KR20180004015A - Method for preparing the immunoglobulin from slaughterhouse blood, and its use for enhancement of oral health - Google Patents

Abstract

The present invention relates to a method for preparing immune protein derived from slaughtered animal blood and uses of immune protein prepared by the method for enhancement of oral health. More specifically, immune protein prepared by a method for preparing immune protein derived from slaughtered animal blood has excellent antibacterial capacity and antigen adherence capacity, thereby being usefully used for a pharmaceutical composition and a food composition for enhancement of oral health.

Description

[0001] The present invention relates to a method for preparing a slaughter blood-derived immunoprotein, and a method for preparing the same,

The present invention relates to a method for producing an immune protein derived from a slaughter blood and a use for improving oral health of an immunoprotein prepared by the method. More specifically, the immune protein produced by the method for producing an immune protein derived from slaughter blood according to the present invention has excellent antibacterial activity and antigen-binding ability, and thus can be usefully used in pharmaceutical compositions and food compositions for improving oral health It is expected to be.

Despite the high content of useful components, the blood that is discarded at the slaughterhouse is only used in very small quantities for procurement and is all disposed of and accounts for most of the cost of slaughterhouse wastewater treatment. Protein, which is a major component of slaughter blood, can be used as a raw material for various products. Despite its high commercial value, hygienic treatment and recycling methods of slaughter blood have not been systematically established and most of them have been disposed of. In particular, the immune proteins present in the slaughtered blood have excellent amino acid composition, which not only excel in nutritional value, but also demonstrate various effects such as suppression of diseases from pathogenic microorganisms in the body and strengthening of the immune system. It is a newly emerging substance.

It is very important to keep your mouth healthy because the mouth is the primary organ for digesting food through chewing. The various microbial strains present in the oral cavity penetrate into the gums, teeth, or mucous membranes, causing gingivitis and periodontal disease, etc., which exacerbates oral health. Since all food enters the body through the oral cavity, But can cause disease in other institutions. Therefore, inhibiting the pathogenicity of microbial strains in the oral cavity is a very important factor to maintain the oral health and can reduce the medical expenses (about 2 trillion won by 2015) consumed by oral treatment such as periodontal diseases.

At present, utilization methods of slaughter blood are suggested both domestically and externally, and they are being developed competitively, but they can be used industrially when the production process of material using slaughter blood is established and the functionality is scientifically proved. In order to improve the oral health, it is necessary to study the technology that can separate the useful components in the slaughter blood according to the domestic reality and to develop materials suitable for the needs of the industry. In particular, Development can be expected to revitalize related industries and secure product competitiveness.

Prior Patent Document

1. Korean Patent Publication No. 10-2017-0022295

The present invention seeks to provide a method for producing an immune protein derived from a slaughter blood and an immune protein produced by the method in use for improving oral health.

According to a first embodiment,

(a) a one-dimensional segregation step for recovering plasma from slaughtered blood;

(b) serum separation step in which calcium chloride is added to the recovered plasma and allowed to stand at room temperature for 30 minutes to 1 hour after stirring;

(c) a two-dimensional segregation step for separating and recovering the serum from the precipitate;

(d) salting out the immune protein by adding ammonium sulfate to the separated supernatant;

(e) a three-dimensional segregation step for removing the separated supernatant;

(f) adjusting pH by adding sodium hydroxide to the precipitate in which the immune protein is precipitated;

(g) an ultrafiltration step of ultrafiltering the pH-adjusted precipitate with a 10 kDa filtration membrane; And

(h) recovering an immune protein solution that has not been filtered by the ultrafiltration, and recovering the immune protein solution.

In the method for producing an immunogenic protein according to the present invention, the slaughter blood of the step (a) can be preferably derived from pig, horse, goat, cattle, sheep, camel or yak, , Chlorine or cow, and most preferably from pigs.

In the method for producing an immunogenic protein according to the present invention, the one-dimensional segregation of step (a) can be preferably performed at a speed of 7000-9000 rpm, more preferably at a speed of 7500-8500 rpm , And most preferably at a rate of 8000 rpm.

In the method for producing an immunogenic protein according to the present invention, the one-dimensional segregation of step (a) can be preferably performed for 1-60 minutes, more preferably for 10-40 minutes, Most preferably 30 minutes.

In the method for producing an immunogenic protein according to the present invention, the one-dimensional segregation of step (a) can be performed preferably at 4-25 ° C, more preferably at 4-15 ° C, Most preferably at < RTI ID = 0.0 > 4 C. < / RTI >

In the method for producing an immunogenic protein according to the present invention, the calcium chloride of step (b) may be added in an amount of preferably 0.01 to 1% by weight, more preferably 0.3 to 0.7% by weight based on the recovered plasma weight, May be added in an amount of 0.5 wt%, and most preferably 0.5 wt%.

In the method for producing an immunogenic protein according to the present invention, the two-dimensional segregation of step (c) can be preferably performed at a speed of 7000-9000 rpm, more preferably at a speed of 7500-8500 rpm , And most preferably at a rate of 8000 rpm.

In the method for producing an immunogenic protein according to the present invention, the two-dimensional segregation of step (c) can be performed preferably for 1-60 minutes, more preferably for 10-40 minutes, Most preferably 30 minutes.

In the method for producing an immunogenic protein according to the present invention, the two-dimensional segregation of step (c) can be performed preferably at 4-25 ° C, more preferably at 4-15 ° C, Most preferably at < RTI ID = 0.0 > 4 C. < / RTI >

In the method for producing an immunogenic protein according to the present invention, the ammonium sulfate in the step (d) may be added in an amount of preferably 10 to 30% by weight, more preferably 15 To 20% by weight, and most preferably 20% by weight.

In the method for producing an immunogenic protein according to the present invention, the three-dimensional segregation of step (e) can be preferably performed at a speed of 7000-9000 rpm, more preferably at a speed of 7500-8500 rpm , And most preferably at a rate of 8000 rpm.

In the method for producing an immunogenic protein according to the present invention, the three-dimensional segregation of step (e) can be preferably performed for 1-60 minutes, more preferably for 10-40 minutes, Most preferably 30 minutes.

In the method for producing an immunogenic protein according to the present invention, the three-dimensional segregation of step (e) can be performed preferably at 4-25 ° C, more preferably at 4-15 ° C, Most preferably at < RTI ID = 0.0 > 4 C. < / RTI >

In the method for producing an immunogenic protein according to the present invention, the pH adjusting step of step (f) can be adjusted preferably to a pH of from 8 to 10, more preferably to a pH of from 8.5 to 9.5, Can be adjusted to pH 9.

In a method for producing an immunogenic protein according to the present invention, the method comprises:

(g) a pH adjusting step of adjusting the pH by adding hydrochloric acid to the immune protein solution; And

(h) spray-drying the pH-adjusted immune protein solution to prepare an immunoprotein powder.

According to a second embodiment,

An oral pharmaceutical composition comprising an immunoprotein prepared according to the first embodiment is disclosed.

In the pharmaceutical composition for oral hygiene according to the present invention, the composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation, and include lactose, textol, sucrose, sorbic, mannitol, starch, acacia rubber, calcium phosphate, Lactone, calcium silicate, microcrystalline cellulose, rosin, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil But is not limited thereto. The pharmaceutical compositions for oral hygiene of the present invention may further comprise lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents and preservatives in addition to the above components. Suitable pharmaceutically acceptable carriers and preparations are described in Remington's Pharmaceutical Sciences 19th ed. , 1995).

In the oral pharmaceutical composition for oral hygiene according to the present invention, the pharmaceutical composition may be orally or parenterally administered.

In the oral pharmaceutical composition for oral hygiene according to the present invention, the appropriate dosage of the pharmaceutical composition may vary depending on factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, And responsiveness. ≪ / RTI >

In the oral pharmaceutical composition for oral hygiene according to the present invention, the pharmaceutical composition may be formulated into a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. , Or may be manufactured by inserting it into a multi-dose container. Here, the formulations may be in the form of solutions, suspensions, syrups or emulsions in an oil or aqueous medium, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to a third embodiment,

Disclosed is an oral care food composition comprising an immunoprotein prepared according to the first embodiment.

In the oral care food composition according to the present invention, the oral hygiene food composition may be in the form of a food such as cream, paste or gel-type toothpaste, mouthwash, oral spray, beverage, chewing gum or candy.

In the oral care composition for oral hygiene according to the present invention, the oral hygiene food composition may be in any form such as a health functional food, a nutritional supplement, a nutrient, a pharmafood, a health food, a nutritional food, a designer food, Of food.

In the oral care composition for oral hygiene according to the present invention, the oral hygiene food composition includes ingredients that are ordinarily added in the manufacture of food, for example, a protein, a carbohydrate, a fat, a nutrient, a seasoning agent, . Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose sucrose, oligosaccharides and the like; And polysaccharides, for example, conventional sugars and sorbies such as methylene chloride, cyclodextrin, and sugar alcohols such as erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavors (saccharin, aspartame, etc.) may be used as flavorings.

Since the immunoprotein prepared by the method of the present invention has excellent antibacterial activity (Experimental Example 1) and ability to adhere antigen (Experimental Example 2), it can be effectively used for improving oral health. More specifically, the immunoprotein produced by the method for producing an immunoprotein according to the present invention is a protein selected from the group consisting of Eubacterium nodatum ATCC 33099) , Staphylococcus aureus ATCC 25923 ), Streptococcus mitis KCTC 5650 ), Streptococcus mutans ATCC 25175 ), Streptococcus sobrinus ATCC 33478 ), Actinomyces viscocus ATCC 15988 , Eikenella < RTI ID = 0.0 > corrodens ATCC 23834 ), Fusobacterium nucleatum ( Fusobacterium < RTI ID = 0.0 > nucleatum ATCC 25586 ), Prevotella intermedia ATCC 49046 ) and Prevotella nigrescens ATCC 33563 ), it is expected to be usefully used in a pharmaceutical composition or a food composition for improving oral health.

Fig. 1 shows a method for producing a hair-softening white material produced according to Example 1. Fig.
Fig. 2 shows the immunoprotein material prepared according to Example 1. Fig.
Figure 3 shows the protein separation pattern of the immunoprotein material according to Example 2 (M, protein molecular weight marker; A, pig IgG; B, pig plasma; C, immunoprotein material; D, serum albumin.
Fig. 4 shows the effect of the immune protein material according to Experimental Example 1 on the inhibition of oral strain growth (p < 0.01). (BSA, bovine serum albumin, PI, porcine immunoglobulin, A, E ubacterium nodatum ATCC 33099 ; B, Staphylococcus aures ATCC 25923 ; C, Streptococcus mitis KCTC 5650 ; D, Streptococcus mutans ATCC 25175 ; E, Actinomyces viscocus ATCC 15988 ; F, Fusobacterium nucleatum ATCC 25586 ; G, Prevotella nigrescens ATCC 33563 ).
Fig. 5 shows the ability of the immune protein material to attach an antigen to an oral strain according to Experimental Example 2 (1-10 corresponds to the number shown in Table 1 below).

Hereinafter, various embodiments are provided to facilitate understanding of the present invention. The following examples are provided to facilitate understanding of the invention and are not intended to limit the scope of the invention.

<Examples>

Example 1. Mass production of immunoprotein material

Pork blood was recovered from the slaughterhouse of Seoul Livestock Quality Assurance Service (Pocheon) and centrifuged (8,000 rpm, 30 minutes, 4 ℃) to separate the plasma. 45 mM calcium chloride was added to the separated plasma, and the mixture was stirred for 5 minutes and reacted at 25 ° C for 1 hour to remove fibrinogen. 20% ammonium sulfate was added thereto and the mixture was continuously stirred at 4 ° C overnight. After centrifugation (8,000 rpm, 30 minutes, 4 ° C), the supernatant containing albumin and the like is removed, and the precipitate is recovered and sterilized water is added at a ratio of 1: 1 (w / w) to dissolve the precipitate. To recover the immune protein, the pH of the solution was adjusted to 9 using 1N sodium hydroxide solution, ultrafiltered with a 10 kD filtration membrane, adjusted to pH 7.4 with 1N hydrochloric acid solution, and dried using a Pilot Spray Dryer (Yoojin) The immunoprotein material was pulverized and mass produced through spray drying (inlet hot air temperature 150 ° C, outlet hot air temperature 90 ° C, injection pressure 18 kgf / cm ² , flow rate 5 L / h). The above process and results are shown in FIGS. 1 and 2, respectively.

Example 2. SDS-PAGE electrophoresis

SDS-PAGE electrophoresis was performed using Mini-Protean® Tetra System (Biorad) and PowerPac ™ HV (Biorad). Samples were equilibrated with 2X sample buffer (0.125 M TrisCl, 4% SDS, 20% glycerol, 2% β-mercaptoethanol, pH 6.8) and heated at 95 ° C for 10 min to use as an electrophoresis sample. 12.5% separating gels and 4% stacking gels were prepared and incubated for 1 hour. 10 μl of each was loaded and electrophoresed at 20 mA per gel for 1 hour. After the electrophoresis gel was photographed an image 2 hours stained with coomassie blue solution, and using a bleaching rear ^{Molecular Imager ⓡ GelDoc TM XR plus Imaging} system (Biorad). The results were analyzed using Image Lab ^TM Software 5.1 (Biorad), and the degree of protein separation and the concentration of immunoglobulin G (IgG) in the immunoprotein material were measured. The results are shown in Fig.

As can be seen from FIG. 3, it was confirmed that most of proteins other than immunological proteins such as fibrinogen and albumin were effectively removed from the plasma of the immunoprotein material. It was also confirmed that about 70% or more of IgG was contained in the prepared immunoprotein material.

< Experimental Example >

Experimental Example 1. Antimicrobial activity

The oral strains used in the antibacterial ability test of the immunoprotein material according to the present invention are shown in Table 1 below.

No. Bacterium Gram positive  One Eubacterium nodatum ATCC 33099  2 Staphylococcus aures ATCC 25923  3 Streptococcus mitis KCTC 5650  4 Streptococcus mutans ATCC 25175  5 Streptococcus sobrinus ATCC 33478 Gram negative  6 Actinomyces viscocus ATCC 15988  7 Eikenella corrodens ATCC 23834  8 Fusobacterium nucleatum ATCC 25586  9 Prevotella intermedia ATCC 49046  10 Prevotella nigrescens ATCC 33563

All oral strains were cultured at 37 ℃ for 3 hours or more at 37 ℃ using tryptic soy broth and brain heart infusion medium. The mixed solution prepared by dissolving the immunoprotein material in buffered peptone water (BPW) was mixed with about 10 ³ cfu / ml oral strains and cultured at 37 ° C for 3 hours. After incubation, the cells were diluted with a number of peptones and cultured at 37 ° C for 48 hours using tryptic soy agar and brain heart infusion agar. Bovine serum albumin (BSA) was used as a positive control, BPW solution without an immunoprotein was used as a control, and antibacterial ability was calculated as follows. The results are shown in FIG.

N _c : the number of bacteria treated with BPW instead of the immunoprotein material

N _m : the number of bacteria cultured for 3 hours after the addition of the immunoprotein material

As can be seen from FIG. 3, it was confirmed that the immunoprotein material prepared according to the present invention significantly inhibited growth of seven kinds of 10 oral strains ( p <0.01). In particular, when 10% of the immune protein (PI 10%) was treated with Streptococcus mutans and Actinomyces viscocus oral strains to about 45 to 55%. On the other hand, considering that the growth of oral strains was not inhibited in the BSA-treated group used as a positive control group, it was proved that the prepared immunoprotein material had antimicrobial ability, not inhibiting the growth of oral strains by pure protein action .

Experimental Example 2. Investigation of antigen attachment ability

Antigen attachment experiments of the immunoprotein material according to the present invention were conducted on the oral strains of Table 1 above. All oral strains were used after 3 passages, centrifuged at 4,000 rpm for 20 minutes and the supernatant was removed. After washing the precipitated strains with PBS twice, the absorbance (610 nm) was adjusted to 1.0 using a spectrophotometer, and the oral strains were attached to a 96 well plate overnight at 4 ° C. The cells were washed four times with PBST (phosphate buffered saline containing 0.05% tween 20), blocked with 3% BSA in PBS at 37 ° C for 1 hour, washed again with PBST, and immune protein material (10 mg / Respectively. After the immune protein material treatment, the cells were incubated at 37 ° C for 2 hours, washed 4 times with PBST, and incubated at 25 ° C for 1 hour. After incubation, each well was washed with PBST. After the substrate solution was dispensed, it was cultured in a dark place for 10 minutes, treated with 0.18 M sulfuric acid, and the absorbance was measured at 450 nm. The control was carried out by attaching each immune protein material instead of the oral strain, and the antigen attachment capacity was calculated as follows. The results are shown in Fig.

As can be seen from FIG. 5, the immunoprotein material prepared according to the present invention was confirmed to exhibit antigen attachment ability to all the oral strains used in the present study. According to the oral strain used, the antigen attachment ability was confirmed to be about 49 ~ 94%, and the ability to attach the antigen to the Fusobacterium nucleatum strain was most effective at 94%.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (10)

(a) a one-dimensional segregation step for recovering plasma from slaughtered blood;
(b) serum separation step in which calcium chloride is added to the recovered plasma and allowed to stand at room temperature for 30 minutes to 1 hour after stirring;
(c) a two-dimensional segregation step for separating and recovering the serum from the precipitate;
(d) a step of salting out the immune protein to which ammonium sulfate is added to the separated supernatant;
(e) a three-dimensional segregation step for removing the separated supernatant;
(f) adjusting pH by adding sodium hydroxide to the precipitate in which the immune protein is precipitated;
(g) an ultrafiltration step of ultrafiltering the pH-adjusted precipitate with a 10 kDa filtration membrane; And
(h) recovering the immune protein solution that has not been filtered by the ultrafiltration, and recovering the immune protein solution.
The method according to claim 1,
Wherein the slaughter blood of step (a) is derived from pig, horse, goat, cattle, sheep, camel or yak.
The method according to claim 1,
Wherein the one-dimensional segregation of step (a) is performed at 4000 rpm at a rate of 8000 rpm for 30 minutes.
The method according to claim 1,
Wherein the two-dimensional segregation of step (c) is performed for 30 minutes at a rate of 8000 rpm at 4 占 폚.
The method according to claim 1,
Wherein the three-dimensional segregation of step (e) is carried out at 4000 rpm at a rate of 8000 rpm for 30 minutes.
The method according to claim 1,
Wherein the pH adjusting step of step (f) is adjusted to a pH of 9.
The method according to claim 1,
The method comprising:
(g) a pH adjusting step of adjusting the pH by adding hydrochloric acid to the immune protein solution; And
(h) spray-drying the pH-adjusted immunoprotein solution to prepare an immunoprotein powder.
8. An oral care composition comprising an immunoprotein produced by the method according to any one of claims 1 to 7.
9. The method of claim 8,
Wherein the oral care composition is in the form of a cream, paste or gel dentifrice, mouthwash, oral spray, beverage, chewing gum or candy.
9. The method of claim 8,
The composition for oral hygiene may be selected from the group consisting of Eubacterium nodatum ATCC 33099) , Staphylococcus aureus ATCC 25923 ), Streptococcus mitis KCTC 5650 ), Streptococcus mutans ATCC 25175 ), Streptococcus sobrinus ATCC 33478 , Actinomyces &lt; RTI ID = 0.0 &gt; viscocus ATCC 15988 ), Eikenella corrodens ATCC 23834 ), Fusobacterium &lt; RTI ID = 0.0 &gt; nucleatum ATCC 25586 ), Prevotella intermedia ATCC 49046 ) or Prevotella nigrescens ATCC 33563 ) having an excellent antimicrobial activity and an antigen-binding ability.

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