KR20210094503A - Method for Manufacturing Spirulina sp. Algae using minimal medium - Google Patents

Abstract

The present invention relates to a method for producing Spirulina sp. Algae using a minimal medium including NaHCO_3, K_2HPO_4, MgSO_4·7H_2O and NaNO_3, Spirulina sp. Algae produced thereby, a pharmaceutical composition for treating or preventing liver diseases including Spirulina sp. Algae, a food composition for improving liver functions, a pharmaceutical composition for treating or preventing type 1 diabetes, and a food composition and feed composition for alleviating type 1 diabetes. By using the method for producing Spirulina sp. Algae provided in the present invention, it is possible to minimize the components contained in the medium, thereby economically producing Spirulina sp. Algae. The Spirulina sp. Algae produced by the method exhibits various pharmacological activities such as alleviation of diabetes symptoms, prevention of liver functional damage, treatment of liver damage, and thus can be widely used in the economical production of Spirulina sp. Algae.

Description

Method for producing algae of the genus Spirulina using a minimal medium {Method for Manufacturing Spirulina sp. Algae using minimal medium}

The present invention relates to a method for producing algae of the genus Spirulina using a minimal medium, and more particularly, the present invention relates to a method for producing algae of the genus Spirulina using a minimal medium containing NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ ·7H ₂ O and NaNO ₃ of the production method, NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ ·7H ₂ O and NaNO ₃ Minimal medium for production of algae of the genus Spirulina, the alga of the genus Spirulina produced by the method, liver disease including the alga of the genus Spirulina It relates to a pharmaceutical composition for treatment or prevention, 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.

Currently, crops cultivated worldwide have been used as a source of stock for mankind, but the yield per farmland area is not large and the utilization rate of solar energy is extremely low. In contrast, microscopic photosynthetic algae that can grow in water with solar energy, carbon dioxide, and a small amount of inorganic salts can grow even in areas where crop growth is impossible, and can obtain 20 times more protein per unit cultivated area compared to crops. In addition, it is possible to produce various kinds of useful substances and natural rare substances that cannot be produced from microorganisms or animal and plant cells. Moreover, algae with a large cell size are easily precipitated so that extraction and separation through various methods are easy, and by using solar energy as the main energy source, it is possible to efficiently use solar energy irradiated on the earth, and carbon dioxide as a carbon source It has a photosynthetic process that uses and releases oxygen as a by-product, so it has the function of lowering air pollution.

Accordingly, various types of microscopic photosynthetic algae have been studied, and studies on microscopic photosynthetic algae such as Chlorella genus, Dunaliella genus, and Spirulina genus are being actively conducted. In particular, micro photosynthetic algae of the genus Spirulina (hereinafter referred to as 'Spirulina algae') have larger cells than other micro photosynthetic algae, and are algae that can easily grow even in an alkaline-contaminated environment. applied to the product. For example, Korean Patent Registration No. 48714 discloses an adsorbent for removing harmful substances or odors containing spirulina algae powder, and Korean Patent Registration No. 353888 discloses a chewing gum composition containing spirulina algae powder with dioxin excretion action. and Korean Patent Registration No. 454224 discloses a health supplement composition comprising spirulina algae powder. Accordingly, research to improve the productivity of spirulina algae is being actively conducted worldwide.

Recently, studies related to the effective production of spirulina algae have been started in Korea as well. Due to the climatic characteristics of the four seasons and the temperature change and the difference in sunlight according to the seasons, studies related to indoor culture of spirulina algae rather than outdoor culture have been conducted. is in progress However, under the conditions in which spirulina algae can grow most actively, because various plant algae or microorganisms other than spirulina algae grow together, the yield of spirulina algae is relatively reduced, and thereby an environment suitable for the growth of spirulina algae is provided indoors. Compared to the cost required to create the algae, the added value obtained from the production of spirulina algae is not large, so research is being conducted in the direction of developing a method that can produce it more economically, but it is not practically utilized. .

For example, Korean Patent Laid-Open No. 2004-0073693 discloses a method of culturing spirulina algae by adding charcoal instead of NaOH to control the pH and at the same time using it as a carbon source, but the charcoal is precipitated in the culture medium to increase the pH control effect. Spirulina algae are adsorbed to the precipitated charcoal, and consequently, there is a problem that productivity is deteriorated, and Korean Patent Publication No. 2006-17033 discloses optimization of growth of spirulina algae and maximum harvest. For this purpose, a medium composition in which the concentration of nitrogen and carbon is adjusted has been disclosed, but the increase in the unit price of the culture medium is higher than the improvement in productivity, and thus it has not been practically utilized. In addition, Republic of Korea Patent No. 0704436 discloses a method for developing a SOT medium specialized for the culture of algae of the genus Spirulina and using it to produce algae of the genus Spirulina, but the SOT medium contains 15 components of the medium. Because the production cost is high, there was a disadvantage that the economical production of spirulina is difficult.

If, compared to the conventional culture method, if a more economical method for producing algae of the genus Spirulina is developed, it is expected that it will bring about a revolutionary change in the commercial use of Spirulina algae.

Under this background, the present inventors made intensive research efforts to develop a more economical method for producing algae of the genus Spirulina, and as a result, developed a minimal medium for production of algae of the genus Spirulina containing four components, and completed the present invention.

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

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

Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of liver disease comprising the algae of the genus Spirulina.

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

Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of type 1 diabetes comprising the algae of the genus Spirulina.

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

Another object of the present invention is to provide a feed composition comprising the algae of the genus Spirulina.

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

The present inventors tried to improve the previously developed SOT medium (Korean Patent No. 10-0704436) for culturing algae of the genus Spirulina while conducting various studies in order to economically develop a method for producing algae of the genus Spirulina. Accordingly, as a result of conducting various studies to select components essential for the culture of algae of the genus Spirulina among the components constituting the SOT medium, NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ ·7H ₂ O and NaNO ₃ Minimal medium containing It was confirmed that algae of the genus Spirulina can be produced when using .

However, when using the minimum medium, if the medium is not circulated at a constant flow rate, the cultured algae of the genus Spirulina are precipitated and then decayed and normal culture does not proceed. Accordingly, as a result of screening the flow rate of a medium capable of culturing algae of the genus Spirulina normally while using the minimal medium, it was confirmed that it is preferable to circulate the medium at a rate of 30 to 45 cm/sec.

On the other hand, it was confirmed that the algae of the genus Spirulina produced by the above method exhibited superior diabetes symptom improvement effect and liver function improvement effect than the conventional algae of the genus Spirulina.

As described above, the technology for producing algae of the genus Spirulina using a medium containing four components has not been previously reported at all, and was first developed by the present inventors.

One embodiment of the present invention for achieving the above object is NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ ·7H ₂ O and NaNO ₃ Inoculated with algae of the genus Spirulina, 3,000 at 32 to 38 ° C. To 9,000Lux light conditions, 12 to 20 (h): 12 to 4 (h) of photoperiod (light: dark) and a flow rate of 30 to 60 cm / s circulating the medium in a tubular incubator comprising the step of culturing , provides a method for producing algae of the genus Spirulina.

As used herein, the term "algae of the genus Spirulina (Spirulina sp. Algae)" refers to a multicellular organism with a thin cell wall as the oldest algae on Earth. It grows mainly in salt lakes in the tropics, and although there are not many worldwide distributions, the optimum water temperature is 32~42℃ and it is known to breed in a strong alkaline environment. The main ingredients contained in Spirulina include protein, carbohydrates, soluble dietary fiber, antioxidant pigments (chlorophyll, carotenoids, phycocyanin), antioxidant enzymes (SOD), gamma-linolenic 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, Spirulina maxima UTEX LB2342 strain was used as the alga of the genus Spirulina, but it is not particularly limited thereto, and all known types of algae of the genus Spirulina can be applied to the method provided in the present invention.

In the method for producing algae of the genus Spirulina provided by the present invention, the culture temperature is not particularly limited thereto, but may be 32 to 38 ° C. as an example, and 34 to 36 ° C. as another example. as 35°C. When the culture temperature is lower than 32 ℃, the growth rate of the algae of the genus Spirulina is greatly reduced, and when it is higher than 38 ℃, the rancidity rate of the algae of the genus Spirulina is increased.

In the method for producing algae of the genus Spirulina provided by the present invention, light conditions are not particularly limited thereto, but may be 3,000 to 9,000 Lux as an example, and may be 5,000 to 7,000 Lux as another example. It can be 6,000 Lux. When the light conditions are 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 conditions without particularly affecting the growth of spirulina is excessive, and thus the production cost is increased.

In the method for producing algae of the genus Spirulina provided by the present invention, the photoperiod is not particularly limited thereto, but as an example, the photoperiod (light: dark) may be 12 to 20 (h): 12 to 4 (h). there is. The photoperiod may be changed depending on the culture purpose of the algae of the genus Spirulina. For the purpose of maintaining the algae of the genus Spirulina, it is preferable to maintain the photoperiod (light: dark) at 12:12, and as another example For the purpose of producing algae of the genus Spirulina, it is preferable to maintain the photoperiod (light: dark) at 20:4.

In the method for producing algae of the genus Spirulina provided by the present invention, as the light conditions included in the photoperiod are increased, the flow rate of the medium may also be increased. can be As an example, when the photoperiod (light:dark) is 12:12, the medium flow rate may be 30 to 45 cm/sec; When the photoperiod (light:dark) is 13:11, the medium flow rate may be 37.5 to 45 cm/sec; When the photoperiod (light:dark) is 14:10, the medium flow rate may be 37.5 to 45 cm/sec; When the photoperiod (light:dark) is 15:9, the medium flow rate may be 37.5 to 52.5 cm/sec; When the photoperiod (light:dark) is 16:8, the medium flow rate may be 45 to 52.5 cm/sec; When the photoperiod (light:dark) is 17:7, the medium flow rate may be 45 to 52.5 cm/sec; When the photoperiod (light:dark) is 18:6, the medium flow rate may be 45 to 60 cm/sec; When the photoperiod (light:dark) is 19:5, the medium flow rate may be 45 to 60 cm/sec; When the photoperiod (light:dark) is 20:4, the medium flow rate may be 52.5 to 60 cm/sec.

When the flow rate of the medium is lower than 30 cm / s, the cultured algae of the genus Spirulina are precipitated and decay, and when it is higher than 60 cm / s, the collision frequency between algae of the genus Spirulina increases, thereby increasing the loss rate of algae of the genus Spirulina. .

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

In the method for producing algae of the genus Spirulina provided by 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 may be included.

In the method for producing algae of the genus Spirulina provided by the present invention, the minimum medium can be artificially prepared by combining the four components constituting it as an example, and as another example, it can be prepared by purifying seawater existing in nature. can For example, as a method of purifying seawater, a method of filtering seawater to remove solids contained in seawater may be used, but is not particularly limited thereto.

Another embodiment of the present invention provides an alga of the genus Spirulina, produced by the above method.

The algae of the genus Spirulina produced by the method for producing algae of the genus Spirulina provided by the present invention are cultured by circulating a minimum medium at a constant flow rate, and have characteristics distinguishing them from algae of the genus Spirulina produced by the conventional method.

As a result of testing the pharmacological activity of the algae of the genus Spirulina produced by the method of the present invention, the present inventors confirmed that it exhibits improved pharmacological activity than the algae of the genus Spirulina produced by the conventional method in terms of improving the symptoms of diabetes and improving liver function. did.

According to one embodiment of the present invention, the inhibitory effect of increasing the level of phosphorylated JNK and the inhibitory effect of increasing the level of phosphorylated p38 in apoptosis-induced insulin tumor cells is superior to that of PC (Phycocyanin), a major component of algae of the genus Spirulina. was confirmed (FIGS. 3a and 3b). In addition, when administered to animals with impaired liver function, it was confirmed that abnormally increased blood cholesterol, AST and ALT levels were effectively reduced ( FIGS. 5a to 7c ).

Therefore, since the algae of the genus Spirulina produced by the method of the present invention exhibit effects such as improvement of diabetes symptoms and improvement of liver function damage, it can be used as an active ingredient in pharmaceutical compositions, food compositions, etc. that can utilize these effects.

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

As described above, since the algae of the genus Spirulina produced by the method of the present invention exhibit an effect of improving liver function, it can be used as an active ingredient in a pharmaceutical composition for treating or preventing liver disease.

In the pharmaceutical composition for the treatment or prevention of liver disease of the present invention, the algae of the genus Spirulina included as an active ingredient may be processed and used in various forms. For example, a powder form prepared by drying a culture of algae of the genus Spirulina, a tablet form prepared by tableting the algae of the genus Spirulina in powder form, and a sludge form prepared by concentrating a culture of algae of the genus Spirulina. can be used

According to an embodiment of the present invention, it was confirmed that there is a difference in the liver function improvement effect according to the processing form of the algae of the genus Spirulina ( FIGS. 5a to 7c ). That is, the algae of the genus Spirulina generally exhibit the effect of improving liver function, but when the algae of the genus Spirulina were processed in the form of sludge, it was found that the algae of the genus Spirulina exhibited superior liver function improvement effect than when processed in the form of powder or tablet.

In addition, the method for processing the culture of algae of the genus Spirulina into the sludge form is not particularly limited thereto, but a filtration method, a centrifugal separation method, a drying method, etc. may be used.

As used herein, the term “liver disease” refers to symptoms in which hepatocytes, liver tissues, etc. do not perform normal functions due to internal, genetic, or extrinsic factors. Specific examples of the liver disease may be cirrhosis, cirrhosis, hepatitis, liver cancer, liver fibrosis, fatty liver, liver tissue hypertrophy, and the like, but is not particularly limited thereto.

As used herein, the term “prevention” refers to any act of inhibiting or delaying liver disease by administering a composition containing the algae of the genus Spirulina.

As used herein, the term "treatment" refers to any action in which the symptoms of liver disease are improved or beneficially changed by administration of the composition containing the algae of the genus Spirulina.

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 of the alga of the genus Spirulina based on the total weight of the composition, but is not limited thereto.

In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition, and the carrier may include a non-naturally occurring carrier. there is. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, 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 composition is prepared according to a conventional method for each tablet, pill, powder, granule, capsule, suspension, internal solution, emulsion, syrup, sterilized aqueous solution, non-aqueous solution, suspension, emulsion, lyophilized formulation, transdermal It can be formulated and used in the form of absorbents, gels, lotions, ointments, creams, patches, cataplasmas, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages or suppositories. Specifically, in the case of formulation, it may be prepared using a diluent or excipient such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, a surfactant, etc. commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, for example, wetting agents, sweetening agents, fragrances, preservatives, etc., in addition to liquids for oral use and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, 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, Witepsol, Macrogol, Tween 61, cacao butter, laurin, glycerogelatin, etc. may be used.

Another embodiment of the present invention provides a method for treating liver disease, comprising administering the pharmaceutical composition to a subject suspected of having liver disease except for humans.

As used herein, the term "administration" refers to the act of introducing a composition comprising the alga of the genus Spirulina to an individual by an appropriate method.

As used herein, the term "individual" refers to all animals, such as rats, mice, and livestock, including humans, that have or can develop 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. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. For example, the algae of the genus Spirulina may be administered at a dose of 0.01 to 500 mg/kg per day, specifically 10 to 100 mg/kg, and the administration may be administered once a day or divided several times.

The pharmaceutical composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with 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, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage may vary depending on the patient's condition and weight, and the degree of disease. , depending on the drug form, administration route and time, but 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 produced algae of the genus Spirulina as an active ingredient.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated by administration of a composition comprising an alga of the genus Spirulina, for example, the degree of symptoms.

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods, and health foods, and includes all foods in a conventional sense.

Since the food composition of the present invention is derived from algae of the genus Spirulina that can be consumed daily, a high liver disease improvement effect can be expected, and thus it can be very usefully used for health promotion purposes.

The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, 'function (sex)' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food can be prepared without limitation as long as it is a formulation recognized as a food.

The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long period of time by using a natural product as a raw material, and it is excellent in portability. The food of the invention can be ingested as an adjuvant for enhancing the effect of improving liver disease.

The health food means a food having an active health maintenance or promotion effect compared to general food, and the health supplement food means a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement are preferred.

Specifically, the health functional food is a food prepared by adding the alga of the genus Spirulina of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, suspension, etc. It means to bring a specific effect, but unlike general drugs, it has the advantage of not having side effects that can occur when taking the drug for a long time using food as a raw material.

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

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

In addition, the food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butyl hydro Loxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, etc.) , sodium, etc.), flavorings (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, improvers, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

As an example of the food composition of the present invention, it may be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatin, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. 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, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected in 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 alga of the genus Spirulina as an active ingredient, and a method of treating type 1 diabetes using the composition.

As described above, since the alga of the genus Spirulina produced by the method of the present invention exhibits an effect of improving diabetes symptoms, it can be used as an active ingredient in a pharmaceutical composition for treating or preventing type 1 diabetes, and using the pharmaceutical composition, the first It can treat type diabetes.

A detailed description of the composition of the pharmaceutical composition and the treatment method for type 1 diabetes is the same as described in the above-mentioned liver disease related item.

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

A detailed description of the food composition for improving type 1 diabetes containing algae of the genus Spirulina as an active ingredient is the same as described in the above-mentioned food composition for improving liver function.

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

As used herein, the term “feed” means any natural or artificial diet, meal, etc., or a component of the meal, intended for or suitable for being eaten, consumed, and digested by an animal. Specifically, the feed containing the algae of the genus Spirulina produced by the method of the present invention can be prepared in various types of feed known in the art, and specifically, a concentrated feed, roughage and/or special feed may be included.

In the enriched feed, seed fruits containing grains such as wheat, oats and corn, bran containing rice bran, bran, barley bran, etc. as by-products obtained by refining grains, soybeans, fluid, sesame, linseed, coco palm, etc. are extracted. It is a concentrated product obtained by concentrating the residues such as residual starch, which is the main component of starch residues, which are the by-products obtained by subtracting starch from sweet potatoes and potatoes, fish meal, fish residues, and fresh liquids obtained from fish. Animal feed such as dried whey, yeast, fish soluble, meat meal, blood meal, feather flour, skim milk powder, milk from cheese, and whey, which is the remainder of the production of casein from skim milk, dried whey, yeast, Chlorella, seaweed, but not limited thereto. For roughage, raw grass feed such as wild grasses, grasses and green cuttings, turnips for feed, beets for feed, root vegetables such as luther beargers, a type of turnip, raw herbs, green crops, grains, etc. are placed in a silo. Examples include, but are not limited to, silage, which is stored feed fermented with lactic acid after filling, wild grasses, hay obtained by cutting and drying grass, straw for breeding crops, and leaves of legumes. Special feed includes mineral feed such as oyster shells and rock salt, urea feed such as urea or its derivative diureide isobutane, supplementing ingredients that are easily lacking when only natural feed raw materials are mixed, or adding to the compounded feed to increase the storability of the feed. There are feed additives and dietary supplements, which are substances added in trace amounts, but are not limited thereto.

The feed containing the algae of the genus Spirulina produced by the method of the present invention can be prepared by adding the alga of the genus Spirulina in an appropriate effective concentration range according to various feed preparation methods known in the art.

The feed provided in 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 feed is applicable. For example, non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, etc., birds and fish, etc., but are not limited thereto, and can be applied to any individual.

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

Using the method for producing algae of the genus Spirulina provided in the present invention, it is possible to minimize the components contained in the medium, so that algae of the genus Spirulina can be economically produced, and the algae of the genus Spirulina produced by the above method can improve diabetes symptoms, liver Since it exhibits various pharmacological activities such as prevention of functional damage and treatment of liver damage, it will be widely used in the economical production of algae of the genus Spirulina showing improved pharmacologically activity.

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

Hereinafter, the present invention will be described in more detail through 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 a medium for culturing algae of the genus Spirulina

Example 1-1: Comparison according to the medium for culturing algae of the genus Spirulina

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

Comparison of components of medium for culturing algae in the genus Spirulina (content per 1 liter) 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 13.61g
4.03g
0.5g
2.5g
1 g
1 g
0.2g
0.04g
-
-
10.00 ml
6.00 ml
1.00 ml
1.00 ml
- 16.8g
-
0.5g
2.5g
1 g
1 g
0.2g
0.04g
0.01g
0.08g
-
-
-
-
- 16.8g
-
0.5g
2.5g
1 g
1 g
0.2g
0.04g
0.01g
0.08g
-
-
-
-
1.00 ml

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

As shown in Figure 1, the growth rate of algae of the genus Spirulina cultured in SOT medium showed a relatively higher level than that cultured in UTEX medium, and it was confirmed that it exhibited the same level as that cultured in Zarrouk medium.

Example 1-2: Development of a minimal medium for culturing algae of the genus Spirulina

Since the SOT medium used in Example 1-1 is composed of various components, it was attempted to select an essential medium composed of essential components for the cultivation of algae of the genus Spirulina among these components.

To this end, algae of the genus Spirulina were cultured under the same conditions as described above, except that each medium composed of a combination of various components constituting the SOT medium was used, and whether they were cultured was confirmed.

As a result, it was confirmed _{that when a medium composed of NaHCO 3} , K ₂ HPO ₄ , MgSO ₄ ·7H ₂ O and NaNO ₃ was used, algae of the genus Spirulina could be normally cultured using a minimum of medium components. In addition, when using a medium containing 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 to the medium composed of the above components, It was confirmed that the growth rate of algae in the genus Spirulina was increased.

Example 1-3: Effect of medium flow rate on the culture of algae of the genus Spirulina

When algae of the genus Spirulina were produced using the minimal medium containing the components determined in Example 1-2, it was confirmed that the production of algae of the genus Spirulina was greatly affected by the flow rate of the medium. In the case of culturing while circulating the medium at a flow rate lower than 30 cm/sec in a tubular incubator through a prior experiment, a part of the algae of the genus Spirulina in which the culture is being performed is precipitated at the bottom of the tubular incubator, decays over time and is cultured It was confirmed that this did not proceed. On the other hand, when culturing using a known medium for culturing Spirulina (UTEX medium, Zarrouk medium or SOT medium), when culturing at a rate of 10 cm/sec or more, even if a portion is precipitated at the bottom of the incubator, the precipitated Spirulina genus It was confirmed that the algae did not decay and proliferated.

Therefore, using the minimal medium containing the components determined in Example 1-2, the culture was performed while changing the flow rate of the medium, and it was attempted to determine how the change in the medium flow rate affects the growth of the Spirulina sp.

Roughly, the algae of the genus Spirulina are inoculated into the minimal medium, and cultured for 57 days under light conditions of 3,000 Lux at 32 ° C. for 12 hours in light conditions / 12 hours in dark conditions, for each section, the flow rate of the medium is 30 to 60 cm / The flow rate was changed to sec. At this time, the daily growth rate of algae of the genus Spirulina for each flow rate change section of the medium was measured and compared (FIG. 2).

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

As shown in FIG. 2 , in the range of 30 to 45 cm/sec, the daily growth rate exhibited an absorbance (565 nm) of 0.02 or more, but in the range of 52.5 and 60 cm/sec, the daily growth rate exhibited a lower absorbance level than 0.02 (565 nm). ) was confirmed. As such, when the medium was circulated at a speed higher than 45 cm/sec, the daily growth rate of algae of the genus Spirulina was reduced. As a result of analyzing the cause, when the medium was circulated at a flow rate that was too fast, the collision frequency between algae of the genus Spirulina was decreased. It was confirmed that this is because the loss rate of algae of the genus Spirulina increases.

Example 1-4: Effect of photoperiod on the culture of algae of the genus Spirulina

From the results of Example 1-3, when culturing while circulating the medium at a flow rate that is too low (30 cm/sec or less) or too high (45 cm/sec or more) in a tubular culture medium, the alga of the genus Spirulina It was found that the culture was not performed normally, and we wanted to check whether the photoperiod could affect the culture result according to the medium flow rate.

Roughly, the algae of the genus Spirulina are inoculated into a minimal medium containing the components determined in Example 1-2, and photoperiod of 3,000 Lux at 32 ° C. and photoperiod of various conditions (light condition (h): dark condition (h) = 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, 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 algae of the genus Spirulina for each flow rate change section of the medium was measured and compared (Table 2).

Comparison of the daily growth rate of algae of the genus Spirulina according to the change of photoperiod and flow rate photoperiod
(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 the photoperiod was increased, the medium flow rate showing the maximum value of the daily growth rate of algae of the genus Sriraulina showed a tendency to increase. It was analyzed that this was because the growth rate of algae of the genus Spirulina according to the time increase in light conditions was relatively higher than the loss rate of algae of the genus Spirulina according to the increase of the medium flow rate.

However, when the light condition time of the photoperiod exceeded 20 hours, it was confirmed that the daily growth rate of algae of the genus Sriraulina was rapidly reduced even when the medium flow rate was increased, which indicates that the growth rate of algae of the genus Spirulina according to the increase of time under light conditions was confirmed. It was analyzed that it was because the algae of the genus Srirulina, which were overgrowth in excess, were deposited and decayed on the bottom of the tubular incubator.

Therefore, it was confirmed that there is a condition in which the productivity of algae of the genus Spirulina is no longer increased even when the light conditions and medium flow rate of the photoperiod are increased. The conditions showing the maximum productivity of algae of the genus Spirulina are light conditions: dark conditions 20 : 4, and it was found that the medium flow rate was 60 cm/sec.

Example 1-5: Comparison of culture maintenance and production of algae of the genus Spirulina

The culture conditions including the minimum medium flow rate for producing the algae of the genus Spirulina identified in Example 1-3 and the culture conditions including the maximum medium flow rate showing the maximum productivity of the algae of the genus Spirulina identified in Examples 1-4 The algae of the genus Spirulina were cultured, and the culture results were compared (Table 3). The culture result is the pH of the culture medium, the OD of the culture, the content of chlorophyll a in the culture, the dry weight of 1L of the culture, the floating activity of the algae of the genus Spirulina, and the number of coils of the algae of the genus Spirulina (No. of Coils). The floating activity is a result of converting the characteristics of algae in the genus Spirulina to a ratio, which is settled in dark conditions and floats in light conditions. As the floating activity increases, it can be determined that the level of activation of biological functions of algae of the genus Spirulina is higher. can In addition, the number of coils wound (No. of Coils) means the number of coil shapes, which is a morphological characteristic of algae in the genus Spirulina, when the number of coils increases, the morphological stability improves, and when the number of coils decreases, the stability of Spirulina is known to decrease.

Comparison of culture results according to medium flow rate Medium flow rate (cm/sec) 30 60 Photoperiod (name: dark)
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 above, under the condition that the medium flow rate was increased and a photoperiod was given to match this, the OD value, chlorophyll a content, dry weight and flotation activity were significantly increased compared to the case where the medium flow rate was decreased, so spirulina 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, so it was confirmed that the morphological stability of the cultured algae of the genus Spirulina was equivalent to that when the medium flow rate was decreased. could

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

Example 2: Verification of pharmacological activity effects of algae in the genus Spirulina

Example 2-1: Preparation of algal samples of the genus Spirulina

After culturing algae of the genus Spirulina using the flow rate range determined in Example 1-2 and the medium determined in Examples 1-3, the following four types of samples were prepared: Sample 1 was cultured Spirulina. It is a powdered sample after complete drying of the genus algae; 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 a culture of algae of the genus Spirulina and repeating the process of suspending the precipitated cells in distilled water through centrifugation; Sample 4 is an extract sample obtained by ethanol extraction of algae of the genus Spirulina. The method for preparing the extract sample is roughly described as follows: The cultured algae of the genus Spirulina are extracted three times with 70% ethanol at 40° C. for 4 hours to obtain an extract, which is lyophilized to powder, and then The powder was again added to 70% ethanol at 40° C. and dissolved 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 freeze-dried.

Example 2-2: Diabetes treatment effect

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

The effect of algae of the genus Spirulina on apoptosis induced by treatment with cytokines (IL-1β and IFN-γ) for 30 minutes in RINm5F cells, which are rat insulin tumor cells, was tested. At this time, the algae of the genus Spirulina used used the spirulina extract (Sample 4) prepared in Example 2-1, and as a comparison group, PC (Phycocyanin), a type of phycobiliprotein, as a known component of algae of the genus Spirulina was used. was used. The PC is known to exhibit antioxidant effects, anti-inflammatory effects, neuroprotective effects, hepatocellular protective effects, and the like.

Roughly, 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) were alone Or it was cultured by complex treatment. Then, RINm5F cells were recovered, extracts were obtained from each cell, and the level of phosphorylated JNK, phosphorylated p38, phosphorylated ERK, and caspase 3 activity were compared by Western blot analysis ( 3a to 3d). In addition, TUNEL staining was performed on each of the recovered RINm5F cells to compare the level of DNA fragmentation (FIG. 3e).

3A is a graph showing the results of comparing the effects of Spirulina extract or PC on the level of phosphorylated JNK expressed in RINm5F cells treated with cytokines (IL-1β+IFN-γ) to induce apoptosis.

As shown in Figure 3a, the level of phosphorylated JNK was increased by the treatment of cytokines, but it was confirmed that the increase in the level of phosphorylated JNK was inhibited when the Spirulina extract or PC was treated with cytokines. In addition, it was confirmed that the inhibitory effect of increasing the level of phosphorylated JNK was relatively high in the spirulina extract compared to that of PC.

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

As shown in FIG. 3b , the level of phosphorylated p38 was increased by the treatment of cytokines, but it was confirmed that the increase in the level of phosphorylated p38 was inhibited by treatment with the spirulina extract or PC together with cytokines. In addition, it was confirmed that the inhibitory effect on the increase of phosphorylated p38 level was relatively high in the spirulina extract compared to PC.

3C is a graph showing the results of comparing the effects of Spirulina extract or PC on the level of phosphorylated ERK expressed in RINm5F cells treated with cytokines (IL-1β+IFN-γ) to induce apoptosis.

As shown in Figure 3c, it was confirmed that the spirulina extract, PC, IL-1β and IFN-γ had no significant effect on 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 RINm5F cells treated with cytokines (IL-1β+IFN-γ) to induce apoptosis.

As shown in FIG. 3d , the activity of caspase 3 was increased by the treatment of cytokines, but it was confirmed that the increase in activity of caspase 3 was inhibited when the spirulina extract or PC was treated with cytokines. In addition, it was confirmed that the activity increase inhibitory effect of caspase 3 was similar to that 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 DNA fragmentation shown in RINm5F cells treated with cytokines (IL-1β+IFN-γ) to induce apoptosis.

As shown in FIG. 3e, TUNEL-positive cells showing DNA fragmentation were increased by the treatment of cytokines, but it was confirmed that the increase of TUNEL-positive cells was inhibited when Spirulina extract or PC was treated with cytokines.

Example 2-2-2: Effect on diabetic animal model

Example 2-2-2-1: Preparation of samples

Since it is known that when streptozotocin (STZ) is orally administered to rats, it is known that type 1 diabetes symptoms can be induced in rats, algae extract of the genus Spirulina for a diabetic animal model in which type 1 diabetes is induced using the STZ. of diabetes was tested.

Roughly, the following six groups were established using rats and reared for 4 weeks (FIG. 4a): a control group that was orally administered with only water during the breeding period; Experimental group 1 (Spi), in which 200 mg of spirulina extract (Sample 4) was orally administered per body weight (Kg) during breeding period; Experimental group 2 (STZ(2 wks)), which was orally administered with streptozotocin at the time of breeding for 2 weeks, and reared for 2 weeks again; During the breeding period, 200 mg of spirulina extract (Sample 4) per body weight (Kg) was orally administered, and streptozotocin was orally administered at the time of breeding for 2 weeks, followed by breeding for another 2 weeks (Spi+STZ); Experimental group 4 (STZ (4 wks)) was administered with streptozotocin and bred for 4 weeks; and experimental group 5 (STZ+Spi) in which streptozotocin and 200 mg of spirulina extract (Sample 4) per body weight (Kg) were orally administered and reared for 4 weeks.

The weight of the rats of each experimental group after breeding was measured and compared with the body weight before the experiment (Table 4).

Weight change in diabetic animal model experimental group Initial weight (g) final weight (g) control
Experimental group 1
Experimental group 2
Experimental 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 above, when streptozotocin (STZ) was administered, body weight was increased to a relatively small level compared to the control group, but when the spirulina extract was treated, compared to the case where streptozotocin (STZ) was administered. It was confirmed that the body weight increased to a relatively high level.

In addition, each rat was sacrificed after anesthesia, and the pancreas was removed from each rat and blood was obtained 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: Pancreatic analysis

Hematoxylin & eosin staining was performed to confirm the cell shape of islets contained in each pancreas extracted in Example 2-2-2-1, and insulin and nitrotyrosine expressed in pancreatic tissues were In order to compare the levels, immunostaining using an anti-insulin antibody and an anti-nitrotyrosine antibody was performed and then observed under a fluorescence microscope (FIG. 4b). In addition, the size of the islets of Langerhans was measured using the IMT i-solution software (Fig. 4c).

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

As shown in FIG. 4b, when streptozotocin was treated, degenerative and necrotic changes of islet cells were observed, but when the spirulina extract was treated, the level of degenerative and necrotic change was reduced. did. In addition, when streptozotocin was treated, the level of insulin in the pancreas was rapidly reduced, but it was confirmed that the decrease in insulin was inhibited when the spirulina extract was treated. Finally, when streptozotocin was treated, the level of nitrotyrosine in the pancreas tissue was increased, but it was confirmed that the increase in nitrotyrosine was inhibited when the spirulina extract was treated.

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

As shown in Fig. 4c, when streptozotocin was treated, the size of the islets in the pancreas was reduced, but when the spirulina extract was treated, it was confirmed that the size reduction of the islets was suppressed.

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 ).

4D is a graph showing the results of comparing changes 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 streptozotocin was treated, the insulin level in the blood was decreased and the level of NO was increased, but it was confirmed that the decrease in insulin level and the increase in No level were suppressed when the spirulina extract was treated.

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

Water preparation of various components in blood (unit: ㎎/㎗) experimental group BG CH TG BUN CR control
Experimental group 1
Experimental group 2
Experimental 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 above, streptozotocin-treated rats showed general diabetes mellitus (increased blood sugar level, decreased insulin level, etc.), but it was confirmed that this diabetes was improved when the spirulina extract was treated.

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 exhibits an effect of alleviating the symptoms of diabetes.

Example 2-3: liver function improvement effect

Various types of spirulina (samples 1 to 3) obtained in Example 2-1 were administered to acute liver disease model animals prepared by administering carbon tetrachloride to mice, and blood cholesterol levels, ALT levels and AST levels were measured. , The liver function improvement effect according to the formulation of algae of the genus Spirulina was compared.

Example 2-3-1: Preparation of the experimental group

Roughly, a primary control group (Nor_Sham) bred for 37 days without any administration to normal mice, an experimental group 11 (Nor_Powder) bred for 37 days with sample 1 (powder) administered to normal mice, and sample 2 (Nor_Powder) to normal mice ( Tablet) was administered to experimental group 12 (Nor_Tablet) and bred for 37 days, sample 3 (sludge) was administered to normal mice and 13 (Nor_Sludge) was bred for 37 days, and sample 3 (sludge) was administered to normal mice 37 Experimental group 14 (CCl4_Pre_Sludge), which was bred for one day, then administered carbon tetrachloride and bred for 3 days again, the secondary control group (CCl4_Sham), which was bred for 3 days without administering any stone to the mice administered with carbon tetrachloride (CCl4_Sham), was administered to mice Experimental group 21 (CCl4_Powder) bred for 3 days after administering sample 1 (powder), experimental group 22 (CCl4_Tablet) bred for 3 days after administration of sample 2 (tablet) to mice administered with carbon tetrachloride, and mice administered with carbon tetrachloride Sample 3 (sludge) was administered and experimental group 23 (CCl4_Sludge) bred for 3 days was prepared, respectively. At this time, 5 male and 5 female mice were assigned to each group.

Example 2-3-2: Comparison of cholesterol levels

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

However, as a result of comparing the cholesterol levels measured in the blood of Control 2, Experimental Group 21, Experimental Group 22, and Experimental Group 23 for acute liver disease model animals (Fig. 5b), the level of blood cholesterol in Control 2 was higher than in Control 1. It was confirmed that the level of blood cholesterol was relatively decreased compared to the control group 2 when the spirulina sample was treated (experimental groups 21 to 23).

In addition, when sludge-form spirulina samples were treated (experimental groups 13, 14 and 23), when comparing (Fig. 5c), blood cholesterol in all acute liver disease model animals (experimental groups 14 and 23) compared to normal mice (experimental group 13). The level increased, and it was confirmed that the blood cholesterol level was further increased when the spirulina sample was administered later (experimental group 23) compared to the case where the spirulina sample was administered in advance (experimental group 14). It was confirmed that the treatment (experimental group 23) exhibited 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 normal mice (FIG. 6a), there was no significant difference for each group.

However, as a result of comparing the AST levels measured in the blood of Control 2, Experimental Group 21, Experimental Group 22, and Experimental Group 23 for acute liver disease model animals (FIG. 6b), the level of AST in the blood in Control 2 was higher than in Control 1. It increased rapidly, and when spirulina samples were treated (experimental groups 21 to 23), the blood AST levels of experimental groups 21 and 23 were relatively decreased compared to control 2, and the blood AST levels of experimental group 23 were relatively lower than those of experimental group 21. It was confirmed that

In addition, when sludge-form spirulina samples were treated (experimental groups 13, 14 and 23), when comparing (Fig. 6c), blood AST in all acute liver disease model animals (experimental groups 14 and 23) compared to normal mice (experimental group 13). The level increased, and it was confirmed that the AST level in the blood was further increased when the spirulina sample was administered later (experimental group 23), compared to the case where the spirulina sample was administered in advance (experimental group 14).

Example 2-3-4: ALT level comparison

First, as a result of comparing the ALT levels measured in the blood of the control group 1, the experimental group 11, the experimental group 12 and the experimental group 13 for normal mice (Fig. 7a), the case where the powdered spirulina sample was treated (experimental group 11) was excluded. And there was no significant difference between each group.

However, as a result of comparing the ALT levels measured in the blood of Control 2, Experimental Group 21, Experimental Group 22 and Experimental Group 23 for acute liver disease model animals (FIG. 7b), the blood ALT level in Control 2 was sharply higher than in Control 1. increased, and when the spirulina sample was treated (experimental groups 21 to 23), it was confirmed that the blood ALT level was relatively decreased compared to the control 2, and among them, when the sludge-type spirulina sample was treated (experimental group 23), the lowest blood level was It was confirmed that it represents the ALT level.

In addition, when sludge-type spirulina samples were treated (experimental groups 13, 14 and 23), when comparing (Fig. 7c), blood ALT in all acute liver disease model animals (experimental groups 14 and 23) compared to normal mice (experimental group 13). The level increased, and it was confirmed that the blood ALT level exhibited a relatively low value when the spirulina sample was administered later (experimental group 23) compared to the case where the spirulina sample was previously administered (experimental group 14).

Therefore, from the results of Examples 2-3-2 to 2-3-4, when a formulation obtained by processing spirulina cultured in the culture solution provided in the present invention in the form of sludge is used, relatively excellent prevention of liver damage and damaged liver It was found to have a functional therapeutic effect.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (2)

NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ ·7H ₂ O and NaNO ₃ A minimal medium for production of algae in the genus Spirulina.
_{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 one or more components selected from the group consisting of a combination thereof, NaHCO ₃ , K ₂ HPO ₄ , MgSO ₄ ·7H ₂ O and NaNO ₃ A minimal medium for the production of algae of the genus Spirulina.

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