KR20190139793A - Method for Manufacturing Spirulina sp. Algae using minimal medium - Google Patents

Abstract

The present invention relates to a method of producing Spirulina sp. Algae by using a minimal medium including NaHCO_3, K_2HPO_4, MgSO_4·7H_2O and NaNO_3; a minimal medium for producing Spirulina sp. Algae, which includes NaHCO_3, K_2HPO_4, MgSO_4·7H_2O and NaNO_3; Spirulina sp. Algae produced by the method; and a pharmaceutical composition for treating or preventing a liver disease, a food composition for improving a liver function, a pharmaceutical composition for treating or preventing type 1 diabetes, and food and feed compositions for ameliorating type 1 diabetes which comprise the Spirulina sp. Algae. According to the present invention, the method of producing Spirulina sp. Algae can economically produce Spirulina sp. Algae by enabling ingredients included in the medium to be minimized, and the Spirulina sp. Algae produced by the method can be widely utilized in economic production of Spirulina sp. Algae exhibiting pharmaceutically improved activity by exhibiting various pharmacological activities including amelioration of diabetic symptoms, prevention of liver functional damages, treatment of liver damages, etc.

Description

Production method of Spirulina algae using minimal medium {Method for Manufacturing Spirulina sp. Algae using minimal medium}

The present invention relates to a method for producing spirulina algae using a minimal medium, and more particularly, the present invention relates to a spirulina genus algae using a minimal medium containing NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ .7H ₂ O and NaNO ₃ . Production method of, NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and the minimum medium for the production of Spirulina algae containing NaNO ₃ , Spirulina algae produced by the above method, Liver disease comprising the spirulina algae The present invention relates to a pharmaceutical composition for treating or preventing, a food composition for improving liver function, a pharmaceutical composition for treating or preventing type 1 diabetes, a food composition for improving type 1 diabetes, and a feed composition.

At present, crops being cultivated all over the world have been used as a stock supply source for humanity, but the yield per farm area is not high and the utilization of solar energy is extremely low. On the other hand, micro photosynthetic algae, which can grow in water with inorganic energy containing solar energy, carbon dioxide, and small amounts, can grow even in areas where crops cannot be grown, and can obtain more than 20 times more protein per acreage area than crops. In addition, it is possible to produce a variety of useful substances and natural rare substances that cannot be produced from microorganisms or plant cells. Moreover, algae with large cell size are easily settled and are easily extracted and separated through various methods, and by using solar energy as a main energy source, efficient use of solar energy irradiated on the earth and carbon dioxide as a carbon source It has photosynthetic process that uses it and releases oxygen as a by-product, and it has the function of lowering air pollution.

Accordingly, various types of micro photosynthetic algae are being studied, and studies on micro photosynthetic algae such as Chlorella genus, Dunaliella genus, and Spirulina genus are being actively conducted. In particular, spirulina microscopic photosynthetic algae (hereinafter referred to as `` spirulina algae '') are larger algae than other micro photosynthetic algae and can easily grow in alkaline contaminated environments. It is applied to a product. For example, Korean Patent Registration No. 48714 discloses an adsorbent for removing toxic substances or odors including spirulina algae powder, and Korean Patent Registration No. 353888 discloses chewing gum composition having a dioxin excretion effect including spirulina algae powder. Korean Patent Registration No. 454224 discloses a health supplement composition comprising spirulina algae powder. Accordingly, researches to improve the productivity of spirulina algae are being actively conducted worldwide.

Recently, studies on the effective production of spirulina algae have been started in Korea. Due to the climatic characteristics of the four seasons and the seasonal temperature fluctuations and the difference in the amount of sunshine, studies related to indoor culture rather than outdoor culture of spirulina algae have been started. It's going on. However, in the conditions in which spirulina algae grow most actively, various vegetable algae or microorganisms other than spirulina algae grow together, so that the yield of spirulina algae decreases relatively, thereby providing an environment suitable for the growth of spirulina algae. Although the added value obtained from the production of spirulina or algae is not large compared to the cost required to produce the phosphate, research is being conducted to develop a method to produce this more economically, but it is not practically used. .

For example, Korean Patent Laid-Open Publication No. 2004-0073693 discloses a method of culturing spirulina or algae using carbon as a carbon source instead of adding NaOH to control pH, but charcoal is precipitated in a culture medium to control pH. It is not shown, and it is not used as a carbon source, and there is a problem that spirulina algae are adsorbed on the precipitated charcoal, resulting in deterioration of productivity, and Korean Patent Publication No. 2006-17033 discloses optimization and growth of spirulina algae. In order to adjust the concentration of nitrogen and carbon, a media composition has been disclosed, but the unit cost increase of the culture solution is higher than the degree of improvement in productivity, and thus, it was not actually utilized. In addition, the Republic of Korea Patent No. 0744360 has developed a SOT medium specialized for the cultivation of Spirulina genus algae, and a method for producing spirulina genus algae using the same, but the SOT medium contains 15 components of the medium Due to the high cost of production, it was difficult to economically produce spirulina.

If the development of the spirulina genus algae more economically compared to the conventional culture method, it is expected that it can bring a significant change in the commercial use of spirulina algae.

Under these circumstances, the present inventors have made intensive studies to develop a method for producing spirulina genus algae more economically, and have developed a minimal medium for producing spirulina genus algae including four components, and completed the present invention.

The main object of the present invention is to provide a method for producing spirulina genus algae using a medium comprising NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and NaNO ₃ .

Another object of the present invention is to provide a spirulina genus alga produced by the above method.

Still another object of the present invention is to provide a pharmaceutical composition for treating or preventing liver disease, including the spirulina genus algae.

Another object of the present invention to provide a food composition for improving liver function comprising the spirulina algae.

Another object of the present invention to provide a pharmaceutical composition for treating or preventing type 1 diabetes, including the spirulina genus algae.

Another object of the present invention to provide a food composition for improving type 1 diabetes comprising the spirulina genus algae.

Still another object of the present invention is to provide a feed composition comprising the spirulina genus algae.

Another object of the present invention is to provide a minimal medium for the production of Spirulina genus alga including NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ .7H ₂ O and NaNO ₃ .

The present inventors have been trying to improve the SOT medium for spirulina genus algae culture (Korean Patent No. 10-0704436) developed above, while performing various studies in order to economically develop a method for producing spirulina genus algae. Therefore, as a result of various studies to select the components necessary for the cultivation of spirulina algae among the components constituting the SOT medium, the minimum medium containing NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and NaNO ₃ It was confirmed that can be used to produce spirulina algae.

However, in the case of using the minimum medium, if the medium is not circulated at a constant flow rate, the cultured spirulina or algae precipitates, and there is a disadvantage that normal culture does not proceed. Therefore, the flow rate of the medium capable of culturing spirulina genus algae while using the minimum medium was selected, and it was confirmed that the medium was circulated at a rate of 30 to 45 cm / sec.

On the other hand, the spirulina genus algae produced by the above method was found to show an effect of improving diabetic symptoms and liver function better than the conventional spirulina algae.

As described above, a technique for producing spirulina algae using a medium containing four components has not been reported in the prior art and was first developed by the present inventors.

One embodiment of the present invention for achieving the above object is inoculated spirulina algae in a medium comprising NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and NaNO ₃ , and 3,000 at 32-38 ℃ Culturing the medium in a tubular incubator at a light condition of 9,000Lux, 12-20 (h): 12-4 (h) photoperiod (light: dark), and a flow rate of 30-60 cm / s. It also provides a method for producing algae, spirulina.

As used herein, the term “Spirulina sp. Algae” refers to a multicellular organism with a thin cell wall as the oldest algae on the planet. It grows mainly in the salt lakes of the tropics and does not have much global distribution, but its optimum water temperature is 32-42 ℃ and it is known to breed in strong alkaline environment. The main components of spirulina include proteins, carbohydrates, water-soluble fiber, antioxidant pigments (chlorophyll, carotenoids, phycocyanin), antioxidant enzymes (SOD), gammalinolenic acid (GLA), beta-carotene, vitamin B1, B2, B6, B12, E, inositol, folic acid, calcium, iron, potassium, magnesium, zinc, manganese, selenium, germanium and the like are known. In the present invention, the Spirulina maxima UTEX LB2342 strain was used as the spirulina genus alga, but is not particularly limited thereto, and all known spirulina genus algae may be applied to the method provided by the present invention.

In the production method of the spirulina genus alga provided by the present invention, the culture temperature is not particularly limited, but may be 32 to 38 ℃ as one example, 34 to 36 ℃ may be another example, another example It can be 35 ℃ as. When the culture temperature is lower than 32 ℃ the growth rate of the spirulina genus algae is greatly reduced, when higher than 38 ℃ the rate of rancidity of the cultured spirulina algae is increased.

In the production method of spirulina genus alga provided by the present invention, the light conditions are not particularly limited, but as an example may be 3,000 to 9,000 Lux, another example may be 5,000 to 7,000 Lux, as another example It can be 6,000 Lux. When the light condition is lower than 3,000 Lux, photosynthesis of spirulina does not proceed normally, and when it is higher than 9,000 Lux, the cost of maintaining the light condition is excessively increased without excessively affecting the growth of spirulina, thereby increasing the production cost.

In the spirulina genus algae production method provided by the present invention, the photoperiod is not particularly limited, but as an example, the photoperiod (light: dark) may be 12 to 20 (h): 12 to 4 (h). have. The photoperiod may be changed according to the purpose of culturing the spirulina genus algae, for example, for maintaining the spirulina genus algae, it is preferable to maintain the photoperiod (light: dark) at 12:12, as another example For the purpose of producing spirulina algae, it is preferable to maintain the photoperiod (light: dark) at 20: 4.

In the production method of the spirulina genus alga provided by the present invention, as the bright conditions included in the photoperiod increases, the flow rate of the medium may also be increased, but is not particularly limited thereto, the speed of 30 to 60 cm / sec Can be. As an example, when the photoperiod (light: dark) is 12:12, the medium flow rate may be a speed of 30 to 45 cm / sec; When the photoperiod (light: arm) is 13:11, the medium flow rate may be 37.5 to 45 cm / sec; When the photoperiod (light: arm) is 14:10, the medium flow rate may be a speed of 37.5 to 45 cm / sec; When the photoperiod (light: arm) is 15: 9, the medium flow rate may be 37.5 to 52.5 cm / sec; When the photoperiod (light: arm) is 16: 8, the medium flow rate may be 45 to 52.5 cm / sec; When the photoperiod (light: arm) is 17: 7, the medium flow rate may be 45 to 52.5 cm / sec; When the photoperiod (light: arm) is 18: 6, the medium flow rate may be 45 to 60 cm / sec; When the photoperiod (light: arm) is 19: 5, the medium flow rate may be 45 to 60 cm / sec; If the photoperiod (light: arm) is 20: 4, the medium flow rate may be 52.5 to 60 cm / sec.

If the flow rate of the medium is lower than 30 cm / s, the cultured spirulina genus algae precipitates and decay, and if it is higher than 60 cm / s, the frequency of collision between the spirulina genus algae increases, thereby increasing the loss rate of the spirulina algae .

According to one embodiment of the present invention, for the purpose of maintaining the spirulina genus algae, it is preferable to maintain the photoperiod (light: cancer) 12:12 and the medium flow rate 30 cm / sec, another example, the spirulina genus algae For the purpose of production, it is preferable to maintain the photoperiod (light: dark) 20: 4 and the medium flow rate 60 cm / sec (Table 3).

In the method for producing spirulina algae provided in the present invention, the minimum medium containing NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and NaNO ₃ is additionally H ₃ BO ₃ , MnSO ₄ · 7H ₂ O, ZnSO ₄ · 7H ₂ O, CoCl ₂ · 6H ₂ O, Na ₂ MoO ₄ · 7H ₂ O and the like.

In the production method of the spirulina genus alga provided by the present invention, the minimum medium can be artificially prepared by combining four components constituting it as an example, and as another example can be produced by purifying seawater present in nature Can be. For example, the method of purifying the seawater may use a method of filtering seawater to remove solids contained in the seawater, but is not particularly limited thereto.

Another embodiment of the present invention provides a spirulina genus alga produced by the above method.

The spirulina genus algae produced by the method for producing genus spirulina algae provided by the present invention are cultured by circulating a minimum medium at a constant flow rate, which is distinguished from the spirulina genus algae produced by a conventional method.

The present inventors have tested the pharmacological activity of the spirulina genus algae produced by the method of the present invention, it was confirmed that the pharmacological activity of the spirulina genus algae produced by the conventional method in terms of improving diabetic symptoms and liver function improvement effect It was.

According to an embodiment of the present invention, the effect of inhibiting the increase of phosphorylated JNK and the increase of phosphorylated p38 in apoptosis-induced insulin tumor cells is superior to PC (Phycocyanin), the main component of the algae of Spirulina It was confirmed (FIGS. 3A and 3B). In addition, when administered to animals with impaired liver function, it was confirmed that effectively reduce the abnormally increased blood cholesterol, AST and ALT levels (Figs. 5a to 7c).

Therefore, the spirulina genus algae produced by the method of the present invention exhibits effects such as diabetic symptom improvement and liver function impairment, and can be used as an active ingredient in pharmaceutical compositions, food compositions and the like which can utilize such effects.

Another embodiment of the present invention provides a pharmaceutical composition for treating or preventing liver disease, comprising the produced spirulina genus algae as an active ingredient.

As described above, the spirulina genus algae produced by the method of the present invention has an effect on improving liver damage, and can be used as an active ingredient of a pharmaceutical composition for treating or preventing liver disease.

In the pharmaceutical composition for treating or preventing liver disease of the present invention, spirulina genus algae included as an active ingredient may be processed and used in various forms. For example, by processing the spirulina genus algae culture in the form of a powder, the tablet form of the spirulina genus algae in the tablet form, the spirulina algae cultured in a concentrated sludge form, etc. Can be used.

According to one embodiment of the present invention, it was confirmed that the difference in the effect of improving liver function according to the processing form of the spirulina genus algae (Figs. That is, the spirulina genus algae generally shows an effect of improving liver function, but when the spirulina algae is processed into a sludge form, it can be seen that it shows an excellent liver function improving effect than the case of processing into a powder or tablet form.

In addition, the method of processing the culture of spirulina genus algae in the form of sludge is not particularly limited thereto, and a filtration method, a centrifugation method, a drying method, and the like may be used.

As used herein, the term "liver disease" refers to a condition in which hepatocytes, liver tissues, and the like do not perform a normal function due to internal, genetic, or exogenous factors. Specific examples of the liver disease may be cirrhosis, cirrhosis, hepatitis, liver cancer, liver fibrosis, fatty liver, liver tissue hypertrophy, but are not particularly limited thereto.

As used herein, the term "prevention" means any action of inhibiting or delaying liver disease by administration of a composition comprising the spirulina genus algae.

As used herein, the term "treatment" means any action in which symptoms of liver disease are improved or beneficially changed by administration of a composition comprising the spirulina genus algae.

The pharmaceutical composition of the present invention may include 0.001 to 80, specifically 0.001 to 70, more specifically 0.001 to 60% by weight based on the total weight of the composition of the spirulina genus algae, but is not limited thereto.

In addition, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition, the carrier may comprise a non-naturally occuring carrier (carrier) have. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical compositions are tablets, pills, powders, granules, capsules, suspensions, solvents, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, transdermals, respectively, according to a conventional method. It can be formulated in the form of absorbents, gels, lotions, ointments, creams, patches, cataplasms, pastes, sprays, skin emulsions, skin suspensions, transdermal patches, drug containing bandages or suppositories. Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, weights, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid form preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. It may be prepared by adding various excipients such as humectants, sweeteners, fragrances, preservatives and the like in addition to liquid oral liquids or liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Another embodiment of the present invention provides a method of treating liver disease comprising administering the pharmaceutical composition to a subject suspected of developing a liver disease other than a human.

As used herein, the term "administration" refers to the introduction of a composition comprising said spirulina algae into a subject in a suitable manner.

As used herein, the term "individual" means all animals, such as rats, mice, and livestock, including humans who may or may have developed liver disease. As a specific example, it may be a mammal including a human.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type and severity of the subject, severity, age, sex, activity of the drug, Sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. For example, the spirulina genus algae may be administered at 0.01 to 500 mg / kg per day, specifically, at a dose of 10 to 100 mg / kg, and the administration may be administered once or several times a day.

The pharmaceutical composition may be administered as a separate therapeutic agent or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

In addition, the pharmaceutical composition may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally, or topically) according to a desired method, and the dosage is based on the condition and weight of the patient, and the degree of disease. Depending on the drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

Another embodiment of the present invention provides a food composition for improving liver function, comprising the spirulina genus algae produced as an active ingredient.

As used herein, the term " improvement " means any action that at least reduces the parameters associated with a condition, e.

The term "food" of the present invention, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinks, tea, drinks, alcoholic beverages , Vitamin complexes, nutraceuticals and health foods, and includes all foods in the usual sense.

Since the food composition of the present invention is derived from the spirulina genus algae that can be ingested daily, it can be expected to have a high liver disease improvement effect, it can be very useful for health promotion purposes.

The functional food (functional food) is the same term as a food for special health use (Food for special health use, FoSHU), in addition to the nutritional supply, the processed food, so that the bioregulatory function appears efficiently, high medical effect Means food. Here, the term 'function (sex)' refers to obtaining a useful effect for health purposes such as nutrient control or physiological action on the structure and function of the human body. The food of the present invention may be prepared by a method commonly used in the art, and the preparation may be performed by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as the formulation is recognized as food.

Food composition of the present invention can be prepared in a variety of dosage forms, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a natural product, and because the portability is excellent, The food of the present invention can be taken as an adjuvant for enhancing the improvement effect of liver disease.

The health food refers to foods having active health maintenance or promotion effect as compared to general foods, and the health supplement food refers to foods for health supplementation purposes. In some cases, the terms nutraceutical, health food, dietary supplement are used.

Specifically, the health functional food is a food prepared by adding the spirulina genus algae of the present invention to food materials, such as beverages, teas, spices, gums, confectionery, or the like, encapsulated, powdered, suspensions, and the like when ingested It means to bring a specific effect, but unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material.

The food composition may further include a physiologically acceptable carrier, and the type of carrier is not particularly limited and may be any carrier that is commonly used in the art.

In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve the smell, taste, time and the like. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu) and chromium (Cr); And amino acids such as lysine, tryptophan, cysteine, valine and the like.

In addition, the food composition is a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetic acid, etc.), fungicides (bleaching powder and highly bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisol (BHA), butylhydride) Oxytoluene (BHT), etc.), colorants (such as tar pigments), colorants (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (such as MSG glutamate), sweeteners (ducin, cyclate, saccharin Foods such as sodium, etc.), fragrances (vanillin, lactones, etc.), swelling agents (alum, potassium D-tartrate, etc.), reinforcing agents, emulsifiers, thickeners (foils), coatings, gum herbicides, foam inhibitors, solvents, modifiers, etc. It may include food additives. The additive may be selected according to the type of food and used in an appropriate amount.

An example of the food composition of the present invention may be used as a health beverage composition, in which case it may contain various flavors or natural carbohydrates and the like as additional ingredients, such as a general beverage. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin, cyclodextrin; Sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumartin, stevia extract; Synthetic sweeteners such as saccharin and aspartame; The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 ml of the health beverage composition of the present invention.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, Alcohol or carbonation agent and the like. Others may contain fruit flesh for the production of natural fruit juices, fruit juice drinks, or vegetable drinks. These components can be used independently or in combination. Although the ratio of such an additive is not critical, it is generally selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

Another embodiment of the present invention provides a pharmaceutical composition for treating or preventing type 1 diabetes, comprising the produced spirulina genus algae as an active ingredient, and a method for treating type 1 diabetes using the composition.

As described above, the spirulina genus algae produced by the method of the present invention exhibits an improvement in diabetic symptoms, and thus can be used as an active ingredient in the pharmaceutical composition for treating or preventing type 1 diabetes mellitus, and using the pharmaceutical composition to form a first It can cure diabetes.

The detailed description of the composition of the pharmaceutical composition and the method of treating type 1 diabetes is the same as described in the above-mentioned liver disease related items.

Another embodiment of the present invention provides a food composition for improving type 1 diabetes, comprising the produced spirulina genus algae as an active ingredient.

Specific description of the food composition for improving type 1 diabetes comprising spirulina genus algae as an active ingredient is the same as described above in the food composition for improving liver function.

Another embodiment of the present invention provides a feed composition comprising the produced spirulina algae as an active ingredient.

The term "feed" of the present invention means any natural or artificial diet, one meal, or the like or any ingredient of the one meal for the animal to eat, ingest and digest. Specifically, feed comprising spirulina genus algae produced by the method of the present invention may be prepared in various forms of feed known in the art, and specifically, may include rich feed, research feed and / or special feed.

The rich feed includes seed fruits containing grains such as wheat, oats and corn, and by-products obtained by refining grains, bran, beans, fluids, sesame seeds, linseed, coco, etc., including rice bran, bran and barley bran. Concentrated fresh liquid from fish, fish meal, fish residues and other fish residues such as by-products such as dried starch, sweet potato, and potato. Animal feeds such as fish soluble, meat meal, blood meal, milk powder, skim milk powder, milk from cheese, and whey, which is a balance when making casein from skim milk, Chlorella, seaweed, but are not limited thereto. Forage, raw vegetables such as grasses, grasses, and green grass, fodder turnips, fodder beets, and root vegetables such as Lutherbearger, a type of turnip, raw grass, green grass crops, and grains Silage (silage), grasses, grasses, and dried hay, straws of breeders, and leaves of legumes, which are filled and fermented by lactic acid fermentation, are not limited thereto. Special feeds are supplemented with mineral feeds such as oyster shells and rock salts, urea feeds such as urea and its derivatives, diureide isobutane, and natural feed ingredients. Feed additives and dietary supplements, which are substances added in small amounts, are not limited thereto.

Feed containing spirulina genus algae produced by the method of the present invention can be prepared by adding the spirulina genus algae in a suitable effective concentration range according to various feed production methods known in the art.

The feed provided by the present invention is not particularly limited as long as it is an individual for the purpose of preventing or treating diabetes or liver disease, and any one may be applied. For example, monkeys, dogs, cats, rabbits, morphotes, rats, mice, cows, sheep, pigs, goats and the like, but not limited to, such as, but not limited to, and can be applied to any individual.

Another embodiment of the invention provides a minimal medium for the production of Spirulina algae comprising NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ .7H ₂ O and NaNO ₃ .

By using the production method of spirulina genus algae provided in the present invention, it is possible to minimize the components contained in the medium, to economically produce spirulina genus algae, the spirulina genus algae produced by the above method improves diabetic symptoms, liver Since it exhibits various pharmacological activities, such as preventing impairment of function and treating liver damage, it may be widely used for economic production of spirulina genus algae showing pharmacologically improved activity.

1 is a graph showing a result of comparing the growth rate of spirulina genus algae cultured using UTEX medium, Zarrouk medium or SOT medium.
Figure 2 is a graph and chart showing the growth rate of the spirulina genus algae according to the culture time and the flow rate of the medium in culturing the spirulina genus algae using the minimum medium provided in the present invention.
Figure 3a is a graph showing the results of comparing the effect of spirulina extract or PC on the level of phosphorylated JNK expressed in cytokines (IL-1β + IFN-γ) treated apoptosis-induced RINm5F cells.
3B is a graph showing the results of comparing the effects of spirulina extract or PC on the level of phosphorylated p38 expressed in cytokines (IL-1β + IFN-γ) treated RINm5F cells induced with apoptosis.
Figure 3c is a graph showing the results of comparing the effect of spirulina extract or PC on the level of phosphorylated ERK expressed in cytokines (IL-1β + IFN-γ) treated RINm5F cells induced with apoptosis.
FIG. 3D is a graph showing the results of comparing the effects of spirulina extract or PC on the caspase 3 activity expressed in cytokines (IL-1β + IFN-γ) treated RINm5F cells induced with apoptosis.
Figure 3e is a fluorescence micrograph showing the result of comparing the effect of spirulina extract or PC on the cytokine (IL-1β + IFN-γ) treated DNA fragmentation in apoptosis-induced RINm5F cells.
4A is a diagram schematically showing an experimental design using an animal model of diabetes.
Figure 4b is a micrograph (top) of the cell shape of the islet of the pancreas according to the combination treatment of streptozotocin (STZ) and spirulina extract; Fluorescence micrograph (interruption) showing the results of immunostaining of pancreatic tissue with anti-insulin antibody; And fluorescence micrographs showing the results of immunostaining of pancreatic tissue with an anti-nitrotyrosine antibody.
Figure 4c is a graph showing the result of comparing the size change of the islet of the pancreas according to the combination treatment of streptozotocin (STZ) and spirulina extract.
Figure 4d is a graph showing the results of comparing the changes in insulin and NO levels in the blood, according to the combination treatment of streptozotocin (STZ) and spirulina extract.
Figure 5a is a graph showing the result of comparing the cholesterol level measured in the blood of normal mice administered three types of spirulina samples.
Figure 5b is a graph showing the result of comparing the cholesterol level measured in the blood of acute liver disease model animals administered three types of spirulina samples.
Figure 5c is a graph showing the result of comparing the cholesterol level measured in the blood of normal mice or acute liver disease model animals administered the sludge-type spirulina sample.
Figure 6a is a graph showing the results of comparing the AST levels measured in the blood of normal mice administered three types of spirulina samples.
Figure 6b is a graph showing the results of comparing the AST levels measured in the blood of acute liver disease model animals administered three types of spirulina samples.
Figure 6c is a graph showing the results of comparing the AST level measured in the blood of normal mice or acute liver disease model animals administered sludge-type spirulina samples.
Figure 7a is a graph showing the results of comparing the ALT levels measured in the blood of normal mice administered three types of spirulina samples.
Figure 7b is a graph showing the results of comparing the ALT levels measured in the blood of acute liver disease model animals administered three types of spirulina samples.
Figure 7c is a graph showing the results of comparing the ALT level measured in the blood of normal mice or acute liver disease model animals administered sludge-type spirulina samples.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Development of Medium for Spirulina Genus Algae Culture

Example 1-1: Comparison according to the medium for spirulina algae culture

Until now, UTEX medium or Zarrouk medium is known as a medium used to culture spirulina genus algae, and recently, a SOT medium improved from the Zarrouk medium has been developed (Korean Patent No. 10-0704436). The growth rate of spirulina genus algae cultured using the SOT medium was compared with the growth rate of spirulina genus algae cultured using UTEX medium or Zarrouk medium (Table 1 and FIG. 1). In this case, Spirulina maxima UTEX LB2342 strain (UTEX Culture Collection of Algae) was used as the spirulina genus algae, and the culture was performed under light conditions of 12 lux / 12 hours at 32 ° C. at 3,000 Lux, and cultured spirulina The individual level of the genus algae was calculated by measuring the absorbance of the culture at 565 nm.

Comparison of Components of Algal Culture Medium of Spirulina Genus (Content per 1L) ingredient UTEX badge Zarrouk Badge SOT Badge NaHCO3
Na2CO3
K2HPO4
NaNO3
K2SO4
NaCl
MgSO4
CaCl2
FeSO4
EDTA
Micronutrient Sol.
PIV Solution
Chu solution
Vitamin b12
Mineral mix 13.61 g
4.03 g
0.5g
2.5g
1 g
1 g
0.2 g
0.04 g
-
-
10.00 ml
6.00 ml
1.00 ml
1.00 ml
- 16.8 g
-
0.5 g
2.5g
1 g
1 g
0.2 g
0.04 g
0.01 g
0.08g
-
-
-
-
- 16.8 g
-
0.5g
2.5g
1 g
1 g
0.2 g
0.04 g
0.01 g
0.08g
-
-
-
-
1.00 ml

1 is a graph showing a result of comparing the growth rate of spirulina genus algae cultured using UTEX medium, Zarrouk medium or SOT medium.

As shown in Figure 1, the growth rate of the spirulina genus algae cultured in SOT medium is relatively higher than that in UTEX medium, it was confirmed that the same as that cultured in Zarrouk medium.

Example 1-2 Development of Minimal Medium for Spirulina Genus Algae Culture

Since the SOT medium used in Example 1-1 is composed of various components, it was intended to select the essential medium consisting of the components necessary for the culture of spirulina genus algae among these components.

To this end, except for using each medium consisting of a combination of the various components constituting the SOT medium, spirulina genus algae were cultured under the same conditions as described above, and their culture was confirmed.

As a result, when using a medium consisting of NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and NaNO ₃ , it was confirmed that the spirulina genus algae can be normally cultured using the minimum medium components. In addition, when using a medium added with H ₃ BO ₃ , MnSO ₄ · 7H ₂ O, ZnSO ₄ · 7H ₂ O, CoCl ₂ · 6H ₂ O or Na ₂ MoO ₄ · 7H ₂ O in the medium consisting of the above components, It was confirmed that the growth rate of algae of spirulina increased.

Example 1-3 Effect of Medium Flow Rate on Culture of Spirulina Genus Algae

When producing spirulina genus algae using the minimum medium containing the component determined in Example 1-2, it was confirmed that the production of spirulina genus algae is greatly affected by the flow rate of the medium. In the case of culturing the medium at a flow rate lower than 30 cm / sec in the tubular incubator through a preliminary experiment, a part of the spirulina genus in which the incubation proceeds is settled at the bottom of the tubular incubator, and decayed over time. It was confirmed that this did not proceed. In contrast, when cultured using a known spirulina culture medium (UTEX medium, Zarrouk medium or SOT medium), when culturing at a rate of 10 cm / sec or more, the spirulina genus that precipitated even if some of the precipitates at the bottom of the incubator It was confirmed that the algae proliferated without decay.

Thus, the culture medium was changed while changing the flow rate of the medium using the minimum medium containing the component determined in Example 1-2, and the change of the medium flow rate was to determine how the effect on the growth of Spirulina spp.

Approximately, inoculate the spirulina genus algae to the minimum medium and incubate for 57 days in 12 hours light condition / 12 hours dark condition at 32 ℃ light conditions of 3,000 Lux, the flow rate of the medium 30 to 60 cm / The flow rate was changed to sec. At this time, the daily growth rate of the spirulina genus algae according to the flow rate change section of the medium was measured and compared (Fig. 2).

Figure 2 is a graph and chart showing the growth rate of the spirulina genus algae according to the culture time and the flow rate of the medium in culturing the spirulina genus algae using the minimum medium provided in the present invention.

As shown in FIG. 2, the daily growth rate showed absorbance (565 nm) of 0.02 or more in the range of 30 to 45 cm / sec, but the absorbance level of lower than 0.02 (565 nm) in the range of 52.5 and 60 cm / sec. Was confirmed. As a result, the daily growth rate of spirulina or algae was decreased when the medium was circulated at a speed higher than 45 cm / sec. However, if the circulating medium was circulated at too high flow rate, the collision frequency between the spirulina and the algae was reduced. It was confirmed that this is due to an increase in the loss rate of spirulina algae.

Example 1-4: Effect of Photoperiod on Culture of Spirulina Genus Algae

From the results of Examples 1-3, when culturing the medium while circulating the medium at too low flow rate (30 cm / sec or less) or too high flow rate (45 cm / sec or more) in the tubular incubator, It was found that the culture could not be performed normally, and to determine whether photoperiod could affect the culture results according to the medium flow rate.

Approximately, inoculate the spirulina algae to the minimum medium containing the component determined in Example 1-2, and the photoperiod (light condition (h): dark condition (h): light condition of 3,000 Lux and various conditions at 32 ° C) = 12:12, 13:11, 14:10, 15: 9, 16: 8, 17: 7, 18: 6, 19: 5, 20: 4, 21: 3, 22: 2, 23: 1 and 24 : 0) While culturing for 57 days under conditions, the flow rate of the medium was changed to a flow rate of 30 to 75 cm / sec for each section. At this time, the daily growth rate of the spirulina algae according to the flow rate change section of the medium was measured and compared (Table 2).

Comparison of Daily Growth Rate of Spirulina Algae with Photoperiod and Flow Rate Gwangju
(Name: cancer) Medium flow rate (cm / sec) 30 37.5 45 52.5 60 67.5 75 12:12
13:11
14:10
15: 9
16: 8
17: 7
18: 6
19: 5
20: 4
21: 3
22: 2
23: 1
24: 0 0.024
0.024
0.022
0.022
0.020
0.019
0.010
0.010
0.010
0.006
0.005
0.003
0.003 0.032
0.033
0.031
0.030
0.028
0.028
0.027
0.027
0.023
0.014
0.010
0.008
0.008 0.029
0.032
0.035
0.038
0.040
0.038
0.038
0.035
0.032
0.027
0.022
0.018
0.014 0.017
0.020
0.024
0.029
0.035
0.036
0.037
0.038
0.039
0.005
0.005
0.005
0.002 0.014
0.016
0.022
0.025
0.029
0.033
0.035
0.037
0.054
0.025
0.022
0.008
0.002 0.011
0.012
0.016
0.020
0.023
0.027
0.029
0.033
0.005
0.005
0.003
0.003
0.002 <0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.003
<0.002

As shown in Table 2, it was confirmed that when the light condition time of photoperiod increased, the medium flow rate showing the maximum daily growth rate of Srirulina genus algae tended to increase. This is because the growth rate of spirulina genus algae is relatively higher than the loss rate of spirulina genus algae with increasing medium flow rate.

However, when the light condition time of photoperiod exceeded 20 hours, it was confirmed that the daily growth rate of Srirulina genus algae drastically decreased even with increasing medium flow rate. The overproliferated Srirulina genus algae were analyzed to settle and rot at the bottom of the tubular incubator.

Therefore, it was confirmed that there is a condition that the productivity of spirulina genus algae no longer increases even if the light condition and the medium flow rate of photoperiod increase, but the condition that indicates the maximum productivity of the genus spirulina algae is 20: : 4, the medium flow rate was found to be 60cm / sec.

Example 1-5: Comparison of culture maintenance and production of spirulina algae

As a culture condition including the minimum medium flow rate for producing the spirulina genus algae identified in Example 1-3 and the maximum medium flow rate representing the maximum productivity of the algae of Spirulina genus confirmed in Example 1-4 Spirulina genus algae were cultured and the culture results were compared (Table 3). The results of the culture were determined by the pH of the culture medium, the OD of the culture, the content of chlorophyll a in the culture, the dry weight of 1 L of culture, the floating activity of Spirulina algae, and the number of coils wound by the Spirulina algae. (No. of Coils). Floating activity is a result of converting the spirulina genus algae into the ratio of the subsidence in the dark conditions and injured in the light conditions as a ratio, the higher the activity level of the activity of the spirulina algae is determined to be higher Can be. In addition, the number of coils coiled (No. of Coils) refers to the number of coil forms, which are the morphological characteristics exhibited by the spirulina genus, the morphological stability is improved as the number of coils increases, the stability of spirulina when the number of coils is reduced It is known that this is reduced.

Comparison of culture results according to medium flow rate Medium flow rate (cm / sec) 30 60 Photoperiod (light: cancer)
Incubation time
Medium pH
OD
Chlorophyll a (ug / mL)
Dry weight (g / L)
Floating activity (%)
No. of Coils 12:12
30 days
9.3
0.171
7.14
0.07
-71%
1.5 20: 4
30 days
9.46
0.526
17.94
0.22
71%
1.3

As shown in Table 3, under the conditions in which the medium flow rate is increased and the photoperiod is applied to meet the above, the OD value, the content of chlorophyll a, the dry weight and the floating activity are significantly increased, compared to the case where the medium flow rate is decreased. It was confirmed that the productivity of the genus algae was significantly increased.

In addition, when the medium flow rate was increased, the number of coils showed a level similar to that when the medium flow rate was decreased. Therefore, it was confirmed that the morphological stability of the cultured spirulina genus algae was equivalent to that when the medium flow rate was decreased. Could.

From the above results, cultured under conditions of 12:12 photoperiod (light: cancer) and medium flow rate 30 m / sec to maintain the spirulina genus algae, and to produce spirulina genus algae, It was found that it is preferable to incubate at a condition of 20: 4 and a medium flow rate of 60 m / sec.

Example 2: Validation of Pharmacological Activity of Algae of Spirulina

Example 2-1 Preparation of Spirulina Genus Algae Samples

Using the flow rate range determined in Example 1-2 and the medium determined in Examples 1-3, the spirulina genus algae were cultured, and then prepared into four types of samples: Sample 1 was cultured spirulina The genus algae were dried and then powdered; Sample 2 is a sample prepared in the form of a tablet by solidifying the powdered sample; Sample 3 is a sample prepared in the form of sludge obtained by centrifuging the culture of algae of spirulina and suspending the cells precipitated through centrifugation in distilled water; Sample 4 is an extract sample obtained by ethanol extraction of spirulina algae. The preparation method of the extract sample is as follows: The cultured spirulina genus algae are extracted three times for 4 hours with 70% ethanol at 40 ° C. three times to obtain an extract, which is lyophilized and powdered. The powder was again added to 70% ethanol at 40 ° C. to dissolve for 1 hour, and the dissolved sample was centrifuged at 3,500 rpm for 5 minutes to obtain a supernatant, and the obtained supernatant was prepared by lyophilization.

Example 2-2: Diabetes Treatment Effect

Example 2-2-1: Effect on apoptosis of insulin tumor cells

The effects of spirulina algae on apoptosis induced by treatment of cytokines (IL-1β and IFN-γ) for 30 minutes on RINm5F cells, which are insulin tumor cells of rats, were tested. At this time, the spirulina genus algae used is the spirulina extract (sample 4) prepared in Example 2-1, the comparative group as a known component of the spirulina genus algae (Phycocyanin) which is a kind of pigment protein (phycobiliprotein) Used. The PC is known to exhibit antioxidant effects, anti-inflammatory effects, neuronal cell protective effect, hepatocyte protective effect and the like.

Approximately, RINm5F cells were cultured, and the spirulina extract (1 μg / ml), PC (1 μg / ml), IL-1β (50 U / ml) and IFN-γ (100 U / ml) alone Or in combination with the culture. RINm5F cells were then harvested, extracts were obtained from each of the cells, and then analyzed by Western blot analysis to compare the level of phosphorylated JNK, the level of phosphorylated p38, the level of phosphorylated ERK and the activity of caspase 3 ( 3A-3D). In addition, TUNEL staining was performed on each of the recovered RINm5F cells to compare DNA fragmentation levels (FIG. 3E).

Figure 3a is a graph showing the results of comparing the effect of spirulina extract or PC on the level of phosphorylated JNK expressed in cytokines (IL-1β + IFN-γ) treated apoptosis-induced RINm5F cells.

As shown in Figure 3a, the level of phosphorylated JNK increased by the treatment of cytokines, it was confirmed that the treatment of spirulina extract or PC with cytokines suppressed the increase of phosphorylated JNK levels. In addition, the inhibitory effect of the phosphorylated JNK level increase was confirmed that the spirulina extract is relatively higher than the PC.

FIG. 3B is a graph showing the results of comparing the effects of spirulina extract or PC on the level of phosphorylated p38 expressed in cytokines (IL-1β + IFN-γ) treated RINm5F cells induced with apoptosis.

As shown in Figure 3b, the level of phosphorylated p38 was increased by the treatment of cytokines, but it was confirmed that the treatment of spirulina extract or PC with cytokines suppressed the increase of the level of phosphorylated p38. In addition, the inhibitory effect of the phosphorylated p38 level increase was confirmed that the spirulina extract is relatively higher than the PC.

Figure 3c is a graph showing the results of comparing the effect of spirulina extract or PC on the level of phosphorylated ERK expressed in cytokines (IL-1β + IFN-γ) treated RINm5F cells induced with apoptosis.

As shown in Figure 3c, it was confirmed that spirulina extract, PC, IL-1β and IFN-γ did not significantly affect the level of phosphorylated ERK.

3D is a graph showing the results of comparing the effects of spirulina extract or PC on the activity of caspase 3 expressed in cytokines (IL-1β + IFN-γ) -treated RINm5F cells induced with apoptosis.

As shown in FIG. 3D, caspase 3 activity was increased by the treatment of cytokines, but it was confirmed that the treatment of caspase 3 was inhibited by treating spirulina extract or PC with cytokines. In addition, it was confirmed that the inhibitory effect of caspase 3 increased the activity of spirulina extract and PC.

Figure 3e is a fluorescence micrograph showing the result of comparing the effect of spirulina extract or PC on the cytokine (IL-1β + IFN-γ) treated DNA fragmentation in apoptosis-induced RINm5F cells.

As shown in FIG. 3E, TUNEL-positive cells showing DNA fragmentation were increased by cytokine treatment, but it was confirmed that the treatment of spirulina extract or PC with cytokines inhibits the increase of TUNEL-positive cells.

Example 2-2-2: Effect on Diabetic Animal Model

Example 2-2-2-1: Preparation of Sample

Since oral administration of streptozotocin (STZ) to rats is known to induce type 1 diabetes symptoms from rats, Spirulina genus algae extracts are used in animal models of type 1 diabetes-induced diabetes. Diabetes treatment effect was tested.

Approximately, the following six groups were set up and reared for 4 weeks using rats (FIG. 4A): control group orally administered only water during the breeding period; Experimental group 1 (Spi) orally administered 200 mg of spirulina extract (sample 4) per body weight (Kg) during the breeding period; Experimental group 2 (STZ (2 wks)) orally administered streptozotocin at the time of breeding for 2 weeks, and breeding again for 2 weeks; Oral administration of streptozotocin at the time of breeding for 2 weeks with oral administration of 200 mg of spirulina extract per sample (Kg) during breeding period (Spi + STZ); Experimental group 4 (STZ (4 wks)) administered streptozotocin and raised for 4 weeks; And experimental group 5 (STZ + Spi), orally administered with 200 mg of spirulina extract (sample 4) per streptozotocin and body weight (Kg).

Rats of each experimental group whose breeding was completed were measured and compared with the weight before the experiment (Table 4).

Weight Change in Diabetic Animal Models Experimental group Initial weight (g) Final weight (g) Control
Experimental group 1
Experiment group 2
Experiment group 3
Experimental Group 4
Experimental group 5 230.0 ± 5.32
235.0 ± 4.20
235.0 ± 5.44
230.0 ± 6.03
260.0 ± 2.56
260.0 ± 5.13 346.6 ± 4.21
353.0 ± 3.65
284.0 ± 7.55
295.3 ± 7.35
234.8 ± 13.01
284.0 ± 6.97

As shown in Table 4, when streptozotocin (STZ) was administered, the body weight was increased to a relatively low level compared to the control group, but when spirulina extract was treated, compared to the case of administration of streptozotocin (STZ) It was confirmed that the weight is increased to a relatively high level.

In addition, each rat was sacrificed after anesthesia, and the pancreas was removed from each rat to obtain blood at the same time. The obtained blood was centrifuged at 3,500 rpm for 5 minutes to obtain a supernatant, which was then used as a blood sample.

Example 2-2-2-2 Pancreas Analysis

Hematoxylin & eosin staining was performed to confirm the cell morphology of Langerhans islet contained in each pancreas extracted in Example 2-2-2-1, and the expression of insulin and nitrotyrosine expressed in pancreatic tissue To compare the levels, immunostaining with anti-insulin and anti-nitrotyrosine antibodies was performed followed by fluorescence microscopy (Figure 4b). In addition, the size of the Langerhans island was measured using IMT i-solution software (FIG. 4C).

Figure 4b is a micrograph (top) of the cell shape of the islet of the pancreas according to the combination treatment of streptozotocin (STZ) and spirulina extract; Fluorescence micrograph (interruption) showing the results of immunostaining of pancreatic tissue with anti-insulin antibody; And fluorescence micrographs showing the results of immunostaining of pancreatic tissue with an anti-nitrotyrosine antibody.

As shown in Figure 4b, when treated with streptozotocin, degenertive and necrotic changes of Langerhans islet cells were observed, but when the spirulina extract was treated, the level of degenerative and necrotic changes was reduced. It was. In addition, when treated with streptozotocin, the level of insulin in the pancreatic tissue was drastically reduced, but when spirulina extract was treated, it was confirmed that the decrease in insulin was suppressed. Finally, when treated with streptozotocin, the level of nitrotyrosine in pancreatic tissue was increased, but when spirulina extract was treated, it was confirmed that the increase of nitrotyrosine was suppressed.

Figure 4c is a graph showing the results of comparing the size change of the islet of the pancreas according to the combination treatment of streptozotocin (STZ) and spirulina extract.

As shown in Figure 4c, when the treatment of streptozotocin was reduced in the size of the island of Langerhans in pancreatic tissue, it was confirmed that the size of the Langerhans island is suppressed when the spirulina extract is treated.

Example 2-2-2-3: Blood Sample Analysis

Using the blood sample in Example 2-2-2-1, insulin levels and NO levels were measured (FIG. 4D).

Figure 4d is a graph showing the result of comparing the change in the level of insulin and NO in the blood, according to the combination treatment of streptozotocin (STZ) and spirulina extract.

As shown in Figure 4d, when treated with streptozotocin, it was confirmed that the insulin level in the blood was reduced and the level of NO was increased, but when the spirulina extract was treated, the insulin level decrease and the No level increase were suppressed.

Meanwhile, blood glucose (BG) levels, total cholesterol (CH) levels, triglyceride (TG) levels, blood urea nitrogen (BUN) levels, and creatinine (CR) levels included in the blood samples were measured and compared (Table 1). 5).

Comparison of Levels of Various Components in Blood (Unit: ㎎ / ㎗) Experimental group BG CH TG BUN CR Control
Experimental group 1
Experiment group 2
Experiment group 3
Experimental Group 4
Experimental group 5 200.5 ± 15.3
252 ± 16.1
522.3 ± 4.36
469.7 ± 19.93
639 ± 7.8
574 ± 16.7 81.0 ± 1.47
77.0 ± 1.05
125.5 ± 3.24
108.0 ± 2.22
129.3 ± 5.31
105.3 ± 1.83 54 ± 6.9
48.0 ± 5.9
414.0 ± 37.28
360.0 ± 14.71
232 ± 34.2
126.8 ± 11.1 20.6 ± 1.0
20.7 ± 0.4
38.8 ± 2.05
31.1 ± 1.10
43.7 ± 3.0
39.9 ± 2.0 0.5 ± 0.01
0.6 ± 0.02
0.5 ± 0.02
0.5 ± 0.02
0.5 ± 0.02
0.5 ± 0.02

As shown in Table 5, the streptozotocin-treated rats showed general diabetes mellitus (increased blood sugar level, decreased insulin level, etc.), but when the spirulina extract was treated, it was confirmed that such diabetes mellitus was improved.

Therefore, from the results of Examples 2-2-1 and 2-2-2, it was found that the spirulina extract cultured with the culture solution provided in the present invention has an effect of alleviating the symptoms of diabetes.

Example 2-3: effect of improving liver function

Acute liver disease model animals prepared by administering carbon tetrachloride to mice were administered with various types of spirulina (samples 1 to 3) obtained in Example 2-1, and blood cholesterol levels, ALT levels, and AST levels were measured. The effects of liver spirulina on algae formulations were compared.

Example 2-3-1 Preparation of Experimental Group

Approximately, the primary control group (Nor_Sham), which was bred for 37 days without administering anything to the normal mice, the experimental group 11 (Nor_Powder), which was administered for 37 days with the sample 1 (powder) to the normal mouse, and the sample 2 (for normal mice) Tablets) and experimental group 12 (Nor_Tablet) incubated for 37 days, sample 3 (sludge) in normal mice, and experimental group 13 (Nor_Sludge) incubated for 37 days, sample 3 (sludge) in normal mice, 37 The experimental group 14 (CCl4_Pre_Sludge), which was fed for 4 days, and then reared for 3 days, the secondary control group (CCl4_Sham), which was raised for 3 days without administration of any stones, and the mice that received carbon tetrachloride. Experiment group 21 (CCl4_Powder) administered with sample 1 (powder) and experimental group 22 (CCl4_Tablet) and carbon tetrachloride administered with sample 2 (tablet) and fed to carbon tetrachloride mice for 3 days Administration of the sample 3 (sludge) in mice administered, respectively, and were prepared to a breeding experiment 23 (CCl4_Sludge) for 3 days. At this time, mice of each group were assigned to 5 males and 5 females.

Example 2-3-2: Cholesterol Level Comparison

First, as a result of comparing the cholesterol level measured in the blood of the control group 1, the experimental group 11, the experimental group 12 and the experimental group 13 for the normal mouse (Fig. 5a), there was no difference in each group.

However, as a result of comparing cholesterol levels measured in blood of control group 2, test group 21, test group 22, and test group 23 in acute liver disease model animals (FIG. 5B), blood cholesterol levels in control group 2 were higher than control group 1. When the spirulina sample was treated rapidly (experimental groups 21 to 23), it was confirmed that the level of blood cholesterol was relatively decreased compared to the control group 2.

In addition, when treated with sludge-type spirulina samples (experimental groups 13, 14 and 23) (Fig. 5c), blood cholesterol in all acute liver disease model animals (experimental groups 14 and 23) compared to normal mice (experimental group 13). Levels increased, and blood cholesterol levels were further increased when spirulina samples were administered in advance (Experimental Group 14), and later when spirulina samples were administered (Experimental Group 23). When treated (experiment 23) it was confirmed that the lowest blood cholesterol level.

Example 2-3-3: AST Level Comparison

First, as a result of comparing the AST levels measured in the blood of the control group 1, the experimental group 11, the experimental group 12 and the experimental group 13 for the normal mice (Fig. 6a), there was no difference in each group.

However, as a result of comparing the AST levels measured in the blood of control group 2, test group 21, test group 22, and test group 23 of the acute liver disease model animals (Fig. 6b), the level of blood AST in control 2 compared to control 1 When the spirulina sample was treated (experimental groups 21 to 23), the blood AST level of the experimental groups 21 and 23 decreased relatively compared to the control group 2, and the blood AST level of the experimental group 23 was lower than that of the experimental group 21. It was confirmed that

In addition, in the case of treatment of sludge-type spirulina samples (experimental groups 13, 14 and 23) (FIG. 6C), blood AST in all acute liver disease model animals (experimental groups 14 and 23) compared to normal mice (experimental group 13). Levels increased, and blood AST levels were further increased when spirulina samples were previously administered (Experimental Group 23) compared to prior administration of spirulina samples (Experimental Group 14).

Example 2-3-4: ALT Level Comparison

First, as a result of comparing the ALT level measured in the blood of the control group 1, the experimental group 11, the experimental group 12 and the experimental group 13 for the normal mouse (Fig. 7a), except when the powdered spirulina sample treated (experimental group 11) Did not show any difference in each group.

However, as a result of comparing the ALT level measured in the blood of the control group 2, experimental group 21, experimental group 22 and experimental group 23 in the acute liver disease model animals (Fig. 7b), the blood ALT level in the control group 2 compared to the control group 1 rapidly In the case of treatment with spirulina samples (experimental groups 21-23), the blood ALT level was relatively decreased compared to control 2, and among the treatment of sludge-type spirulina samples, the lowest in blood It was confirmed that the ALT level.

In addition, when the sludge-type spirulina samples were treated (experimental groups 13, 14, and 23) (FIG. 7C), acute liver disease model animals (experimental groups 14 and 23) were compared to normal mice (experimental group 13). The level was increased, and compared with the case where the spirulina sample was previously administered (Experimental Group 14), the blood ALT level was relatively lower when the spirulina sample was administered later (Experiment 23).

Therefore, from the results of the above Examples 2-3-2 to 2-3-4, when using a formulation processed in the form of sludge of spirulina cultured with the culture solution provided in the present invention, relatively excellent liver damage prevention and damaged liver It can be seen that it shows a functional therapeutic effect.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (14)

Inoculate Spirulina algae to a medium containing NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O, and NaNO ₃ , and light conditions of 3,000 to 9,000 Lux at 32 to 38 ° C., 12 to 20 (h): 12 And culturing while circulating the medium in a tubular incubator at a photoperiod (light: dark) and a flow rate of 30 to 60 cm / s of 4 to 4 h.
The method of claim 1,
The medium adds a component selected from the group consisting of H ₃ BO ₃ , MnSO ₄ · 7H ₂ O, ZnSO ₄ · 7H ₂ O, CoCl ₂ · 6H ₂ O, Na ₂ MoO ₄ · 7H ₂ O and combinations thereof To include, Spirulina genus algae production method.
The method of claim 1,
The medium is purified seawater, spirulina genus algae production method.
The method of claim 3,
The purified seawater is obtained by filtration of seawater, the production method of the genus Spirulina algae.
The method of claim 3,
The purified seawater is the seawater from which the solids contained in the seawater has been removed, Spirulina genus algae production method.
Spirulina genus alga produced by the method of any one of claims 1 to 5.
Claim 6 spirulina genus algae comprising as an active ingredient, liver disease treatment or prevention pharmaceutical composition.
The method of claim 7, wherein
The spirulina genus algae is in the form of sludge, liver disease treatment or prevention pharmaceutical composition.
The method of claim 8,
The sludge is prepared by a concentration method using centrifugation, liver disease treatment or prevention pharmaceutical composition.
Claim 6 spirulina genus algae comprising as an active ingredient, liver food improvement food composition.
Claim 6 spirulina genus algae comprising as an active ingredient, type 1 diabetes treatment or prevention pharmaceutical composition.
Spirulina genus alga of claim 6 as an active ingredient, type 1 diabetes improvement food composition.
Feed composition comprising a spirulina genus alga of claim 6 as an active ingredient.
Minimal medium for Spirulina genus algae comprising NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and NaNO ₃ .

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