JPH0723774A - Lactic acid bacterium containing docosahexaenoic acid and method for producing the same - Google Patents

Abstract

PURPOSE:To produce lactic bacterium containing docosahexaenoic acid as exclusive higher unsaturated fatty acid in high efficiency and short time. CONSTITUTION:Lactic bacterium containing docosahexaenoic acid is produced by culturing lactic bacterium in a medium incorporated with autolyzate or extract of the cells of marine microalga.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing a lactic acid bacterium, which comprises culturing a lactic acid bacterium as a highly unsaturated fatty acid substantially containing docosahexaenoic acid in a medium containing an extract of marine microalgae. Regarding Such a lactic acid bacterium substantially contains only docosahexaenoic acid as a highly unsaturated fatty acid, and is useful in fields such as foods, cosmetics, marine products, and chemical products.

[0002]

2. Description of the Related Art Lactic acid bacteria have, as their inherent pharmacological actions, 1) growth in the intestine, production of substances that prevent colonization of harmful bacteria, and occupation of colonization sites of harmful bacteria, 2) intestine. Although it does not proliferate in the body, its bacterial component acts as an active substance, directly enhances the immunity of the living body, blocks the absorption of cholesterol, or acts on the improvement of intestinal flora.
3) The fact that the production of endotoxins, enterotoxins, amines, ammonia and the like produced by harmful enterobacteria in the intestine is suppressed is widely known. Therefore, although more widely used, the components of the fatty acid composition in the cells of lactic acid bacteria are palmitic acid, palmitooleic acid,
It is mainly oleic acid, and these fatty acids have poor nutritional and pharmacological values, and they do not contain any polyunsaturated fatty acids.

On the other hand, the ω-3 series of docosahexaenoic acid is a precursor of the ω-6 series of highly unsaturated fatty acids, and has anti-tumor, anti-arteriosclerotic, anti-inflammatory and other effects, as well as a lipid-lowering effect and a cholesterol-lowering effect. It is known to suppress the deterioration of visual acuity and improve memory. The ω-6 series of highly unsaturated fatty acids are contained in terrestrial animal fats and oils, plant roots, etc., and can be ingested sufficiently in the present dietary life.
Docosahexaenoic acid, which is a series of highly unsaturated fatty acids, is a fatty acid specific to marine products, and tends to be insufficient for the generations who are carnivorous. A major factor that tends to avoid foods derived from marine products is their unique odor. Docosahexaenoic acid is also a component of the odor of marine products, but the main odor of marine products is derived from oxidative decomposition products such as aldehydes. Therefore, it is preferable that docosahexaenoic acid can be added to food in a form that is difficult to oxidize.

[0004]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The purpose of the present invention is to maintain the usefulness inherent in lactic acid bacteria and to facilitate the intake of docosahexaenoic acid, which is of nutritional and pharmacological value, to make it highly unsaturated fatty acid. Another object is to provide a lactic acid bacterium containing docosahexaenoic acid.

[0005]

[Means for Solving the Problems] As a result of earnest research for the above-mentioned purpose, the present inventors have produced marine microalgae that produce docosahexaenoic acid at a high concentration and accumulate / contain it in algal cells, especially crypts. Regarding lipids of lactic acid bacteria cultivated by adding the extract of tecodinium cohnii: 1) Even if the lactic acid bacteria cells containing docosahexaenoic acid were washed with distilled water and a surfactant, It was found that the amount of docosahexaenoic acid is as high as 30% or more of the total fatty acid, 2) the growth rate of the lactic acid bacterium used in the present invention is fast, and a large amount of microbial cells can be obtained in a short time, leading to the present invention. It was

That is, the present invention provides a lactic acid bacterium containing docosahexaenoic acid containing substantially only docosahexaenoic acid as a highly unsaturated fatty acid. Further provided is a method for producing a lactic acid bacterium containing docosahexaenoic acid, which comprises culturing a lactic acid bacterium in a medium to which an autolysate of an alga body of a marine microalga is added. Also provided is a method for producing a lactic acid bacterium containing docosahexaenoic acid, which comprises culturing a lactic acid bacterium in a medium to which an extract from an algal body of a marine microalga is added. The marine microalgae is preferably Crypthecodinium cohnii. The extract is extracted with one or more solvents of ethanol, ethanol / water mixture, hexane, ethanol / hexane mixture, methanol / chloroform mixture or methanol / water mixture. preferable.

The present invention was completed on the basis of the above findings, and is based on the self-administration of marine microalgae containing lactic acid bacteria as polyunsaturated fatty acids and containing docosahexaenoic acid alone at a high concentration, especially Crypthecodinium cornii. A lactic acid bacterium containing docosahexaenoic acid, which comprises culturing in a medium to which a digested product or an extract is added, and a method for producing the same.

(Operation) The marine microalgae used in the present invention may be any algae that produces docosahexaenoic acid as a highly unsaturated fatty acid, and for example, algae belonging to the genus Crypthecodinium, particularly, crypt Crypthecodinium cohn
ii) There are species. These are known algae, and are available from preservation organizations such as ATCC (American Type Culture Collection). As a specific example, Crypthecodinium cornie ATCC 30021,30
543, 30556, 30571, 30572, 305
75, 50051, 50053, 50005, 5005
6,50058,50060 and the like. In particular, the Crypthecodinium cohnii ATCC30021 strain is preferable because it produces a high amount of DHA.

The algal bodies of marine microalgae used in the present invention can be obtained by the following culture method. As a medium for culturing the marine microalgae, a medium having any composition can be used as long as the medium can grow well. The culture method may be any culture method such as static culture, liquid shaking culture, liquid tank culture, batch culture, sulphate culture, and continuous culture. Culture conditions are culture temperature 1
Culturing is performed at 5 to 30 ° C. and a culturing time of 2 to 8 days. The algal cells to be used include a wet algal cell obtained by extraction from the culture solution after completion of the culture, or filtration or recovery by centrifugation or the like,
It may be an algal cell paste, or a dried algal cell such as a heat-dried product, a freeze-dried product or a spray-dried product obtained by further heating, freeze-drying or spraying.

The present invention uses the self-digested product obtained by self-digesting the obtained algal cells or the extract extracted from the algal cells.

The self-digested product of algal cells is obtained by covering the liquid surface of an algal cell suspension obtained by suspending wet algal cells or dried algal cells with a small amount of sterile water with p-xylene, toluene, etc. ~ 50 ° C
Warm up to gradually self-extinguish. 2 to self-digestion
It takes 48 hours. After completion of the digestion, p-xylene or toluene and algal cell residue are removed by centrifugation or the like, and concentrated by vacuum distillation, freeze-drying or the like to prepare.
The prepared self-digested product of algal cells is mainly a lipid and contains 10 to 80% by weight of docosahexaenoic acid.

The extract from algal cells is a culture solution of algal cells, or an algal cell suspension obtained by suspending wet algal cells or dried algal cells with a small amount of sterilized water using the following extraction solvent: 20-60 ° C
It can be obtained by extraction in the usual way. The extract from algal cells is mainly a lipid (or ester) and contains 20 to 90% by weight of docosahexaenoic acid. Examples of the extraction solvent include any one solvent or two or more solvents of ethanol, methanol, hexane, an ethanol / water mixture, an ethanol / hexane mixture, a methanol / chloroform mixture, or a methanol / water mixture. When used in a mixture, for example, the mixing ratio of ethanol: water is
The mixing ratio of 2:98 to 98: 2 and ethanol: hexane is 1:99 to 99: 1.

The extract can be used for culturing lactic acid bacteria without removing the water-insoluble extraction solvent, such as hexane and chloroform, by leaving the water-soluble solvent such as ethanol as it is. When the extract (the solvent is distilled off if necessary) is used for culturing lactic acid bacteria, p or p
H is preferably 6 to 8.5.

The term "polyunsaturated fatty acid" as used herein means an unsaturated fatty acid having a degree of unsaturation of 2 or more, and mixed fats and oils of highly unsaturated fatty acids, for example, fats and oils derived from sardines, mackerel, horse mackerel, and tuna, and the like. Fats and oils in which the proportion of highly unsaturated fatty acids in the total fatty acids is 10% or more, and as highly unsaturated fatty acids, linoleic acid, γ-linolenic acid, arachidonic acid, α-
Linoleic acid, eicosapentaenoic acid, docosahexaenoic acid and their salts, esters, triacylglycerols, diacylglycerols, monoacylglycerols, glycerophospholipids, glyceroglycolipids, sphingophospholipids, glycosphingolipids, etc. A method for simultaneously accumulating various polyunsaturated fatty acids in lactic acid bacteria is already known (JP-A-4-341180). However, in this method, various polyunsaturated fatty acids are contained and a specific polyunsaturated fatty acid production ratio is also included. Since it is difficult to control docosahexaenoic acid alone, it is difficult to contain only docosahexaenoic acid alone, and using only docosahexaenoic acid alone requires great labor and cost for its separation, fractionation and purification. There is a problem that the use is limited. A feature of the present invention is that substantially only docosahexaenoic acid as a polyunsaturated fatty acid is selectively accumulated in a lactic acid bacterium at a high concentration, which enables efficient use in applications where only docosahexaenoic acid is desired to be used. became.

The lactic acid bacterium used in the present invention may be any bacterium as long as it is a lactic acid-fermenting bacterium of sugar,
Although the species or strain is not limited, for example, Enterococcus genus, Lactobacillus genus, Bifidobacterium (Bifidob)
acterium), especially Enterococcus
Fecalis (Enterococcus faecalis) species may be mentioned.
These lactic acid bacteria can be easily obtained from microbial deposit institutions such as IFO (Institute for Fermentation, Osaka).

In the method for producing lactic acid bacteria of the present invention, the lactic acid bacteria are cultivated in a medium containing an autolyzed product or an extract obtained from the algal bodies of marine microalgae. As a medium used in the method for producing lactic acid bacteria of the present invention, a medium in which a normal lactic acid bacterium can grow and which is added with an autolysate or an extract derived from an algal body of a marine microalga is used. Here, as a medium in which ordinary lactic acid bacteria can grow, for example, the medium having the composition shown in Table 1 can be used.

[0017]

The concentration of the self-digesting substance derived from the algal bodies of the marine microalgae is 0.001 to 10% by weight, preferably 0.002 to 0.1% by weight, calculated as DHA in the medium. It is desirable to do so from the viewpoint of the growth of lactic acid bacteria and the uptake rate of docosahexaenoic acid. In addition, the amount of addition when using an extract derived from algal cells is 0.01 to 2.0 in the medium.
It is preferably wt%. Further, an emulsifier such as Tween 80 may be added to the medium of lactic acid bacteria.

In the method for producing lactic acid bacteria of the present invention, the lactic acid bacteria are usually cultured by anaerobic culture. In order to prevent the lactic acid bacteria from coming into contact with molecular oxygen in the incubator, i) in order to prevent the lactic acid bacteria from coming into contact with the molecular oxygen, i) after adding the lactic acid bacteria after heating the medium to remove dissolved oxygen, ii) A method such as culturing in a pressure-resistant container under vacuum or reduced pressure, and iii) adding a reducing agent to the medium may be used. The amount of lactic acid bacteria added at the start of culture is 1 ml of medium
The cells of lactic acid bacteria may be added so that 10 ⁶ to 10 ⁷ cells are obtained. A culture solution can be obtained by anaerobically culturing a mixture containing a lactic acid bacterium culture medium, an extract derived from an algal body of a lactic acid bacterium and a marine microalga or an autolysate under the following culture conditions. The culture conditions for lactic acid bacteria are p
H changes depending on the acid such as lactic acid produced by the culture in the course of the culture, but it is preferably 6.0 to 7.5 at the start of the culture. The culturing temperature is around 35 ° C., and the culturing time is about 16 hours. The obtained culture solution can be directly used as a lactic acid bacterium drink or a lactic acid bacterium-added food. In addition, lactic acid bacteria are separated from the obtained culture broth by filtration, centrifugation, etc., and washed with distilled water or 2% glycerin monooleate. For example, dried cells may be used.

After freeze-drying the cells of lactic acid bacteria cultured in such a medium and methyl-esterifying them with boron trifluoride methanol by a conventional method, the fatty acid composition of all fatty acid derivatives ester-bonded in the cells is obtained. Can be analyzed by gas chromatography. Alternatively, wet or dry cells can be extracted with an organic solvent or the like, and after fractionating the lipids by silica gel thin layer chromatography, the constituent fatty acids of the respective lipids can be similarly analyzed. The polyunsaturated fatty acid in the cells of the lactic acid bacterium of the present invention analyzed by the above method is
Substantially only docosahexaenoic acid.

[0021]

EXAMPLES The present invention will be described in more detail below with reference to examples. (Example 1) Crypthecodinium cornie ATCC
100 g of the freeze-dried product of 30021 was added to 1 liter of ethanol / hexane (1/1) at 40 ° C., and the mixture was extracted for 1 hour with stirring.
It was centrifuged for 15 minutes to obtain 0.82 liter of supernatant.
This was concentrated with an evaporator, and an extract of Crypthecodinium cornii (20.2 g, DHA content of 9.
7 g).

Example 2 Enterococcus faecalis was added to the medium composition shown in Table 1 in an amount of 0.2% by weight of the extract of Kuptecodinium cornie obtained in Example 1 (1.0 g in terms of DHA). ) Was used for anaerobic culture at a culture temperature of 35 ° C. for 16 hours. The pH at the start of culture was 6.5. Cells collected from the culture solution by centrifugation, washed with distilled water three times,
Further, the bacterial cells washed three times with a 0.1% glycerin monooleate aqueous solution were dried to obtain lactic acid bacteria powder. 1.0 g of dry powder of lactic acid bacteria under each washing condition was suspended in 14% boron trifluoride (BF ₃ ) methanol, reacted at 80 ° C. for 30 minutes, and the produced fatty trimethyl ester was analyzed by GLC.
The content of docosahexaenoic acid was quantified.

In the bacterial cells that were washed three times with distilled water, the ratio of docosahexaenoic acid to the total fatty acids of the bacterial cells (content per 1 g of dry bacterial cells) was 45%, and no other unsaturated fatty acids were contained. It was

Similarly, the docosahexaenoic acid content was 43% even in the bacterial cells washed three times with a 0.1% glycerin monooleate aqueous solution, and the content of docosahexaenoic acid did not decrease due to the washing, and other than docosahexaenoic acid. No other polyunsaturated fatty acids were found.

[0025]

INDUSTRIAL APPLICABILITY According to the method of the present invention, lactic acid bacteria are cultivated in a medium supplemented with an autolyzate or extract of marine microalgae, particularly Crypthecodinium cornii, to give polyunsaturated fatty acids. A lactic acid bacterium containing only docosahexaenoic acid was obtained, and this lactic acid bacterium can be efficiently produced in a short time.

Claims (5)

[Claims] 1. A lactic acid bacterium containing docosahexaenoic acid, which contains substantially only docosahexaenoic acid as a highly unsaturated fatty acid. 2. A method for producing a lactic acid bacterium containing docosahexaenoic acid, which comprises culturing the lactic acid bacterium in a medium containing an autolysate of an alga body of marine microalgae. 3. A method for producing a lactic acid bacterium containing docosahexaenoic acid, which comprises culturing the lactic acid bacterium in a medium to which an extract from an algal body of a marine microalga is added. 4. The method for producing a lactic acid bacterium containing docosahexaenoic acid according to claim 2 or 3, wherein the marine microalgae is Crypthecodinium cohnii. 5. The extract is ethanol, ethanol /
The lactic acid bacterium containing docosahexaenoic acid according to claim 3, wherein the lactic acid bacterium contains docosahexaenoic acid, which is extracted with one or more solvents selected from a water mixture, hexane, an ethanol / hexane mixture, a methanol / chloroform mixture, and a methanol / water mixture. Production method.