogen substitution packaging, oxygen absorbent treatment, low-temperature storage, and sealed storage.

8. A method according to claim 1, characterized in that it further comprises a step of mixing an antioxidant into the carotenoid extract and formulating the same.

9. A method according to claim 8, characterized in that the antioxidant is tocopheryl acetate or Tinoguard TT.

10. A method according to claim 1, characterized in that the carotenoid is phytoene or phytofluene.

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