KR100768757B1 - Process for the production of Chlorella containing omega-3 fatty acids - Google Patents

Abstract

The present invention provides an omega-3 type of omega-3 family, which is representative of chlorera supplied to rotifers and altemia, which are zooplankton, which are fed to feed fish for feeding omega-3 fatty acids having excellent physiological activity. A new way of accumulating unsaturated fatty acids, eicosapentaenoic acid (hereinafter referred to as EPA) and docosahexaenoic acid (hereinafter referred to as DHA), ie omega- to Chlorella capable of chemo-heterotrophic culture EPA or DHA is added intracellularly by adding unsaturated fatty acid monoglyceride (MAG) form containing EPA or DHA or sugar-fatty acid monoester (sugarester) form in which EPA or DHA is bound to reducing sugars to deliver fatty acids. Provided is a method for producing chlorella, which selectively contains only a high concentration of omega-3 fatty acids.

Omega-3 Polyunsaturated Fatty Acids, Eicosapentaenoic Acid (EPA), Docosahexaenoic Acid (DHA), Chlorella

Description

Process for the production of chlorella containing omega-3 fatty acids

Recently, our society is facing the age of population aging due to the advancement of the industry, and the dietary pattern is changing due to the intake of fat component in the westernization of the diet, and thus the thrombosis that causes ischemic heart disease, cerebral infarction, etc. Cardiovascular diseases such as arteriosclerosis are increasing, and the social interest in prevention, treatment, and diet is increasing. Fat provides about two times more calories as an energy source than carbohydrates and proteins, and it is important not only as a simple energy source but also as a source of essential fatty acids and fat-soluble vitamins. In the 1970s, epidemiological studies showed that Eskimos living in Denmark's Greenland, such as Dyerberg and Bang in Denmark, have a very low incidence of ischemic heart disease.Eskimo consumes 35-40% of its total calories from fat. The low incidence of thrombosis suggests that the sources of fat they eat are due to fatty acids of the omega-3 family rich in EPA or DHA.

Polyunsaturated fatty acids (PUFAs), which play a physiologically important function in vivo, contain omega-6 (n6) fatty acids and omega-3 (n3) depending on the position of the carbon where double bonds appear from the terminal methyl group. It is largely divided into fatty acids. As these essential fatty acids, unsaturated fatty acids include linoleic acid, linolenic acid, EPA, DHA, and arachidonic acid. In terrestrial animals, n6 arachidonic acid is abundant in the lipids in the body, and 8-10% is contained in soybean oil or canola oil, and 50% is contained in perilla oil or linseed oil. It may be supplied. However, it has been reported that arachidonic acid is difficult to be converted into EPA or DHA in vivo, and in particular, the conversion efficiency is lowered in the grown animals (Shin-Hyung et al., 1988).

Omega-3 polyunsaturated fatty acids are representative of EPA and DHA. In vivo, they are made mainly from alpha-linoleic acid (ALA). Since EPA and DHA are hardly synthesized in the human body, and the interaction between the omega-3 and omega-6 families does not occur very well, the EPA and DHA contained in the living body can be found in the intake of foods containing them, especially fish. There is no choice but to depend. Differences in the intake of the polyunsaturated fatty acid groups classified into the omega-3 omega-6 series can have a significant effect on the development of brain and cardiac thrombotic diseases, and the mechanisms are gradually improved by recent epidemiological and nutritional studies. It's getting clearer. In view of the prophylactic medical conditions, such dispersible polyunsaturated fatty acids as EPA and DHA have been attracting attention.

Among these essential fatty acids, omega-3 essential fatty acids, such as EPA and DHA, which are frequently found in blue fish, have recently attracted attention for their physiological activity in animals. It is known to play an essential role in boosting immunity. In addition, in the case of aquaculture fish, if omega-3 fatty acid, ie, EPA or DHA, is not supplied in sufficient quantity for the fry, immune function is reduced, anti-history is high, and mortality is high. This has been reported to drop significantly. Therefore, EPA and DHA are one of the essential fatty acids in marine fish farming, and have been investigated as essential nutrients for larvae (Watanabe Takeshi et al., Bull. Jap. Soc. Sci. Fisheries 45 ( 7): 883-889, 1979)

 Currently, there are two main ways to supply EPA and DHA to fry. First, the direct method of supplying the fry to the fry through the nutritional step of directly supplying unsaturated fatty acids of high omega-3 fatty acid content to yunchung or althemia, the zooplankton used as the primary feeding organisms of the fry. After feeding chlorella or yeast, which is a secondary food organism that already contains omega-3 fatty acids, to zooplankton, which is a primary food organism, it is supplied again to the feed organisms of fry to feed EPA and DHA to fry. There is an indirect way to do this. So far, direct methods have been widely used, but recently, freshwater chlorella, which accumulates omega-3 fatty acids, and seawater chlorella, which is naturally rich in omega-3 fatty acids, can be produced. Indirect direction is favored.

  However, seawater chlorella requires photo-autotrophic culture to contain omega-3 fatty acids in its own cells, which makes it difficult to mass produce industrially and some imported Japanese products are distributed. Very high situation. On the other hand, several techniques for accumulating omega-3 fatty acids in the freshwater chlorella, which are widely used, have been developed recently, but the supply of low omega-3 content and low culturing yield of the entire chlorella has not been achieved.

If it is possible to mass-produce sea chlorella such as Chlorella minutissima, which contains more than 40% of the total fatty acid in the cell, direct fermentation techniques or fermentation techniques that accumulate unsaturated fatty acids in chlorella and yeast will have no useful value. Unfortunately, at present, these seawater chlorella are difficult to cultivate and have high productivity, and their productivity is very low due to the inability to cultivate high concentrations in fermenters like conventional freshwater chlorella (Medina AR et al. 1998).

Therefore, in such a difficult aquaculture market, these products are not yet economical, and the most economical method of supplying omega-3 fatty acids to fry is known to be unsaturated fatty acid by using freshwater chlorella which can be mass cultured. Will be accumulated and produced.

In Japanese Patent Publication No. 59-44020, one of the prior art methods uses yeast to strengthen omega-3 fatty acids, but the yeast itself has a lower feed value than chlorella, resulting in an effective supply of EPA and DHA to fry. It does not have the means to pollute the tank rather has a disadvantage. In addition, Japanese Patent Application Laid-Open No. 1988-080312 converts free fatty acids, fatty acid salts, or fatty acid ester forms into chlorella using omega-3 fatty acids to enhance chlorella EPA and DHA. However, when only squid liver oil was added, EPA or DHA did not accumulate in chlorella cells at all, and in this method, since most fatty acids obtained in nature exist in the form of triglyceride (TG), free fatty acids and fatty acid salts or concentrated In order to obtain a fatty acid ester, there is a disadvantage that a chemical hydrolysis step using strong alkali such as acid or caustic soda and an esterification step and concentration step using methanol or ethanol must be preceded. In addition, in 1977-004307, only the high concentration production of chlorella by heterotrophic culture method is proposed, but the chlorella produced by this method is emulsified in the culture stage of zooplankton, which is extremely low in EPA and DHA in the fry. A separate nutrient fortification step is needed to feed the omega-3 fatty acids.

The present invention has been made in view of the above problems, and provides a method of selectively delivering high concentrations of omega-3 fatty acids such as EPA and DHA in chlorella cells and accumulating them.

The present invention is not primarily in the form of free fatty acids or salts used in the prior invention, but rather in the form of free fatty acids or salts used in the prior invention to devise a method for selectively accumulating omega-3 fatty acids in chlorella. The present invention has been completed by devising monoglycerides and sugar-esters of unsaturated fatty acids including EPA or DHA in omega-3 fatty acids, which can be easily passed.

In order to add omega-3 fatty acids in natural fat or fatty acid form to the culture medium and use them as substrates or auxiliary substrates of the microorganisms, an emulsification process is necessary to enhance the dispersion effect in the culture medium except for the form of fatty acid salts having water solubility. In the absence of emulsification, due to the interfacial separation between the fatty acid and the culture medium, the microorganism becomes difficult to easily break down or ingest fatty acids or fatty acids. Therefore, in the fermentation culture process that needs to add fatty acids, nonionic surfactants such as Tween should be added in the range of 10 to 100 ppm. However, from a process simplification or an economic point of view, it would be preferable to prepare and supply the fatty acid to be supplied in the form of an emulsifier from the beginning or in the form of an emulsifier rather than adding oil and emulsifier separately. In the present invention, a method for preparing chlorella containing omega-3 fatty acids was devised by feeding the chlorella culture medium using an EPA / DHA monoglyceride and a sugar-ester form which do not require emulsification or addition of an emulsifier.

Most microorganisms have a selective transport structure to a relatively easy-to-use carbon source such as glucose or monosaccharides such as glucose and specific to the cell membrane, so that fatty acids can be delivered into chlorella cells in a short time when the fatty acid is supplied in this form. . In addition, high concentrations of omega-3 fatty acids can be accumulated even when a fatty acid is supplied at the end of the culture compared to the existing invention. The disadvantages of the culture and the accumulated fatty acids were able to mass cultivate chlorella containing high concentration of omega-3 fatty acid without the problem that the concentration is reduced by the magnetization of the cells.

<Preparation of EPA / DHA Monoglycerides and Sugar-Ester>

Since glycerin has three hydroxyl groups, there are mono, di, and triglycerides depending on the number of fatty acids bound, but monoglycerides having high emulsifier function are advantageous. In the present invention, since a high-purity omega-3 fatty acid is required, glycerin which is reacted under reduced pressure after being filled with an inert gas in the presence of an alkali catalyst using high-purity ethyl-EPA (75%) or ethyl-DHA (65%) and Prepared by esterification method of fatty acid. The concentration of monoglyceride produced by the reaction was about 40-60% and was used as an auxiliary substrate for chlorella culture after removing unreacted glycerin. In addition, since monoglycerides have a poor dispersibility in water, fatty acid salts were used in the form of self-emulsifying monoglycerides in order to improve dispersibility. For the preparation of sugar-ester, glucose and alkyl fatty acids were finely microemulsified using a large amount of emulsifiers to improve the reactivity. The sugars were dissolved in propylene glycol, and approximately 10% of the total amount as alkyl fatty acids and emulsifiers was used. When soap is added and heated while stirring, a transparent emulsion is formed. At this time, the reaction is terminated while propylene glycol is distilled off under reduced pressure with a calcium carbonate catalyst. After the reaction, unreacted sugar and soap were removed to recover sugar-ester. The type of reducing sugar used may be any reducing sugar such as sugar and glucose, but monosaccharides such as glucose are more preferable for fermentation culture of chlorella. The fatty acids used may be in the form of free fatty acids or alkyl esters, but more preferably in the form of methyl or ethyl esters (Hidaka Toru, 1996).

Features of the present invention

An object of the present invention is to provide a method for producing chlorella cells containing a high concentration of omega-3 fatty acid EPA or DHA, but culturing in a fermenter using a Chlorella sp. Strain capable of heterotrophic culture Even in the method for producing fermentation of chlorella, there are differences from the prior arts in the method of delivering omega-3 fatty acids into chlorella cells, and in particular, prior art (method of preparing chlorella containing no omega-3 fatty acids) 10-0193748 and the method of Published Patent Publication No. 1998-08031 which must separately supply the form of free fatty acids or free fatty acid salts to accumulate EPA and DHA.

Embodiment of the present invention

Chlorella used in the present invention was crossed between light and dark at 3,000-5,000 lux conditions at 25 ~ 30 ℃ from fresh water samples collected from sub-nutrient reservoirs and streams in several places in Jeju Island and the South Coast. -6 hours) Purely isolated species of chlorella capable of heterotrophic cultivation from the culture method, and selected a single colony (colony) with fast growth rate and dark green color, named Chlorella sp. CP-5192. For preservation, the product was refrigerated using calcium-alginate bead immobilization. Hereinafter, the conditions of the present invention will be described in detail with reference to Examples, but this does not limit the scope of the present invention.

Example-1

In order to isolate the superior strain, chlorella immobilized on alginic acid was treated with sodium citrate or sodium hexametaphosphate solution to release immobilized chlorella, and glucose-2%, yeast extract-0.3%, and malt extract- 50 to 100 agar plates containing 0.3, tryptone-0.5%, monophosphate-0.2%, potassium nitrate-0.2%, magnesium sulfate-0.02%, mineral solution-1 ml / L The colonies were diluted in sterile saline solution to form colonies and plated and incubated in a thermostat at 30 ° C. After 5-7 days, only the top 5% (about 5 out of 100) of the dark green, large clusters were screened and inoculated into a 500 lm flask containing 150 ml of liquid medium of the same composition as 72-96. Time incubation.

Spawn cultures for large-scale cultivation were glucose-4%, potassium nitrate-0.4%, potassium monophosphate-0.2%, yeast extract-0.5%, magnesium sulfate-0.2%, EDTA-iron-0.01%, vitamin solution- The cells were incubated for 72 hours in a 20 L working medium containing 1 ml / L.

In this fermentation tank for production for mass production, in a 200L fermenter tank, glucose-2%, citric acid-1%, urea-0.45%, carbophosphate-0.2%, yeast extract-0.5%, calcium chloride-0.02%, magnesium sulfate-0.15 %, Iron sulfate -0.015%, Na-EDTA -0.02%, vitamin solution -2 ml / L, mineral solution -3 ml / L (boric acid-2.86 g, manganese chloride-1.81 g, zinc sulfate-0.22 g, copper sulfate-0.079 g, molybdenum acid-0.0212 g was dissolved in 1 L of distilled water and hydrochloric acid was added until the precipitate disappeared. During the incubation period, glucose concentration is measured every 3 hours using a glucose analyzer, and a certain amount of glucose concentrate (70%, w / v) and 10% urea concentrate is maintained so that the glucose concentration in the culture is maintained at 20 ± 10 g / L. Incubation was carried out for 72 hours.

As the culture conditions of the species fermentation and the main fermentation, the culture pH was performed at 6.5, the fermentation temperature of 32-35 ° C, the stirring speed of 200-350rpm, the aeration amount of 0.8-1vvm, and the pressure of 0.5-1.0kgf / cm2. The growth of chlorella was measured as the relative volume (WCV: wet cell volume) of chlorella cells in the culture obtained after centrifugation at 3,000 rpm for 10 minutes.

Example-2

The culture was carried out under the same conditions as in the fermentation method of Example 1, and after 60 hours of incubation, the glucose concentration in the culture solution was maintained at 5-10 g / L, and 1% of EPA-monoglyceride was added for 12 hours.

Example-3

The culture was carried out under the same conditions as in Example 2, and after 60 hours of incubation, 1% of DHA-monoglyceride was added thereto for 12 hours.

Example-4

The culture was carried out under the same conditions as in the fermentation method of Example 1, and after 66 hours of culture, 2% of EPA-monoglyceride was added to incubate for 6 hours.

Example-5

The culture was carried out under the same conditions as in Example 4, and after 66 hours of incubation, 1% of EPA-sugar ester was added to incubate for 6 hours.

<Example-6>

Example 5 The culture was carried out under the same conditions as the fermentation method. After 66 hours of incubation, 2% of DHA-sugar ester was added to incubate for 6 hours.

<Example-7>

Example 6 The culture was carried out under the same conditions as the fermentation method. After 60 hours of cultivation, 1% of DHA-sugar ester was added to incubate for another 12 hours.

Comparative Example

The culture was carried out under the same conditions as in Example 2, and after 48 hours of culture, the concentration of glucose in the culture medium was maintained at 5-10 g / L and the free fatty acid of fish oil having a high content of omega-3 lipids was 0.1-0.5%. After Tween-80 was supplied 0.05% to the culture volume as a surfactant, the culture temperature was maintained at 37 ~ 39 ℃.

Example Number One 2 3 4 5 6 7 Comparative Example Omega-3 Fatty Acid Addition Form - EPA-mono glyceride DHA-mono glyceride EPA-mono glyceride (2%) EPA-sugar ester (2%) DHA-sugar ester (2%) DHA-sugar ester Free fatty acids Incubation time after addition - 12 12 6 6 6 12 24 WCV (%) (after 72h culture) 12 11 11 12 12 12 11 8 EPA content in total lipid (%) ND 46.4 1.2 38.6 34.4 1.8 1.7 16.1 DHA content in total lipids (%) ND 4.1 37.6 3.8 3.4 36.1 34.6 18.3

* ND: non detected

Chlorella, produced only by high concentration production of chlorella by heterotrophic culture method, provides extra nutrition for supplying emulsified omega-3 fatty acid at the cultivation stage of zooplankton, which is extremely low in EPA and DHA in the fry. A step is necessary. However, the present invention has been made in view of the above problems, and provides a method of selectively delivering high concentrations of omega-3 fatty acids such as EPA and DHA in chlorella cells and accumulating them.

Claims (3)

In culturing chlorella, monoglyceride form and sugar were used as auxiliary substrates after maintaining the culture until the wet cell concentration (WCV,%) was 10-15 wt% and the concentration of glucose in the culture medium was 5-10 g / L. Emulsified omega-3 fatty acids in ester form, separately or mixed, are added to the culture so as to be 0.5 to 5% by weight, and further cultured for 3 to 15 hours. A method for producing fermentation of chlorella containing omega-3 fatty acids (EPA / DHA), characterized in that 1 to 50% by weight of omega-3 fatty acids in the total lipid content of the. The method according to claim 1, Fatty acids which can be added to the above auxiliary substrates are emulsified omega-3 fatty acids in the form of monoglycerides. EPA monoglycerides or DHA monoglycerides may be used alone, and EPA monoglycerides and DHA monoglycerides may be used alone. It is also possible to use in a mixed form, and another fatty acid which can be added to the above auxiliary substrate is an emulsified omega-3 fatty acid in the form of sugar-ester, which can be used alone in the form of EPA sugar-ester or DHA sugar-ester, respectively. And an omega-3 fatty acid (EPA / DHA) fermentation method, characterized in that it can be used in the form of a mixture of EPA sugar-ester and DHA sugar-ester. delete