CN109730006A - 提高动物黑色素含量的方法 - Google Patents
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Abstract
本发明涉及一种提高动物黑色素的方法,属于生物技术领域。本发明的提高动物黑色素含量的方法包括:将动物用6‑苄基腺嘌呤处理,并光照培养,所述动物含有黑色素细胞。本发明通过活体生物实验提供的诱导黑色素生成的方法,能提高体内酪氨酸酶活性,有效促进黑色素生成,简单易操作,成本低,无毒副效应。
Description
技术领域
本发明涉及一种提高动物黑色素的方法,属于生物技术领域。
背景技术
黑色素是脊椎动物皮肤、毛发、羽毛和皮毛的主要色素,用来保护皮肤免受紫外线照射造成损伤。黑色素由黑素细胞生成。紫外线照射会激活黑素细胞中的酪氨酸酶。酪氨酸在酪氨酸酶作用下,相继生成多巴胺、多巴醌等物质,进而转化成黑色素,最后聚集成色素颗粒,均匀地分布在表皮基底层中。大量研究表明,乌骨鸡黑色素具有抗诱变、防癌、抗氧化、延缓衰老、增强机体免疫力等功能。作为紫外线吸收剂、抗氧化剂和新型的天然的药物载体,黑色素可以用来治疗某些与黑色素缺乏有关的神经系统疾病,如着色性干皮病、帕金森氏症、老年性痴呆症、亨廷氏舞蹈病等。因此,它在医疗、食品和化妆品工业中的应用潜力巨大。
传统研究黑色素的模型是黑色素瘤细胞。但细胞、组织、个体在代谢上有一定差异。斑马鱼是热带脊椎动物,具有与人类相似的黑色素细胞和黑色体,并且与人类基因有87%的相似度,是对黑色素研究很好的生物模型。目前斑马鱼胚胎已被用于快速筛选美白成分。
6-苄基腺嘌呤,又称6-苄氨基嘌呤,英文名为6-Benzylaminopurine或N6-Benzyladenine,简称6-BA或6-BAP,CAS登记号为1214-39-7,是一种能促进细胞分裂的人工合成的植物生长调节剂,被广泛应用在农业生产的各环节,用以提高农作物的产量,增强抗逆性,改善外观,缩短周期等。体外研究表明,6-苄基腺嘌呤能增强心肌功能,抗氧化。
发明内容
本发明要解决的第一个技术问题是提供一种提高动物黑色素含量的方法。
为了解决上述第一个技术问题,本发明的提高动物黑色素含量的方法包括:将动物用6- 苄基腺嘌呤处理,并光照培养,所述动物含有黑色素细胞。
优选的,所述处理为口服。
优选的,所述动物为斑马鱼或乌骨鸡。
优选的,所述动物为斑马鱼的幼鱼或成鱼,所述方法包括:
a.将6-苄基腺嘌呤用有机溶剂溶解,再配置6-苄基腺嘌呤水溶液,所述水溶液中6-苄基腺嘌呤的浓度c为0<c≤25mg/L,优选c为12.5≤c≤25mg/L;
b.将斑马鱼放入a步骤配好的6-苄基腺嘌呤水溶液中,22.0~29.0℃,光暗比14~16:10~ 8h处理47~49h。
优选的,所述动物为乌骨鸡,所述方法包括:
a.将6-苄基腺嘌呤用有机溶剂溶解,再添加到鸡粮中,所述6-苄基腺嘌呤的浓度c为0 <c≤0.2mg/kg,优选为0.10≤c≤0.2mg/kg;
b.将乌骨鸡用a步骤配置的鸡粮培养4周,培养湿度55%~65%,起始培养温度34~35℃,每周温度下降至少2℃,最后保持在22~24℃;0~2周每日光照24h,以后每周缩短2h。
本发明要解决的第二个技术问题是提供6-苄基腺嘌呤在制备提高动物黑色素的食品中的应用。
为解决上述第二个技术问题,将6-苄基腺嘌呤用于制备提高动物黑色素的食品。
进一步地,所述食品为动物饲料或微生物发酵的培养基;优选为鱼饲料或乌骨鸡饲料。
本发明通过活体生物实验提供的诱导黑色素生成的方法,能提高体内酪氨酸酶活性,有效促进黑色素生成,简单易操作,成本低,无毒副效应,可添加到动物饲料中增加动物体内黑色素的含量,改善动物体色。
附图说明
图1斑马鱼经6-苄基腺嘌呤处理48h后的色素变化;其中A是斑马鱼胚胎,B是斑马鱼幼鱼,C是斑马鱼成鱼,图中6-BA指6-苄基腺嘌呤;
图2不同浓度6-苄基腺嘌呤处理斑马鱼胚胎、幼鱼和成鱼48h的酪氨酸酶活性的检测结果;其中,胚胎处理时间从受精后6h开始,幼鱼处理时间从受精后60天开始,成鱼处理时间从受精后180天开始;重复三次;one-way ANOVA分析,与对照组比较显著性差异用星号表示,*p<0.05;
图3不同浓度6-苄基腺嘌呤处理斑马鱼胚胎、幼鱼和成鱼48h的黑色素生成量的检测结果;其中,胚胎处理时间从受精后6h开始,幼鱼处理时间从受精后60天开始,成鱼处理时间从受精后180天开始;重复三次;one-way ANOVA分析,与对照组比较显著性差异用星号表示,*p<0.05;
具体实施方式
为了解决上述第一个技术问题,本发明的提高动物黑色素含量的方法包括:将动物用6- 苄基腺嘌呤处理,并光照培养,所述动物含有黑色素细胞。
优选的,所述处理为口服。
优选的,所述动物为斑马鱼或乌骨鸡。
优选的,所述动物为斑马鱼的幼鱼或成鱼,所述方法包括:
a.将6-苄基腺嘌呤用有机溶剂溶解,再配置6-苄基腺嘌呤水溶液,所述水溶液中6-苄基腺嘌呤的浓度c为0<c≤25mg/L,优选c为12.5≤c≤25mg/L;
b.将斑马鱼放入a步骤配好的6-苄基腺嘌呤水溶液中,22.0~29.0℃,光暗比14~16:10~8h处理47~49h。
优选的,所述动物为乌骨鸡,所述方法包括:
a.将6-苄基腺嘌呤用有机溶剂溶解,再添加到鸡粮中,所述6-苄基腺嘌呤的浓度c为0 <c≤0.2mg/kg,优选为0.10≤c≤0.2mg/kg;
b.将乌骨鸡用a步骤配置的鸡粮培养4周,培养湿度55%~65%,起始培养温度34~35℃,每周温度下降至少2℃,最后保持在22~24℃;0~2周每日光照24h,以后每周缩短2h。
本发明要解决的第二个技术问题是提供6-苄基腺嘌呤在制备提高动物黑色素的食品中的应用。
为解决上述第二个技术问题,将6-苄基腺嘌呤用于制备提高动物黑色素的食品。
进一步地,所述食品为动物饲料或微生物发酵的培养基;优选为鱼饲料或乌骨鸡饲料。
下面结合实施例对本发明的具体实施方式做进一步的描述,并不因此将本发明限制在所述的实施例范围之中。
实施例1
斑马鱼动物实验
实验方法
(一)饲养斑马鱼及收集鱼卵
斑马鱼养殖系统采用上海海圣生物设备有限公司。平时野生型斑马鱼成鱼雌雄混养,在进行实验前1~2周,将雌雄鱼分开。养殖条件为:28.5±1℃,pH6.8~7.2,电导率500~550μs,曝气灭菌循环水,光暗比14:10h,每天喂人工小薄片和新鲜丰年虾两次。实验前一天,选雌雄比1:2用隔板隔开放在交配盒黑暗过夜,第二天清晨抽走隔板,光照诱导受精。收集鱼卵,用Holfretor培养液(0.05g/L KCl,3.5g/L NaCl,0.1g/L CaCl2和0.025g/LNaHCO3)洗净,6hpf (受精后小时)时体视显微镜(SZX10,Olympus,Japan)下挑选发育正常受精卵备用。
(二)6-苄基腺嘌呤处理斑马鱼胚胎
将6-苄基腺嘌呤先用微量DMSO溶解(DMSO终浓度不超过0.1%),再用超纯水定容制成1g/L母液。分别取一定体积母液,用Holfretor培养液稀释定容制成0,12.5mg/L,25mg/L 共3组处理液。每组挑选正常受精卵30枚置于6孔板,分别暴露于5mL的6-苄基腺嘌呤处理液,28±0.5℃恒温培养48h,分别测定斑马鱼胚胎黑色素和酪氨酸酶活性。重复3次。处理期间每天更换新鲜处理液。
(三)6-苄基腺嘌呤处理斑马鱼幼鱼
将6-苄基腺嘌呤先用微量DMSO溶解(DMSO终浓度不超过0.1%),再用超纯水定容制成1g/L母液。分别取一定体积母液,用曝气灭菌循环养殖水稀释定容制成0,12.5mg/L,25mg/L 共3组处理液。挑选发育正常健康的60天龄斑马鱼幼鱼,平均体重为0.1±0.03g,平均体长为2.4±0.2cm。每组8条幼鱼,放入2L烧杯中在26.0±1℃,光暗比14:10h恒温光照培养箱中处理48h,分别测定幼鱼黑色素和酪氨酸酶活性。重复3次。处理期间每天更换新鲜处理液。
(四)6-苄基腺嘌呤处理斑马鱼成鱼
将6-苄基腺嘌呤先用微量DMSO溶解(DMSO终浓度不超过0.1%),再用超纯水定容制成1g/L母液。分别取一定体积母液,用曝气灭菌循环养殖水稀释定容制成0,12.5mg/L,25mg/L 共3组处理液。挑选发育正常健康的180天龄斑马鱼成鱼,平均体重为1.1±0.09g,平均体长为3.8±0.2cm。每组4条成鱼,放入3L容器中在26.0±1℃,光暗比14:10h恒温光照培养箱中处理48h,分别测定成鱼黑色素和酪氨酸酶活性。重复3次。处理期间每天更换新鲜处理液。
(五)测定酪氨酸酶活性
收集处理48h的斑马鱼胚胎、幼鱼或成鱼,其中,每一处理50条仔鱼,2条幼鱼或2条成鱼,先用Holfreter水洗两遍,再用PBS洗两遍。分别加入200μL、1000μL和2000μL PBS 缓冲液,在冰浴条件下,匀浆。12 000rpm、4℃离心5min,收集上清液。以牛血清白蛋白为标准蛋白,用考马斯亮蓝法测定上清液蛋白质含量。以每孔含有100μg蛋白质的上清液转移到96孔板,每孔再加入100μL 1mM L-多巴胺溶液和100μL PBS溶液,37℃水浴避光反应1h,490nm测吸光值A490。对照组酪氨酸酶活性设定为100%,其它组酪氨酸酶活性以对照的相对百分率来表示。
(六)测定黑色素含量
收集处理48h的斑马鱼胚胎、幼鱼或成鱼,其中,每一处理50条仔鱼,2条幼鱼或2条成鱼,先用Holfreter水洗两遍,再用PBS洗两遍。加入1N NaOH(体积依照蛋白质含量进行调整),匀浆,100℃避光加热1h。10 000rpm、4℃离心5min,收集上清液,405nm测吸光值A405。对照组黑色素含量设定为100%,其它处理组的黑色素含量以对照的相对百分率来表示。
实验结果
如图1A所示,斑马鱼胚胎经12.5mg/L 6-苄基腺嘌呤处理后,体色与对照比较没有差异, 25mg/L 6-苄基腺嘌呤处理的斑马鱼胚胎体色较对照浅;如图1B和1C所示,斑马鱼幼鱼和成鱼经6-苄基腺嘌呤处理后体色显著加深。
如图2所示,与对照相比,12.5mg/L 6-苄基腺嘌呤对斑马鱼胚胎酪氨酸酶活性没有影响,25mg/L 6-苄基腺嘌呤降低斑马鱼胚胎酪氨酸酶活性,而12.5mg/L和25mg/L 6-苄基腺嘌呤能提高斑马鱼幼鱼和成鱼酪氨酸酶活性。
图3所示,与对照相比,12.5mg/L 6-苄基腺嘌呤对斑马鱼胚胎黑色素含量没有影响, 25mg/L 6-苄基腺嘌呤降低斑马鱼胚胎黑色素含量,而12.5mg/L和25mg/L 6-苄基腺嘌呤能提高斑马鱼幼鱼和成鱼黑色素含量,与酪氨酸酶活性一致。
因此,25mg/L及以下浓度6-苄基腺嘌呤可以提高斑马鱼幼鱼和成鱼黑色素含量,但不能提高斑马鱼胚胎黑色素含量并且随浓度增大有抑制作用。实验中,25mg/L 6-苄基腺嘌呤对斑马鱼幼鱼和成鱼没有毒害。
实施例2
乌骨鸡动物实验
实验方法和结果
(一)乌骨鸡饲养管理
参考2008年农业部颁布的我国地方肉用黄鸡饲养标准,基础日粮配方和营养水平见表1。 6-苄基腺嘌呤先用少量乙醇溶解,再添加到日粮中。刚出生的仔鸡在对照组的基础上分别饲喂添加0、0.1和0.2mg/kg 6-苄基腺嘌呤。每一处理重复三次,每一重复15只鸡,各组间初始体重相似。各组饲养条件相同,第一周温度34~35℃,每周下降至少2℃,最后保持在22~ 24℃,湿度55%~65%。0~2周每日光照24h,以后每周缩短2h,定期清理打扫鸡舍卫生并消毒。按照正常免疫程序进行免疫。4周后测定酪氨酸酶活性和黑色素含量。
表1基础日粮组成及营养水平(风干基础)
注:预混料含维生素、微量元素、赖氨酸、蛋氨酸和苏氨酸。
(二)样品的采集与制备
4周龄小鸡从每个重复中选取2只健康、接近平均体重的乌骨鸡,空腹称重后放血宰杀,收集血液,室温静置30min,3500rpm离心3min分离血清,-20℃保存备用。打开腹腔,分别取胸肌、肝脏、肾脏和皮肤,用冰冷的生理盐水漂洗,去除血液,用滤纸拭干,-20℃冰箱保存备用。
(三)测定酪氨酸酶活性
取解冻后组织0.5g加入1mL PBS冰浴中匀浆2min,用1mL冰冷的PBS液清洗匀浆器,合并匀浆液和清洗液,4℃,12 000rpm离心15min,取上清液待用。以牛血清白蛋白为标准蛋白,用考马斯亮蓝法测定上清液蛋白质含量。以每孔含有100μg蛋白质的上清液转移到96孔板,每孔再加入100μL 1mM L-多巴胺溶液和100μL PBS溶液,37℃水浴避光反应1h,490nm测吸光值A490。对照组酪氨酸酶活性设定为100%,其它组酪氨酸酶活性以对照的相对百分率来表示。结果见表2。
表2 6-苄基腺嘌呤添加水平对乌骨鸡血清和组织酪氨酸酶活性的影响
注:one-way ANOVA分析,表中同列数值不同字母表示显著差异(P<0.05)
从表2可见,日粮添加6-苄基腺嘌呤显著影响乌骨鸡血清和组织酪氨酸酶活性(P<0.05),日粮添加6-苄基腺嘌呤均能不同程度地提高乌骨鸡血清、皮肤、胸肌、肝脏和肾脏酪氨酸酶的活性。
(四)测定黑色素含量
准确称取50mg组织样品,加入1mL 2N NaOH溶液,100℃避光加热1h。10 000rpm、4℃离心5min,收集上清液,405nm测吸光值A405。对照组黑色素含量设定为100%,其它处理组的黑色素含量以对照的相对百分率来表示。结果见表3。
从表3可见,日粮添加6-苄基腺嘌呤显著影响乌骨鸡组织黑色素含量(P<0.05),日粮添加6- 苄基腺嘌呤均0.2mg/kg以下能不同程度地提高乌骨鸡皮肤、胸肌、肝脏和肾脏黑色素含量,其中肝脏的含量最高,其次是肾脏和胸肌,皮肤含量最低。
表3 6-苄基腺嘌呤添加水平对乌骨鸡组织黑色素含量的影响
注:one-way ANOVA分析,表中同列数值不同字母表示显著差异(P<0.05)。
Claims (10)
1.提高动物黑色素含量的方法,其特征在于,所述提高动物黑色素含量的方法包括:将动物用6-苄基腺嘌呤处理,并光照培养,所述动物含有黑色素细胞。
2.根据权利要求1所述提高动物黑色素含量的方法,其特征在于,所述处理为口服。
3.根据权利要求1或2任一项所述提高动物黑色素含量的方法,其特征在于,所述动物为斑马鱼或乌骨鸡。
4.根据权利要求3所述提高动物黑色素含量的方法,其特征在于,所述动物为斑马鱼的幼鱼或成鱼,所述方法包括:
a.将6-苄基腺嘌呤用有机溶剂溶解,再配置6-苄基腺嘌呤水溶液,所述水溶液中6-苄基腺嘌呤的浓度c为0<c≤25mg/L;
b.将斑马鱼放入a步骤配好的6-苄基腺嘌呤水溶液中,22.0~29.0℃,光暗比14~16:10~8h处理47~49h。
5.根据权利要求4所述提高动物黑色素含量的方法,其特征在于,所述水溶液中6-苄基腺嘌呤的浓度c为10≤c≤25mg/L。
6.根据权利要求3所述提高动物黑色素含量的方法,其特征在于,所述动物为乌骨鸡,所述方法包括:
a.将6-苄基腺嘌呤用有机溶剂溶解,再添加到鸡粮中,所述6-苄基腺嘌呤的浓度c为0<c≤0.2mg/kg;
b.将乌骨鸡用a步骤配置的鸡粮培养4周,培养湿度55%~65%,起始培养温度34~35℃,每周温度下降至少2℃,最后保持在22~24℃;0~2周每日光照24h,以后每周缩短2h。
7.根据权利要求6所述提高动物黑色素含量的方法,其特征在于,所述6-苄基腺嘌呤的浓度c为0.10≤c≤0.2mg/kg。
8.6-苄基腺嘌呤在制备提高动物黑色素的食品中的应用。
9.根据权利要求8所述的6-苄基腺嘌呤在制备提高动物黑色素的食品中的应用,其特征在于,所述食品为动物饲料或微生物发酵的培养基。
10.根据权利要求9所述的6-苄基腺嘌呤在制备提高动物黑色素的食品中的应用,其特征在于,所述食品为为鱼饲料或乌骨鸡饲料。
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