CN111227090A - 抗龋齿咀嚼片及其制备方法 - Google Patents
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Abstract
本发明公开了抗龋齿咀嚼片及其制备方法,所述的抗龋齿咀嚼片由下述重量份的原料制备而成:明胶粉100份、去离子水100份、甘油30份、还原糖3份、食用色素0.1份、抗龋齿卵黄抗体5‑10份。所述的制备方法包括步骤S1:制备抗龋齿卵黄抗体;S2、制备咀嚼糖果;S3、制备咀嚼片;S4、检验、包装、入库。本发明的抗龋齿咀嚼片抗龋齿效果好、制造和使用方便、无毒副作用;制备的卵黄抗体可直接识别细菌表面抗原,对完整的、有活性的细菌有靶向抑制和杀灭作用。
Description
技术领域
本发明涉及抗龋齿咀嚼片及其制备方法。
背景技术
目前,随着人民生活水平的普遍提高及饮食中糖含量及其摄入量的增加,使得龋病得以流行,尤其是儿童乳牙患龋齿更为明显,随着人口寿命的延长,老年人根面龋亦在增长,这些都严重影响人们的口腔和身体健康,从而严重影响人们的生活质量。目前世界卫生组织(WHO)已经将龋齿列为是仅次于心血管疾病、癌症的第三大非传染性疾病,并且作为身体健康与否的10项指标之一。
龋齿是一种常见病、多发病,其发病率高,分布广,对人体危害大。经过科学家们不懈的努力,现在普遍认为细菌是龋齿发生的主要原因,其中变形链球菌是主要致病菌,其形成的菌斑粘附于牙齿表面,并在牙齿表面繁殖生长、产酸,腐蚀牙齿,最终形成龋齿。
目前市场上应用的防龋产品大致分为以下几类:一为含氟制剂,其防龋原理为促进牙齿钙化,减少酸性腐蚀。此为间接作用,疗效有限;二是抗菌药物的应用,如含洗必汰、甲硝唑等的漱口水,因其有明显的副作用,应用受到限制;三是由植物提取物制成的防龋产品,如中药提取物制成的含漱液、口胶片等,其对龋齿防治有一定作用,但其均为药物,需在医生指导下使用,不宜长期使用;四是目前市场上被广泛接受的利用代糖生产的用于防龋护齿类的产品,如木糖醇等,它却没有从根本上解决防龋护齿的实质性问题。中国专利公开号CN1307061A于2001年8月8日公开了一种名称为“抗龋齿菌IgY的制备及防龋齿组合物”的专利。该专利抗龋齿菌IgY的制备分以下几步:制备抗原,制备抗体,配置药物。其抗体的生产过程是将细菌的细胞直接进行裂解后作为抗原,导致制备出来的抗体主要是针对细菌内的抗原成份起作用,当面对完整的活性细菌时,由于抗体无法直接进入细菌体内起作用,而其与活体细菌表面的结合能力又弱,因此其灭菌的效果很差。故现有产品存在效价低、使用不方便、效果不明显的缺点。
发明内容
针对上述存在的技术不足,本发明所要解决的技术问题是克服现有技术不足,提供一种抗龋齿咀嚼片,该抗龋齿咀嚼片效果好、制造和使用方便、无毒副作用。本发明所要解决的另一技术问题是提供一种抗龋齿咀嚼片的制备方法,该抗龋齿咀嚼片的制造方法针对引起龋齿的主要病原菌-变形链球菌进行深入分析研究而制成卵黄抗体,制备的卵黄抗体可直接识别细菌表面抗原,对完整的、有活性的细菌有靶向抑制和杀灭作用。
为解决上述技术问题,本发明采用如下技术方案:
本发明提供抗龋齿咀嚼片,由下述重量份的原料制备而成:明胶粉100份、去离子水100份、甘油30份、还原糖3份、食用色素0.1份、抗龋齿卵黄抗体5-10份。
本发明还提供抗龋齿咀嚼片的制备方法,包括步骤S1:制备抗龋齿卵黄抗体,还包括以下步骤:
S2、制备咀嚼糖果:将配方量的去离子水、明胶粉、甘油、还原糖、食用色素均匀混合后进行溶胶,得到溶胶混合物;再将制得的抗龋齿卵黄抗体灌注与溶胶混合物中,得到咀嚼糖果;
S3、制备咀嚼片:对步骤S2得到的咀嚼糖果进形压制成型、干燥吹洗后得到咀嚼片成品;
S4、检验、包装、入库。
优选地,制备抗龋齿卵黄抗体步骤S1具体包括:
S1.1、制备抗原:取血清型为C型和D型的变形链球菌,分别以固体TYCSB琼脂斜面传代,在37℃厌氧条件下培养48小时,经PCR鉴定得到所述变形链球菌的纯种后,在液体TYCSB培养基中扩大培养48~72小时收集菌体,所述菌体用0.5%福尔马林25℃灭活24小时后,冻成干粉后作为抗原;
S1.2、制备抗体:
S1.2.1、佐剂的选用和产卵母鸡的免疫及取卵:初次免疫选用弗氏完全佐剂与所述抗原按照重量比1∶1混合乳化后作为初次免疫抗原,加强免疫均选用弗氏不完全佐剂与所述抗原按照重量比为1∶1混合乳化后作为加强免疫抗原;选用健康的白种来行母鸡隔离饲养并进行鸡肌肉多点注射所述初次免疫抗原,此为初次免疫;隔2周后开始加强免疫,对初次免疫的母鸡隔离饲养并进行鸡肌肉多点注射所述加强免疫抗原,以后每隔一周加强免疫一次,其初次免疫和加强免疫共三次,从初次免疫后算起,第三十天开始收集鸡卵,4℃条件下储存备用;
S1.2.2、卵黄抗体的提取:将鸡卵中的卵黄抗体提取,冷冻干燥,即得抗体。
更优选地,所述的卵黄抗体的提取采用水稀释法:从收集的免疫的鸡卵中分离得到蛋黄,用所述去离子水稀释,调节pH值在4.0-5.0范围内,在4℃条件下搅拌4小时,用离心机加工后弃去沉淀,将上清浓缩、冷冻干燥,即可。
更优选地,所述的卵黄抗体的提取采用PEG沉淀法:从收集的免疫的鸡卵中分离得到蛋黄,加入体积为所述蛋黄3倍体积的浓度为4%的PEG进行稀释,充分搅拌混匀后在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,取上清,然后向所述上清内逐滴滴加1/6所述上清体积的浓度为40%的PEG,充分搅拌混匀后在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,收集沉淀,冷冻干燥,即可。
更优选地,所述的卵黄抗体的提取采用膜过滤法:从收集的免疫的鸡卵中分离得到蛋黄,用所述去离子水稀释,在温度为4℃条件下静置12小时后,用分子量为200KD和150KD的膜分别过滤,收集150KD-200KD的蛋白物质,冷冻干燥,即可。
更优选地,所述的卵黄抗体的提取采用硫酸铵沉淀法:从收集的免疫的鸡卵中分离得到蛋黄,重复提取3次:第一次,取所述蛋黄等体积的生理盐水稀释并混合均匀后,缓慢滴加2倍蛋黄体积且浓度为50%的饱和硫酸铵,在温度为4℃条件下作用3小时,并在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,弃上清,留沉淀;第二次,用所述生理盐水溶解沉淀,然后加入2/3所述生理盐水体积且浓度为40%的饱和硫酸铵,在温度为4℃条件下作用3小时,并在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,弃上清,留沉淀;第三次,用所述生理盐水溶解沉淀,然后加入1/2所述生理盐水体积且浓度为33%的饱和硫酸铵,在温度为4℃条件作用3小时,将沉淀直接冷冻干燥,即可。
本发明的有益效果在于:抗龋齿效果好、制造和使用方便、无毒副作用;制备的卵黄抗体可直接识别细菌表面抗原,对完整的、有活性的细菌有靶向抑制和杀灭作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例提供的抗龋齿咀嚼片及其制备方法的流程图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
抗龋齿咀嚼片,由下述重量份的原料制备而成:明胶粉100g、去离子水100g、甘油30g、还原糖3g、食用色素0.1g、抗龋齿卵黄抗体5g。
如图1所示,本发明还提供抗龋齿咀嚼片的制备方法,包括步骤S1:制备抗龋齿卵黄抗体,还包括以下步骤:
S2、制备咀嚼糖果:将配方量的去离子水、明胶粉、甘油、还原糖、食用色素均匀混合后进行溶胶,得到溶胶混合物;再将制得的抗龋齿卵黄抗体灌注与溶胶混合物中,得到咀嚼糖果;
S3、制备咀嚼片:对步骤S2得到的咀嚼糖果进形压制成型、干燥吹洗后得到咀嚼片成品;
S4、检验、包装、入库。
进一步的,制备抗龋齿卵黄抗体步骤S1具体包括:
S1.1、制备抗原:取血清型为C型和D型的变形链球菌,分别以固体TYCSB琼脂斜面传代,在37℃厌氧条件下培养48小时,经PCR鉴定得到所述变形链球菌的纯种后,在液体TYCSB培养基中扩大培养48~72小时收集菌体,所述菌体用0.5%福尔马林25℃灭活24小时后,冻成干粉后作为抗原;
S1.2、制备抗体:
S1.2.1、佐剂的选用和产卵母鸡的免疫及取卵:初次免疫选用弗氏完全佐剂与所述抗原按照重量比1∶1混合乳化后作为初次免疫抗原,加强免疫均选用弗氏不完全佐剂与所述抗原按照重量比为1∶1混合乳化后作为加强免疫抗原;选用健康的白种来行母鸡隔离饲养并进行鸡肌肉多点注射所述初次免疫抗原,此为初次免疫;隔2周后开始加强免疫,对初次免疫的母鸡隔离饲养并进行鸡肌肉多点注射所述加强免疫抗原,以后每隔一周加强免疫一次,其初次免疫和加强免疫共三次,从初次免疫后算起,第三十天开始收集鸡卵,4℃条件下储存备用;
S1.2.2、卵黄抗体的提取:将鸡卵中的卵黄抗体提取,冷冻干燥,即得抗体;
更进一步的,所述的卵黄抗体的提取可采用以下四种方法之一:
a、水稀释法:从收集的免疫的鸡卵中分离得到蛋黄,用所述去离子水稀释,调节PH值在4.0-5.0范围内,在4℃条件下搅拌4小时,用离心机加工后弃去沉淀,将上清浓缩、冷冻干燥,即可。
b、PEG沉淀法:从收集的免疫的鸡卵中分离得到蛋黄,加入体积为所述蛋黄3倍体积的浓度为4%的PEG进行稀释,充分搅拌混匀后在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,取上清,然后向所述上清内逐滴滴加1/6所述上清体积的浓度为40%的PEG,充分搅拌混匀后在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,收集沉淀,冷冻干燥,即可。
c、膜过滤法:从收集的免疫的鸡卵中分离得到蛋黄,用所述去离子水稀释,在温度为4℃条件下静置12小时后,用分子量为200KD和150KD的膜分别过滤,收集150KD-200KD的蛋白物质,冷冻干燥,即可。
d、硫酸铵沉淀法:从收集的免疫的鸡卵中分离得到蛋黄,重复提取3次:第一次,取所述蛋黄等体积的生理盐水稀释并混合均匀后,缓慢滴加2倍蛋黄体积且浓度为50%的饱和硫酸铵,在温度为4℃条件下作用3小时,并在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,弃上清,留沉淀;第二次,用所述生理盐水溶解沉淀,然后加入2/3所述生理盐水体积且浓度为40%的饱和硫酸铵,在温度为4℃条件下作用3小时,并在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,弃上清,留沉淀;第三次,用所述生理盐水溶解沉淀,然后加入1/2所述生理盐水体积且浓度为33%的饱和硫酸铵,在温度为4℃条件作用3小时,将沉淀直接冷冻干燥,即可。
实施例2
抗龋齿咀嚼片,由下述重量份的原料制备而成:明胶粉100g、去离子水100g、甘油30g、还原糖3g、食用色素0.1g、抗龋齿卵黄抗体8g。
其制备方法同实施例1。
实施例3
抗龋齿咀嚼片,由下述重量份的原料制备而成:明胶粉100g、去离子水100g、甘油30g、还原糖3g、食用色素0.1g、抗龋齿卵黄抗体10g。
其制备方法同实施例1。
本发明所采用的技术机理是:首先制备一种抗龋齿的卵黄抗体,它按照非变性聚丙烯凝胶电泳进行检测,经过考马斯亮蓝R250染色后,图谱检测可以在分子量170~190KD处呈现单一条带,Western Bloting检测可以在分子量170~190KD处发现一条单一的特异性条带;或按照变性聚丙烯凝胶电泳进行检测,经过考马斯亮蓝R250染色后,图谱呈现两条条带,Western Bloting检测可以在分子量约65KD和25KD处发现两条特异性条带,采用紫外分光光度法测定蛋白质浓度为0.1~20mg/ml;经过酶联免疫吸附实验测定其效价为1∶50-1∶100000。
抗龋齿效果统计:
1.体外抑菌试验
试验方法:
分别取实施例中的咀嚼片粉碎后加10ml无菌水配成混合溶液。另外取市售无糖木糖醇1g粉碎后加10ml无菌水配成混合溶液和市售漱口水,作为阴性对照。
抑菌片的制备:取无菌并干燥的滤纸片,每片滴加20μl,然后将滤纸片平放于清洁的无菌平面皿内,开盖置温箱(37℃)中烤干,或置室温下自然干燥后备用。阴性对照组抑菌片的制备:取无菌干燥滤纸片,每片滴加无菌蒸馏水20μl,干燥后备用。
接种变形链球菌:用无菌棉拭子蘸取浓度为5×105~5×106cfu/ml变形链球菌菌悬液,在营养琼脂培养基平板表面均匀涂抹3次,每涂抹一次,平板转动60°,最后将棉拭子绕平板边缘涂抹一周,盖好平皿,置室温干燥5分钟。
抑菌片贴放:每次贴放一个染菌平板,每个平板贴放抑菌片和1片阴阳对照抑菌片。用无菌镊子取样片贴放于平板表面,各样片中心之间相距25mm以上,与平板的周缘相距15mm以上。贴放好后,用无菌镊子轻压样片,使其紧贴于平板表面,盖好平皿,置于37℃温箱内培养16~18h,用游标卡尺测量抑菌环的直径(包括贴片)并记录,实验重复三次。
2、实验结果
表1体外抑菌实验
抑菌圈大小(直径mm) | |
实施例 | 18.2 |
对照-木糖醇口香糖 | 9.5 |
对照-漱口水 | 10.6 |
2.志愿者实验
实施例和对比例药剂抗龋齿的效果:
志愿者选择标准:随机选择患者100例,选择的标准是:符合西医诊断标准和中医辩证标准、年龄在18~55岁之间,其中,女性50例,男性50例。随机将上述病例分成实施例组和对比例组,各50人,男女各半,实施例和对比例药剂抗龋齿的效果见表2。
用法用量:饭后立即咀嚼一粒,饭后3~4小时再次咀嚼1粒,连续使用7天。
疗效判定标准:有效:龋洞不再扩大,疼痛减轻;治愈:龋洞不再扩大,疼痛消失。
表2:实施例和对比例药剂抗龋齿的效果
类目 | 患者数目 | 有效(%) | 治愈(%) |
实施例 | 826 | 86.73 | 72.64 |
对比例 | 858 | 45.24 | 39.84 |
从表2可以看出,实施例为抗龋齿咀嚼片,实施例的有效率为86.73%,治愈率为72.64%,作用效果快,说明实施例抗龋齿咀嚼片具有明显的抗龋齿效果。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (7)
1.抗龋齿咀嚼片,其特征在于,由下述重量份的原料制备而成:明胶粉100份、去离子水100份、甘油30份、还原糖3份、食用色素0.1份、抗龋齿卵黄抗体5-10份。
2.如权利要求1所述的抗龋齿咀嚼片的制备方法,包括步骤S1:制备抗龋齿卵黄抗体,其特征在于,还包括以下步骤:
S2、制备咀嚼糖果:将配方量的去离子水、明胶粉、甘油、还原糖、食用色素均匀混合后进行溶胶,得到溶胶混合物;再将制得的抗龋齿卵黄抗体灌注与溶胶混合物中,得到咀嚼糖果;
S3、制备咀嚼片:对步骤S2得到的咀嚼糖果进形压制成型、干燥吹洗后得到咀嚼片成品;
S4、检验、包装、入库。
3.如权利要求2所述的抗龋齿咀嚼片的制备方法,其特征在于,制备抗龋齿卵黄抗体步骤S1具体包括:
S1.1、制备抗原:取血清型为C型和D型的变形链球菌,分别以固体TYCSB琼脂斜面传代,在37℃厌氧条件下培养48小时,经PCR鉴定得到所述变形链球菌的纯种后,在液体TYCSB培养基中扩大培养48~72小时收集菌体,所述菌体用0.5%福尔马林25℃灭活24小时后,冻成干粉后作为抗原;
S1.2、制备抗体:
S1.2.1、佐剂的选用和产卵母鸡的免疫及取卵:初次免疫选用弗氏完全佐剂与所述抗原按照重量比1∶1混合乳化后作为初次免疫抗原,加强免疫均选用弗氏不完全佐剂与所述抗原按照重量比为1∶1混合乳化后作为加强免疫抗原;选用健康的白种来行母鸡隔离饲养并进行鸡肌肉多点注射所述初次免疫抗原,此为初次免疫;隔2周后开始加强免疫,对初次免疫的母鸡隔离饲养并进行鸡肌肉多点注射所述加强免疫抗原,以后每隔一周加强免疫一次,其初次免疫和加强免疫共三次,从初次免疫后算起,第三十天开始收集鸡卵,4℃条件下储存备用;
S1.2.2、卵黄抗体的提取:将鸡卵中的卵黄抗体提取,冷冻干燥,即得抗体。
4.如权利要求3所述的抗龋齿咀嚼片的制备方法,其特征在于,所述的卵黄抗体的提取采用水稀释法:从收集的免疫的鸡卵中分离得到蛋黄,用所述去离子水稀释,调节pH值在4.0-5.0范围内,在4℃条件下搅拌4小时,用离心机加工后弃去沉淀,将上清浓缩、冷冻干燥,即可。
5.如权利要求3所述的抗龋齿咀嚼片的制备方法,其特征在于,所述的卵黄抗体的提取采用PEG沉淀法:从收集的免疫的鸡卵中分离得到蛋黄,加入体积为所述蛋黄3倍体积的浓度为4%的PEG进行稀释,充分搅拌混匀后在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,取上清,然后向所述上清内逐滴滴加1/6所述上清体积的浓度为40%的PEG,充分搅拌混匀后在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,收集沉淀,冷冻干燥,即可。
6.如权利要求3所述的抗龋齿咀嚼片的制备方法,其特征在于,所述的卵黄抗体的提取采用膜过滤法:从收集的免疫的鸡卵中分离得到蛋黄,用所述去离子水稀释,在温度为4℃条件下静置12小时后,用分子量为200KD和150KD的膜分别过滤,收集150KD-200KD的蛋白物质,冷冻干燥,即可。
7.如权利要求3所述的抗龋齿咀嚼片的制备方法,其特征在于,所述的卵黄抗体的提取采用硫酸铵沉淀法:从收集的免疫的鸡卵中分离得到蛋黄,重复提取3次:第一次,取所述蛋黄等体积的生理盐水稀释并混合均匀后,缓慢滴加2倍蛋黄体积且浓度为50%的饱和硫酸铵,在温度为4℃条件下作用3小时,并在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,弃上清,留沉淀;第二次,用所述生理盐水溶解沉淀,然后加入2/3所述生理盐水体积且浓度为40%的饱和硫酸铵,在温度为4℃条件下作用3小时,并在温度为4℃、离心机转数为每分钟1300转条件下离心10分钟,弃上清,留沉淀;第三次,用所述生理盐水溶解沉淀,然后加入1/2所述生理盐水体积且浓度为33%的饱和硫酸铵,在温度为4℃条件作用3小时,将沉淀直接冷冻干燥,即可。
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