Composition comprising neoagarooligosaccharide as active ingredient, for prevention or treatment of sepsis or septic shock

Abstract

The present invention relates to a composition for prevention or treatment of sepsis or septic shock, in which the composition includes neoagarooligosaccharide as an active ingredient, and the neoagarooligosaccharide according to the present invention has an excellent effect in terms of immune enhancement by effectively suppressing inflammation, and also exhibits a good effect in preventing sepsis, and therefore can be effectively used in pharmaceuticals and functional foods for prevention or treatment of sepsis or septic shock and immune enhancement.

Claims

1. A method for treating sepsis or septic shock in a subject, the method comprising administering to the subject a neoagarooligosaccharide, wherein the neoagarooligosaccharide is prepared from agar or agarose by an agarase gene (DagA) enzyme reaction wherein the DagA enzyme is obtained from Streptomyces coelicolor.

2. The method of claim 1, wherein the DagA enzyme is obtained from Streptomyces coelicolor and has the amino acid residues 31-309 of the amino acid sequence of SEQ ID NO: 2.

3. The method of claim 2, wherein the DagA enzyme is encoded by a nucleotide sequence of SEQ ID NO: 1.

4. The method of claim 1, wherein the neoagarooligosaccharide is selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose, and any combination thereof.

5. The method of claim 1, wherein the neoagarooligosaccharide is administered at about 0.001 mg/kg to about 1000 mg/kg per day.

6. The method of claim 1, wherein the neoagarooligosaccharide is administered orally, rectally, intravenously, intramuscularly, subcutaneously, or intracerebrally.

7. The method of claim 1, wherein the neoagarooligosaccharide is administered in the form of an injection.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a map of pUWL201pw vector.

(2) FIG. 2 shows a map of a recombination vector according to an embodiment of the present invention.

(3) FIG. 3 shows graphs illustrating results of an HPLC-ELSD analysis of an enzyme reaction product of the present invention.

(4) FIG. 4 is a graph showing the survival rate over time of animal models in which neoagarooligosaccharide of the present invention is pre-treated and sepsis is induced.

(5) FIG. 5 is a graph showing the secretion amount of inflammatory cytokines in the serum of animal models in which neoagarooligosaccharide of the present invention is pre-treated and sepsis is induced.

(6) FIG. 6 is a graph showing AST, ALT and BUN concentrations in the serum of animal models in which neoagarooligosaccharide of the present invention is pre-treated and sepsis is induced.

(7) FIG. 7 is a graph showing the survival rate of animal models in which neoagarooligosaccharide of the present invention is pre-treated and LPS is administered.

(8) FIG. 8 is a graph showing the secretion amount of inflammatory cytokine in serum of animal models in which neoagarooligosaccharide of the present invention is pre-treated and LPS is administered.

(9) FIG. 9 is a graph showing AST, ALT and BUN concentrations in the serum of animal models in which neoagarooligosaccharide of the present invention is pre-treated and LPS is administered.

(10) FIG. 10 is a graph showing the degree of infiltration of inflammatory inducing cells in the lung tissue of animal models in which neoagarooligosaccharide of the present invention is pre-treated and LPS is administered.

(11) FIG. 11 is a graph showing the degree of apoptosis in the splenic tissue of animal models in which neoagarooligosaccharide of the present invention is pre-treated and LPS is administered.

BEST MODES OF THE INVENTION

(12) Hereinafter, the present invention will be described in detail.

(13) The present invention provides a composition for prevention or treatment of sepsis or septic shock, in which the composition includes neoagarooligosaccharide, which is prepared from agar or agarose by a DagA enzyme reaction, as an active ingredient.

(14) The composition includes a pharmaceutical composition and a food composition.

(15) DagA of the present invention is a -agarase protein, which means the amino acid sequence of SEQ ID NO: 2. DagA includes a protein produced from Streptomyces coelicolor which is actinomycetes, or a heterologous strain. Further, DagA includes a recombinant protein according to conventional gene recombinant methods within the range that the function is not changed to be completely different or the beta-agarase activity is not lost, i.e., a method of including a labeled amino acid for advantageous purification, modifying the amino acid sequence for heterologous expression, etc.

(16) More specifically, DagA of the present invention is produced with a molecular weight of about 35 kDa, having 309 amino acids of SEQ ID NO: 2 when it is translated from a gene of beta-agarase prepared by Streptomyces coelicolor. DaeA is secreted in the form of the completed extracellular protein (about 32 kDa) in which 30 N-terminal amino acid signal peptides are cleaved to have the amino acid sequence 31 to 309 of SEQ ID NO: 2.

(17) The dagA gene of Streptomyces coelicolor may encode the DagA, and the dagA gene may be represented by the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the nucleotide sequence of the gene present in the genome of Streptomyces coelicolor A3 (2) and is named SC03471 on database of National Center for Biotechnology Information (NCBI) in U.S. As confirmed through in vitro experiments, the transcription of the dagA gene is regulated by 4 or 5 other promoters that are recognized by at least 3 other holoenzymes of the RNA polymerase. Through an analysis of transcription step of the dagA gene, the transcription was shown to be initiated at the 32nd, 77th, 125th, and 220th bases of the encoding sequence.

(18) DagA of the present invention may be prepared through the culture of Streptomyces coelicolor, which is a producing strain of DagA, but the expression system of Streptomyces lividans, a heterologous strain is preferably used to increase production efficiency. A method is used in which the dagA gene is inserted into a vector for actinomyces to prepare a recombinant vector, then Streptomyces lividans is transformed with the recombinant vector, and then the transformant is cultured. In this case, the recombinant vector is preferably configured in which the transcription of the dagA gene can be regulated by a promoter derived from actinomycetes.

(19) When preparing the recombinant vector, the promoter derived from actinomycetes can be selected and used since the promoter derived from actinomycetes includes various promoters such as a sgtR promoter (sgtRp), an ermE promoter (ermEp), and a tipA promoter (tipAp). Several kinds of vectors configured in which the transcription may be regulated by these promoters have been developed so that SC03471 is cloned into this vector to produce a recombinant vector having a structure in which the transcription is regulated by the promoter derived from actinomycetes.

(20) A host strain may be transformed with a recombinant vector to prepare the transformant. Since there are various methods for transformation according to the host strain, an appropriate method can be selected and used. For example, when Streptomyces lividans is used as a host strain, a transformation method can use PEG (polyethylene glycol) as a medium.

(21) A DagA producing strain such as a transformant may be cultured in a liquid medium to prepare DagA. High purity DagA can be prepared using a conventional protein purification method such as ultrafiltration for the obtained culture medium. At this time, agar or agarose is included in the liquid medium so as to prepare DagA more efficiently.

(22) The polysaccharide such as agar and agarose may be converted into relatively small oligosaccharides through the enzyme reaction of DagA. The enzyme reaction is preferably performed at a temperature of 35 C. to 45 C. and a pH of 6 to 8, but is not limited thereto.

(23) The neoagarooligosaccharide is prepared as a product obtained by the enzyme reaction, and the neoagarooligosaccharide may be each of neoagarobiose, neoagarotetraose, and neoagarohexaose, and they may be in a mixed state thereof, but is not limited thereto.

(24) The neoagarooligosaccharide according to the present invention shows preventive or therapeutic effects of sepsis by reducing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, and significantly lowering the mortality due to sepsis or septic shock.

(25) In the present invention, sepsis refers to a state in which infection causes a serious inflammatory reaction with a microorganism in a whole body. Systemic inflammatory response syndrome (SIRS) is called when there are two or more kinds of symptoms such as fever with body temperature of 38 C. or more, hypothermia down to 36 C., respiratory rate increased more than 24 times per minute (tachypnea), at least 90 heartbeats per minute (tachycardia), and an increase or marked decrease in leukocyte count on the blood test. Sepsis is called when the systemic inflammatory response syndrome is caused by microbial infection. Pathogens consistently or intermittently enter the bloodstream from the body's infectious focuses, settle in various organs, create lesions, and show severe systemic symptoms. The causative bacteria include staphylococcus, streptococcus, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, pneumococcus, fungus, and anaerobic bacteria.

(26) Further, the present invention provides a composition for reinforcement of immunization, in which the composition includes neoagarooligosaccharide, which is prepared from agar or agarose by a DagA enzyme reaction, as an active ingredient.

(27) The composition includes a pharmaceutical composition and a health supplement food.

(28) The neoagarooligosaccharide according to the present invention exhibits an effect of reinforcement of immunization by inhibiting the secretion of pro-inflammatory cytokines and increasing the secretion of anti-inflammatory cytokines.

(29) The composition may be used for an immunological disease or the prevention of the immunological disease. The immunological disease includes various inflammatory diseases, allergic diseases and cancers caused by abnormal immune system of living body. Examples of the immunological diseases include cold, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergies, rheumatoid arthritis, Alzheimer's disease, autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, lupus erythematosus, and psoriasis, but are not limited thereto.

(30) Since the neoagarooligosaccharide according to the present invention shows excellent effects of not only preventing sepsis, but also enhancing immunity, the neoagarooligosaccharide is useful for medicines and functional foods for prevention or treatment of sepsis or septic shock and reinforcement of immunization.

(31) The composition of the present invention may further include suitable carriers, excipients, and diluents which are conventionally used in the preparation of pharmaceutical compositions. The pharmaceutical composition according to the present invention may be formulated and used in the form of oral formulations, external preparations, suppositories, and sterilized injections such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols. Suitable formulations known in the art may preferably include those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton Pa.

(32) The pharmaceutical composition of the present invention may include carriers, excipients and diluents such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

(33) When the pharmaceutical composition of the present invention is formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid formulations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration may include suspensions, solutions, emulsions, syrups and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like in addition to water and liquid paraffin which are simple diluents commonly used. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried formulations, and suppositories. The non-aqueous solvent and suspension may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Bases of suppository may include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

(34) The term administration as used herein refers to provide any composition of the present invention to a subject in any suitable manner.

(35) The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the subject, the degree of disease, the type of drug, the administration route, and the period of time, but may be appropriately selected by those skilled in the art. In order for a desired effect, the composition of the present invention may be administered at 0.001 mg/kg to 1000 mg/kg per one day. The administration may be carried out once a day or divided into several times. The dose does not limit the scope of the present invention in any way.

(36) The pharmaceutical composition of the present invention may be administered to a subject through various pathways. All ways of administration may be expected to include, for example, oral, rectal or intravenous, intramuscular, subcutaneous, uterine duramater, or intracerebral injection administration. The pharmaceutical composition of the present invention may preferably be administered in the form of an injection.

(37) The pharmaceutical composition according to the present invention may further include the above-mentioned effective ingredient as well as at least one known substance having effects of preventing or treating sepsis or septic shock or enhancing immunization.

(38) The pharmaceutical composition according to the present invention may further include a bronchodilator, an antihistamine agent or an anti-inflammatory agent in addition to the above-mentioned active ingredients.

(39) For example, the bronchodilator may include 3-agonist, an anticholinergic agent, a methylantanine, etc. The antihistamines may include acrivastine, cetirizine, desloratadine, fexofenadine, levocertirizine, loratadine, mizolastine, ailmemazine, chlocertirizine, clemastine, cypropheptadine, hydroxyzine, ketotifen, promenthazine, and the like. The anti-inflammatory agent may include aspirin, diclofenac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, naproxen, piroxicam, sulindac, celecoxib, valdecoxib, rofecoxib, and the like.

(40) In the present invention, the term functional food refers to a food group in which food is imparted with added value to allow the function of the food to function or express for particular purpose using physical, biochemical, biotechnological techniques or the like, or food which is designed and manufactured so that the body control function related to the regulation of the bio-defense rhythm of the food composition, disease prevention and recovery, etc. is sufficiently expressed in the living body.

(41) The functional food may include a food supplement additive which is acceptable in sitology, and may further include suitable carriers, excipients and diluents conventionally used in the production of functional foods.

(42) When the composition of the present invention is used as a food additive, the composition may be added individually or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (e.g., prevention, health or therapeutic treatment). In the production of food or beverage, the composition of the present invention is generally added in an amount of 15% or less by weight, preferably 10% or less by weight based on the raw material. However, it may be less than the above range on long-term intake for the purpose of health and hygiene, or for the purpose of controlling health. The active ingredient may be used in an amount exceeding the above range due to no problem in aspects of safety.

(43) In addition to the above, the composition of the present invention may further include various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salt, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonates, and the like. In addition, the composition of the present invention may include flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives does not matter, but is generally selected in the range of 0.01 parts by weight to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

MODES OF THE INVENTION

(44) Hereinafter, the present invention will be described in more detail with reference to Embodiments. However, it should be noted that the following Embodiments are provided to illustrate the present invention to allow a more easily understanding, but the present invention is not limited to Embodiments.

Embodiment 1: Production of DagA

(45) PCR was carried out using the following Asm-F and Asm-R primers with the chromosomal DNA of Streptomyces coelicolor A3 (2) as a template to amplify a dagA gene fragment (a 947 bp fragment in which a signal peptide and a complete peptide of DagA were encoded, and a partial sequence of the primer was included in SEQ ID NO: 1). The dagA gene was cloned into the pUWL201pw vector shown in FIG. 1 using the restriction enzyme site (NdeI/BamHI) of the fragment, and thus a recombinant vector was prepared such that the transcription of the dagA gene was regulated by the ermE promoter. Such configuration is illustrated in FIG. 2.

(46) TABLE-US-00001 Asm-Fprimer: (SEQIDNO:3) 5-GACATATGGTGGTCAACCGACGTGATC-3 (NdeI) Asm-Rprimer: (SEQIDNO:4) 5-GGTGGATCCCTACACGGCCTGATACG-3 (BamHI)

(47) The transformant strain obtained by transforming Streptomyces lividans TK24 with the recombinant vector was used for the preparation of DagA.

(48) The strain was inoculated on R2YE liquid medium containing 0.3% (w/v) of agar and cultured and shaken at 120 rpm and 28 C. for 48 hours to 72 hours. After the pre-culture, the culture was performed, and each time of the culture was 2.5 days. The culture solution obtained through the culture was centrifuged to remove microbial cells, and the supernatant was filtered through an ultrafiltration membrane (5 kDa cut-off membrane) to isolate and purify proteins that did not pass through the filtration membrane. The obtained concentrate (enzyme solution) was used while being stored by freeze drying.

Embodiment 2: Measurement of Agarase Activity of DagA

(49) The agarase activity of DagA in Embodiment 1 was measured using estimation of reducing sugar by DNS method. 0.5 ml of a 20 mM Tris-HCl solution (pH 7) containing 0.5% (w/v) of agarose was mixed with 10 l of the enzyme solution obtained in Embodiment 1, and the mixture was reacted at 40 C. for 15 minutes. Then, 0.5 ml of DNS reagent (dinitrosalicylic acid 6.5 G, 2M NaOH 325 ml, glycerol 45 ml/1 l distilled water) was added thereto, and the mixture was boiled for 10 minutes. Then, the mixture was cooled, and the absorbance thereof was measured at 540 nm. 1 U of the enzyme was defined as an activity that shows an absorbance (540 nm) of 0.001 after 15 minutes of reaction.

Embodiment 3. Degradation of Agar or Agarose by DagA Enzyme Reaction

(50) 1 L of a 20 mM Tris-HCl solution in which 0.5% to 5% (w/v) of agar or agarose was dissolved was prepared and heated at 100 C. for about 10 minutes to dissolve it sufficiently. Then, the mixture was cooled down to 40 C. and then was treated with 2,000 U to 50,000 U of DagA enzyme to perform the enzyme reaction for 24 hours.

(51) In order to remove undegraded agarose from the enzyme reaction product, supernatant was recollected by centrifugation, and then the enzyme reaction product was confirmed using thin layer chromatography (TLC). The recollected supernatant was partially purified by ultrafiltration with a 5 kDa cut-off membrane.

Embodiment 4: Confirmation of DagA Enzyme Reaction Product Composition by HPLC-ELSD Analysis

(52) HPLC-ELSD analysis was performed to confirm the composition of the enzyme reaction product obtained in Embodiment 3 as described above. NH2 P-50 4E multimode column (2504.6 mm) was used, and a mixed solution of acetonitrile and water (acetonitrile:water=65:35) was used as a mobile phase. The result is illustrated in FIG. 3.

(53) As shown in FIG. 3, the total amount of neoagarooligosaccharides composed of neoagarobiose (hereinafter referred to as DP2), neoagarotetraose (hereinafter referred to as DP4), and neoagarohexaose (hereinafter referred to as DP4) of the reaction products was showed to account for 6520% (by weight). DP2, DP4, and DP6 respectively were showed to account for 0% to 10%, 50% to 70%, and 30% to 50% (by weight) based on the total amount of the neoagarooligosaccharides.

Experimental Example 1: Analysis of Preventive Effect on Sepsis by Administration of Neoagarooligosaccharide

(54) In order to analyze the preventive effect of neoagarooligosaccharide administration on sepsis, the following experiment was conducted.

(55) 1-1. Experimental Design and Survival Rate Analysis

(56) Six-week-old female BALB/c mice were used as experimental animals. The mice were pretreated intraperitoneally with neoagarooligosaccharide on mouse abdomen. One hour later, 10.sup.7 CFU/mice of E. coli K1 (dissolved in LB broth) was injected to induce sepsis. FIG. 4 shows the survival rate over time of each mouse in the experimental group and the control group in which sepsis was induced.

(57) As shown in FIG. 4, the control group died 18 hours after the induction of sepsis. On the other hand, the survival rate of the group which was pretreated with neoagarooligosaccharide was significantly increased compared to that of the control group.

(58) 1-2. Analysis of Serum

(59) The serum was separated from each mouse 12 hours after the induction of sepsis to analyze levels of inflammatory cytokines (TNF-, IL-6, IL-1, IL-12p70, and IL-10), AST, ALT, and BUN, respectively, in the serum. At this time, the concentration of the cytokine was measured using an ELISA kit (eBiosciences, San Diego, Calif.), and the result are shown in FIGS. 5 and 6.

(60) As shown in FIG. 5, when pre-treated with neoagarooligosaccharide, the amounts of TNF-, IL-6, IL-1 and IL-12p70, which are pro-inflammatory cytokines increased by sepsis in the mouse serum, were significantly inhibited. On the other hand, the secretion of IL-10, an anti-inflammatory cytokine, was increased by pre-treatment of neoagarooligosaccharide.

(61) Further, as shown in FIG. 6, the concentrations of AST, ALT, and BUN in the serum, which were associated with hepatic and renal toxicity, were increased by sepsis, but were significantly inhibited when the mouse was pre-treated with neoagarooligosaccharide.

Experimental Example 2: Analysis of Effect of Neoagarooligosaccharide on the Immune Enhancement

(62) In order to analyze the effect of neoagarooligosaccharide on the immunity enhancement, the following experiment was performed.

(63) 2-1. Experimental Design and Survival Rate Analysis

(64) Six-week-old female BALB/c mice were used as experimental animals. The mice were pretreated intraperitoneally with 100 mg/kg of neoagarooligosaccharide on mouse abdomen and injected with 20 mg/kg of lipopolysaccharide (LPS) after one hour. FIG. 7 shows the survival rate over time of each mouse in the experimental group and the control group.

(65) As shown in FIG. 7, the control group died 24 hours after administration of LPS. On the other hand, the survival rate of the group pretreated with neoagarooligosaccharide was significantly increased compared to that of the control group. The survival rate was increased depending on the concentration of neoagarooligosaccharide.

(66) 2-2. Analysis of Serum

(67) The serum was separated from each mouse 12 hours after LPS administration to analyze levels of inflammatory cytokines (TNF-, IL-6, IL-1, IL-12p70, and IL-10), AST, ALT, and BUN, respectively, in the serum. At this time, the concentrations of the cytokines were measured using an ELISA kit (eBiosciences, San Diego, Calif.), and the result are shown in FIGS. 8 and 9.

(68) As shown in FIG. 8, when pre-treated with neoagarooligosaccharide, the amounts of TNF-, IL-6, IL-1, and IL-12p70, which are pro-inflammatory cytokines increased by LPS administration in the mouse serum, were significantly inhibited. On the other hand, the secretion of IL-10, an anti-inflammatory cytokine, was increased by pre-treatment of neoagarooligosaccharide.

(69) Further, as shown in FIG. 9, the concentrations of AST, ALT, and BUN in the serum, which were associated with hepatic and renal toxicity, were increased by sepsis, but were significantly decreased when the mouse was pre-treated with neoagarooligosaccharide.

(70) 2-3. Analysis of Organization

(71) The lung and spleen tissues were extracted from each mouse after 12 hours of the administration of LPS. Paraffin section and H&E staining were performed according to a conventionally known method to observe them through a microscope. The results are shown in FIGS. 10 and 11.

(72) As shown in FIG. 10, the infiltration of inflammatory cells in the lung tissue, in which the neoagarooligosaccharide was pretreated was significantly inhibited compared to that of the control group.

(73) Further, as shown in FIG. 11, cell death in the spleen tissue, in which the neoagarooligosaccharide was pretreated was significantly inhibited compared to that of the control group.

(74) Through the experimental results, it was confirmed that the neoagarooligosaccharide has excellent effects of preventing sepsis and septic shock and reinforcing immunization by effectively suppressing the expression of inflammation.

(75) Hereinafter, formulation examples of the pharmaceutical composition and the food composition of the present invention will be described, but the present invention is not limited thereto, but is specifically described.

Formulation Example 1: Preparation of Pharmaceutical Composition

(76) 1-1. Preparation of Powder

(77) 20 mg of neoagarooligosaccharide

(78) 100 mg of lactose

(79) 10 mg of talc

(80) The components were mixed and packed in an airtight bag so as to prepare powders.

(81) 1-2. Preparation of Tablet

(82) 10 mg of neoagarooligosaccharide

(83) 100 mg of corn starch

(84) 100 mg of lactose

(85) 2 mg of magnesium stearate

(86) The components were mixed and tableted according to a conventional method of preparing tablets so as to prepare tablets.

(87) 1-3. Preparation of Capsule

(88) 10 mg of neoagarooligosaccharide

(89) 3 mg of crystalline cellulose

(90) 14.8 mg of lactose

(91) 0.2 mg of magnesium stearate

(92) The components were mixed and filled in gelatin capsules according to a conventional method of preparing capsules so as to prepare capsules.

(93) 1-4. Preparation of Injection

(94) 10 mg of neoagarooligosaccharide

(95) 180 mg of Mannitol

(96) 2974 mg of sterilized water for injection

(97) 26 mg of Na.sub.2HPO.sub.4.2H.sub.2O

(98) According to a conventional method of preparing injections, 2 ml per ample was prepared as an amount of the components.

(99) 1-5. Preparation of Liquid Agent

(100) 10 mg of neoagarooligosaccharide

(101) 10 g of isomerized glucose

(102) 5 g of mannitol

(103) appropriate amount of purified water

(104) According to a conventional method of preparing liquid agents, each component was added and dissolved in the purified water, and the lemon flavor was added in an appropriate amount. Then, the components were mixed, and then the purified water was added thereto. The whole mixture was then adjusted to 100 ml by adding the purified water and was filled in a brown bottle. Then the mixture was sterilized to prepare liquid agents.

Formulation Example 2: Preparation of Food Composition

(105) 2-1. Preparation of Health Food

(106) 100 mg of neoagarooligosaccharide

(107) appropriate amount of vitamin mixture

(108) 70 g of vitamin A acetate

(109) 1.0 mg of vitamin E

(110) 0.13 mg of vitamin B1

(111) 0.15 mg of vitamin B2

(112) 0.5 mg of vitamin B6

(113) 0.2 g of vitamin B12

(114) 10 mg of vitamin C

(115) 10 g of biotin

(116) 1.7 mg of nicotinic acid amide

(117) 50 g of Folic acid

(118) 0.5 mg of calcium pantothenate

(119) appropriate amount of inorganic mixture

(120) 1.75 mg of ferrous sulfate

(121) 0.82 mg of zinc oxide

(122) 25.3 mg of magnesium carbonate

(123) 15 mg of potassium phosphate monobasic

(124) 55 mg of potassium phosphate dibasic

(125) 90 mg of potassium citrate

(126) 100 mg of calcium carbonate

(127) 24.8 mg of magnesium chloride

(128) Although the composition ratio of the vitamin and mineral mixture is configured to be mixed with comparatively suitable ingredients for health food, the composition ratio may be arbitrarily modified. After the components were mixed according to conventional methods of preparing health foods, granules were prepared and used in the preparation of a health food composition according to conventional methods.