Anti-carious tooth chewing gum and preparation method thereof
Technical field
The present invention relates to a kind of anti-carious tooth chewing gum and preparation method thereof.
Background technology
At present, along with living standards of the people generally improve and diet in the increase of sugared content and intake thereof, it is popular to make that the dental caries disease is able to, especially children's deciduous teeth trouble carious tooth is more obvious, along with the population life-time dilatation, the elderly's root face dental caries are also increasing, and these all have a strong impact on people's oral cavity and healthy, thereby have a strong impact on people's quality of life.It is the third-largest NCD that is only second to angiocardiopathy, cancer that present WHO (WHO) has classified carious tooth as, and as one of 10 whether healthy indexs.
Carious tooth is a kind of common disease, frequently-occurring disease, its incidence of disease height, and it is wide to distribute, big to human body harm.Through the scientists unremitting effort, generally believe that now bacterium is the main cause that carious tooth takes place, wherein streptococcus mutans is main pathogenic bacteria, the bacterial plaque of its formation adheres to dental surface, and in dental surface breeding growth, product acid, the corrosion tooth finally forms carious tooth.
The preventing decayed tooth product of Ying Yonging roughly is divided into following a few class in the market: one for containing fluoride preparation, and its preventing decayed tooth principle reduces sour corrosion for promoting the tooth calcification, and this is an indirectly-acting, and curative effect is limited; The 2nd, the application of antibacterials, as contain wash must eliminate, the mouthwash of metronidazole etc., because of it has significant side effects, application is restricted; The 3rd, the preventing decayed tooth product of making by plant extracts, the GARG of making as Chinese medical extract, chewing-gum sheet etc., control has certain effect to carious tooth for it, but it is medicine, need under physician guidance, to use and mouthfeel relatively poor, should not use for a long time; The 4th, widely accepted in the market utilize generation sugar produce be used for the product that preventing decayed tooth protects the tooth class, as xylitol etc., it does not but fundamentally solve the question of substance that preventing decayed tooth protects tooth.Chinese patent publication number CN1307061A discloses the patent that a kind of name is called " preparation of dental caries resistant bacteria IgY and composite for preventing dental caries " August 8 calendar year 2001.The preparation of this patent dental caries resistant bacteria IgY divides following several steps: preparation antigen, preparation antibody, configuration medicine.The production process of its antibody is that the cell with bacterium directly carries out after the cracking as antigen, the antibody that causes preparing mainly is that the interior antigen composition of directed toward bacteria works, when facing complete activated bacterial, because antibody can't directly enter in the bacterial body and work, and a little less than the binding ability on itself and live bacteria surface, so the poor effect of its sterilization.So there is the shortcoming of anti-carious tooth weak effect, manufacturing and use inconvenience, mouthfeel difference in existing product.
Summary of the invention
Technical problem to be solved by this invention is the deficiency that overcomes on the prior art, and a kind of anti-carious tooth chewing gum and preparation method thereof is provided, the anti-carious tooth of this anti-carious tooth chewing gum is effective, manufacturing and easy to use, mouthfeel good.But the antigen of the anti-carious tooth Yolk antibody Direct Recognition The main pathogenic fungi-Streptococcus mutans in the anti-carious tooth chewing gum of the present invention has stronger killing action to complete, activated bacterium.
The technical solution adopted in the present invention is: the anti-carious tooth chewing gum of the present invention is to be made by following materials of weight proportions:
Anti-carious tooth Yolk antibody 0.01-2%
Matrix 77.29-13.5%
Xylitol 10-50%
Arabic gum 2-10%
Sweet mellow wine 10-20%
Citric acid 0.1-0.5%
Lecithin 0.1-2%
Flavoring essence 0.5-2%
The percentage by weight sum of said components is absolutely.
The preparation method of the anti-carious tooth chewing gum of the present invention may further comprise the steps:
One, the preparation of anti-carious tooth Yolk antibody:
(1) preparation antigen: get the streptococcus mutans that serotype is C type and D type, go down to posterity with solid TYCSB agar slant respectively, under 37 ℃ of anaerobic conditions, cultivated 48 hours, through PCR identify obtain described streptococcus mutans purebred after, in liquid TYCSB culture medium, enlarge to cultivate after 48~72 hours and collect thalline, described thalline is frozen into dry powder as antigen with the deactivation under 25 ℃ of conditions of 0.5% formalin after 24 hours;
(2) preparation antibody:
1., selecting for use of adjuvant:
Initial immunity is selected Freund's complete adjuvant for use, with described Freund's complete adjuvant and described antigen according to 1: 1 ratio mixing and emulsifying of weight ratio after as initial immunity antigen; Booster immunization is selected incomplete Freund for use, is as booster immunization antigen according to weight ratio with incomplete Freund and described antigen behind 1: 1 the ratio mixing and emulsifying;
2., to the immunity of the hen that lays eggs and get ovum:
Select for use healthy white race to go hen isolated rearing and carry out the described initial immunity antigen of chicken muscle multi-point injection, this is an initial immunity; To the hen isolated rearing of initial immunity and carry out the described booster immunization antigen of chicken muscle multi-point injection, this is a booster immunization every 2 week backs, later on week about booster immunization once, its initial immunity and booster immunization totally three times; Count behind initial immunity, the 30 day begins to collect ovum gallinaceum, stores for future use under 4 ℃ of conditions;
3., the extraction of Yolk antibody: adopt the water dilution method or the PEG precipitation method or membrane filter method or ammonium sulfate precipitation method from described ovum gallinaceum, to extract anti-carious tooth Yolk antibody;
Two, with described matrix melting under 60 ℃ temperature of described weight proportion, adding the described xylitol of described weight proportion, described Arabic gum, described sweet mellow wine, described citric acid, described lecithin and described flavoring essence fully stirs, when temperature reaches 40 ℃, the described anti-carious tooth Yolk antibody that adds described weight proportion again, after stirring, go into mould, compressing tablet, section, moulding, packing, get final product.
The method that described employing water dilution method extracts anti-carious tooth Yolk antibody is: separate obtaining yolk from the ovum gallinaceum of the immunity of collecting, dilute with deionized water, regulate pH value in 4.0~5.0 scopes, under 4 ℃ of conditions, stirred 4 hours, with discarding precipitation after the centrifuge processing, supernatant is concentrated, freeze drying get final product.
The method that the described employing PEG precipitation method are extracted anti-carious tooth Yolk antibody is: separate obtaining yolk from the ovum gallinaceum of the immunity of collecting, the adding volume is 3 times of volumes of described yolk, concentration is that 4% PEG dilutes, it is 4 ℃ in temperature behind the stirring and evenly mixing fully, centrifuge speed is under the per minute 1300 commentaries on classics conditions centrifugal 10 minutes, get supernatant, in described supernatant, dropwise drip volume then and be described supernatant 1/6 volume, concentration is 40% PEG, it is 4 ℃ in temperature behind the stirring and evenly mixing fully, centrifuge speed is under the per minute 1300 commentaries on classics conditions centrifugal 10 minutes, collecting precipitation, freeze drying gets final product.
The method that described employing membrane filter method extracts anti-carious tooth Yolk antibody is: separate obtaining yolk from the ovum gallinaceum of the immunity of collecting, dilute with deionized water, after temperature is to leave standstill 12 hours under 4 ℃ of conditions, with molecular weight is that the film of 200KD and 150KD filters respectively, collect the protein substance of 150KD-200KD, freeze drying gets final product.
The method that described employing ammonium sulfate precipitation method extracts anti-carious tooth Yolk antibody is: separate obtaining yolk from the ovum gallinaceum of the immunity of collecting, repeat to extract 3 times: for the first time, get with the dilution of the isopyknic physiological saline of described yolk and mix after, slowly drip volume and be 2 times of volumes of described yolk, concentration is 50% saturated ammonium sulfate, in temperature is to act on 3 hours under 4 ℃ of conditions, and be that 4 ℃, centrifuge speed are under the per minute 1300 commentaries on classics conditions centrifugal 10 minutes in temperature, abandon supernatant, stay precipitation; For the second time, precipitate with physiological saline solution, add volume then and be described physiological saline 2/3 volume, concentration is 40% saturated ammonium sulfate, in temperature is to act on 3 hours under 4 ℃ of conditions, and be that 4 ℃, centrifuge speed are under the per minute 1300 commentaries on classics conditions centrifugal 10 minutes in temperature, abandon supernatant and stay precipitation; For the third time, with described physiological saline solution precipitation, add volume then and be described physiological saline 1/2 volume, concentration is 33% saturated ammonium sulfate, is effect 3 hours under 4 ℃ of conditions in temperature, will precipitate direct freeze drying, get final product.
The invention has the beneficial effects as follows: owing to the anti-carious tooth Yolk antibody in the anti-carious tooth chewing gum of the present invention is to analyse in depth research at the The main pathogenic fungi-streptococcus mutans that causes carious tooth to make, when facing complete activated bacterial, the binding ability on itself and live bacteria surface is stronger, can directly enter in the bacterial body and work, bacterium there is stronger killing action, so the anti-carious tooth of this anti-carious tooth chewing gum is effective, and easy to use; Again since the present invention make by the anti-carious tooth Yolk antibody and matrix, xylitol, Arabic gum, sweet mellow wine, citric acid, lecithin and the flavoring essence that from the ovum gallinaceum after the immunity of collecting, extract, so it have no side effect, mouthfeel is good; Again since among the preparation method of the anti-carious tooth chewing gum of the present invention the extraction of Yolk antibody be to adopt methods such as water dilution method, the PEG precipitation method, membrane filter method, ammonium sulfate precipitation method, so that the preparation method is health, environmental protection, convenience, cost is low.
The specific embodiment
Embodiment one:
The technology used in the present invention mechanism is: the Yolk antibody that at first prepares a kind of anti-carious tooth, it detects according to non-sex change polyacrylate hydrogel electrophoresis, after coomassie brilliant blue R250 dyeing, collection of illustrative plates detects can present single band at molecular weight 170~190KD place, and Western Bloting detects and can find a single specific band at molecular weight 170~190KD place; Or detect according to sex change polyacrylate hydrogel electrophoresis, after coomassie brilliant blue R250 dyeing, collection of illustrative plates presents two bands, Western Bloting detects and can find two specific bands at about 65KD of molecular weight and 25KD place, and adopting the determined by ultraviolet spectrophotometry protein concentration is 0.1~20mg/ml; Measuring through enzyme-linked immunosorbent assay that it tires is 1: 50-1: 100000.
The anti-carious tooth chewing gum of the present invention is made by following materials of weight proportions:
Anti-carious tooth Yolk antibody 0.01%
Matrix 77.29%
Xylitol 10%
Arabic gum 2%
Sweet mellow wine 10%
Citric acid 0.1%
Lecithin 0.1%
Flavoring essence 0.5%
The percentage by weight sum of said components is absolutely.
The preparation method of the anti-carious tooth chewing gum of the present invention may further comprise the steps:
One, the preparation of anti-carious tooth Yolk antibody:
(1) preparation antigen: get the streptococcus mutans that serotype is C type and D type, go down to posterity with solid TYCSB agar slant respectively, under 37 ℃ of anaerobic conditions, cultivated 48 hours, through PCR identify obtain described streptococcus mutans purebred after, in liquid TYCSB culture medium, enlarge to cultivate after 48~72 hours and collect thalline, described thalline is frozen into dry powder as antigen with the deactivation under 25 ℃ of conditions of 0.5% formalin after 24 hours;
(2) preparation antibody:
1., selecting for use of adjuvant:
Initial immunity is selected Freund's complete adjuvant for use, with described Freund's complete adjuvant and described antigen according to 1: 1 ratio mixing and emulsifying of weight ratio after as initial immunity antigen; Booster immunization is selected incomplete Freund for use, is as booster immunization antigen according to weight ratio with incomplete Freund and described antigen behind 1: 1 the ratio mixing and emulsifying;
2., to the immunity of the hen that lays eggs and get ovum:
Select for use healthy white race to go hen isolated rearing and carry out the described initial immunity antigen of chicken muscle multi-point injection, this is an initial immunity; To the hen isolated rearing of initial immunity and carry out the described booster immunization antigen of chicken muscle multi-point injection, this is a booster immunization every 2 week backs, later on week about booster immunization once, its initial immunity and booster immunization totally three times; Count behind initial immunity, the 30 day begins to collect ovum gallinaceum, stores for future use under 4 ℃ of conditions;
3., the extraction of anti-carious tooth Yolk antibody: the extraction of anti-carious tooth Yolk antibody can be adopted one of following four kinds of methods:
A) water dilution method: from the ovum gallinaceum of the immunity of collecting, separate obtaining yolk,, regulate pH value in 4.0~5.0 scopes, under 4 ℃ of conditions, stirred 4 hours with the deionized water dilution, with discarding precipitation after the centrifuge processing, with supernatant concentrate, freeze drying, get final product.
B) the PEG precipitation method: from the ovum gallinaceum of the immunity of collecting, separate obtaining yolk, add volume and be 3 times of volumes of described yolk, concentration is that 4% PEG dilutes, be that 4 ℃, centrifuge speed are under the per minute 1300 commentaries on classics conditions centrifugal 10 minutes in temperature behind the stirring and evenly mixing fully, get supernatant, in described supernatant, dropwise drip volume then and be described supernatant 1/6 volume, concentration is 40% PEG, be that 4 ℃, centrifuge speed are under the per minute 1300 commentaries on classics conditions centrifugal 10 minutes in temperature behind the stirring and evenly mixing fully, collecting precipitation, freeze drying gets final product.
C) membrane filter method: from the ovum gallinaceum of the immunity of collecting, separate obtaining yolk, with the deionized water dilution, after temperature is to leave standstill 12 hours under 4 ℃ of conditions, with molecular weight is that the film of 200KD and 150KD filters respectively, collect the protein substance of 150KD-200KD, freeze drying gets final product.
D) ammonium sulfate precipitation method: from the ovum gallinaceum of the immunity of collecting, separate obtaining yolk, repeat to extract 3 times: for the first time, get with the dilution of the isopyknic physiological saline of described yolk and mix after, slowly drip volume and be 2 times of volumes of described yolk, concentration is 50% saturated ammonium sulfate, in temperature is to act on 3 hours under 4 ℃ of conditions, and be that 4 ℃, centrifuge speed are under the per minute 1300 commentaries on classics conditions centrifugal 10 minutes in temperature, abandon supernatant, stay precipitation; For the second time, precipitate with physiological saline solution, add volume then and be described physiological saline 2/3 volume, concentration is 40% saturated ammonium sulfate, in temperature is to act on 3 hours under 4 ℃ of conditions, and be that 4 ℃, centrifuge speed are under the per minute 1300 commentaries on classics conditions centrifugal 10 minutes in temperature, abandon supernatant and stay precipitation; For the third time, with described physiological saline solution precipitation, add volume then and be described physiological saline 1/2 volume, concentration is 33% saturated ammonium sulfate, is effect 3 hours under 4 ℃ of conditions in temperature, will precipitate direct freeze drying, get final product.
Two, with matrix melting under 60 ℃ temperature of 77.29%, adding 10% xylitol, 2% Arabic gum, 10% sweet mellow wine, 0.1% citric acid, 0.1% lecithin and 0.5% flavoring essence fully stirs, when temperature reaches 40 ℃, add 0.01% anti-carious tooth Yolk antibody again, after stirring, go into mould, compressing tablet, section, moulding, packing gets final product (percentage wherein is weight percentage).
The chewing gum of anti-carious tooth of the present invention can also be used the collaborative effect that increases anti-carious tooth with the pharmaceutical preparation and the decayed tooth prophylactic of prevention mouth disease.
Embodiment two:
The anti-carious tooth chewing gum of the present invention is made by following materials of weight proportions:
Anti-carious tooth Yolk antibody 2%
Matrix 13.5%
Xylitol 50%
Arabic gum 10%
Sweet mellow wine 20%
Citric acid 0.5%
Lecithin 2%
Flavoring essence 2%
The percentage by weight sum of said components is absolutely.
The preparation method of the anti-carious tooth chewing gum of the present invention may further comprise the steps:
One, the preparation of anti-carious tooth Yolk antibody: with embodiment one;
Two, with matrix melting under 60 ℃ temperature of 13.5%, adding 50% xylitol, 10% Arabic gum, 20% sweet mellow wine, 0.5% citric acid, 2% lecithin and 2% flavoring essence fully stirs, when temperature reaches 40 ℃, add 2% anti-carious tooth Yolk antibody again, after stirring, go into mould, compressing tablet, section, moulding, packing gets final product (percentage wherein is weight percentage).
Remainder is with embodiment one.
Embodiment three:
The anti-carious tooth chewing gum of the present invention is made by following materials of weight proportions:
Anti-carious tooth Yolk antibody 1%
Matrix 45.4%
Xylitol 30%
Arabic gum 6%
Sweet mellow wine 15%
Citric acid 0.3%
Lecithin 0.8%
Flavoring essence 1.5%
The percentage by weight sum of said components is absolutely.
The preparation method of the anti-carious tooth chewing gum of the present invention may further comprise the steps:
One, the preparation of anti-carious tooth Yolk antibody: with embodiment one;
Two, with matrix melting under 60 ℃ temperature of 45.4%, adding 30% xylitol, 6% Arabic gum, 15% sweet mellow wine, 0.3% citric acid, 0.8% lecithin and 1.5% flavoring essence fully stirs, when temperature reaches 40 ℃, add 1% anti-carious tooth Yolk antibody again, after stirring, go into mould, compressing tablet, section, moulding, packing gets final product (percentage wherein is weight percentage).
Remainder is with embodiment one.