CN107177635A - 一种血红铆钉菇黑色素生产方法 - Google Patents
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Abstract
本发明属于天然色素发酵生产的技术领域，提供了一种血红铆钉菇发酵生产天然黑色素的方法，其包括血红铆钉菇一级种子培养、二级种子培养、发酵液制备，酸沉结合有机溶剂洗剂法提取天然黑色素。本发明提供的方法新颖且易于操作，生产的天然黑色素具有良好的稳定性及较高抗氧化能力，在食品和医疗等方面具有重要的实用价值。
Description
技术领域：
本发明属于天然黑色素制备工艺技术领域，特别涉及一种以铆钉菇为菌种生产天然黑色素的工艺，具体涉及为铆钉菇黑色素发酵技术、微生物源天然黑色素提取制备技术。
背景技术：
黑色素是一类广泛存在于动植物和微生物的进化过程中，通过多羟基酚氧化而成的构造不规则的类多酚聚合体。黑色素具有广泛的生物活性，可为生物体提供结构性强度以及保护生物体免受紫外线、电离辐射、重金属污染、低温和高温等环境胁迫的伤害。黑色素不仅可以作为紫外线吸收剂、抗氧化剂、自由基清除剂、阳离子螯合剂、磁共振成像造影剂、抗癌药物和癌症的光动力学治疗及新型天然的药物载体等，还可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。此外，黑色素可以显著促进脾脏生长，空斑形成细胞数量明显增加，在免疫促进治疗方面有着重要的应用价值。可溶性黑色素还具有体外抗HIV病毒的作用，干扰HIV诱导的合胞体的形成，阻止HIV-1被膜表面的糖蛋白和T细胞特异单抗与淋巴母细胞的结合的能力。近年来，随着毒理学及生物学的发展，已陆续发现多种合成色素对人体有潜在的危害，因此越来越多的天然色素应用于食品和化妆品。在国际市场上天然色素的产量正以每年10％的速度增长。此外，源自动植物的天然色素由于受到季节、气候、地理区域以及生产成本的限制等，已远远不能满足国内外的实际需求，微生物源天然色素将是未来发展的趋势。在已开发及应用的天然色素中，红色、黄色、绿色等品种较多，蓝色较少、黑色则极为稀缺。当前使用的食用黑色素主要为合成色素亮黑BN及植物炭黑等。因此，开发天然功能性黑色素具有现实的经济价值。目前微生物源天然黑色素研究较多，但由于大多数微生物源黑色素为胞内色素，且多为微生物细胞壁的组成成分，通过发酵生产，产量极低且不易提取，限制了微生物源黑色素的产品的工业化生产。本发明目的是通过提供一种以铆钉菇为菌种生产天然黑色素的工艺，然后对发酵产物-发酵液进行提取纯化，提供直接用于食品、化妆品以及药品等领域的天然黑色素原料。
发明内容
本发明是通过血红铆钉菇一级种子培养、二级种子培养、发酵液制备，酸沉结合有机溶剂洗剂法提取天然黑色素。本发明提供的方法新颖且易于操作，生产的天然黑色素水具有良好的稳定性及较高抗氧化能力，在食品和医疗等方面具有重要的实用价值。
本发明所述血红铆钉菇接种前首先在试管斜面上进行28℃培养7天，培养使用的斜面培养基组成为：NaNO32g，K2HPO41g，KCl 0.5g，MgSO40.5g，FeSO40.01g，蔗糖30g，琼脂15-20g，水1000mL，然后用无菌水洗下一支斜面的菌体接入含50mL种子培养液的三角瓶(300mL)中，进行一级种子培养(28℃培养3天)，培养基为NaNO32g，K2HPO41g，KCl 0.5g，MgSO40.5g，FeSO40.01g，蔗糖30g，水1000mL，取一级种子按10％的接种量接装有300mL种子培养液的三角瓶(1000mL)中，进行二级种子培养(28℃培养48小时)，二级种子培养基成分同一级种子培养基。
本发明所述血红铆钉菇菌丝体发酵过程为取培养48小时经扩大培养的种子液，接种量5％，接入发酵培养基中，培养基配比为：NaNO32g，K2HPO41g，KCl 0.5g，MgSO40.5g，FeSO40.01g，蔗糖50g，水1000mL，28℃培养9天终止发酵。
本发明所述一种天然黑色素提取过程为将菌丝体过滤后除去后，留下发酵液，加入磷酸调整pH至2-3，于10-20℃环境内进行静置过夜，过滤收集沉淀，沉淀依次用乙醇、乙酸乙酯、石油醚洗涤后，挥去有机溶液，低温烘干至水分含量小于10％，然后用粉碎机将其粉碎得到产品。
具体实施方式
配制斜面培养基，斜面培养基为NaNO32g，K2HPO41g，KCl 0.5g，MgSO40.5g，FeSO40.01g，蔗糖30g，琼脂15-20g，水1000mL，pH自然，121℃灭菌20min后，取铆钉菇菌丝于斜面培养基中活化一次，活化条件为28℃，8d，菌体斜面活化完成后，用无菌水洗下一支斜面的菌体接入各含50mL种子培养液的两只三角瓶(300mL)中，进行一级种子培养(28℃培养3天)，培养基NaNO32g，K2HPO41g，KCl 0.5g，MgSO40.5g，FeSO40.01g，蔗糖30g，水1000mL，取一级种子按10％的接种量接入二级种子培养基中进行二级种子培养(28℃培养48小时)，二级种子培养基成分同一级种子培养基。然后取培养48小时的菌种二级种子，接种量为5％，接入10L发酵罐中，培养基配比为：：NaNO32g，K2HPO41g，KCl 0.5g，MgSO40.5g，FeSO40.01g，蔗糖50g，水1000mL，发酵温度28℃，搅拌转速150转/分钟，发酵时间9天，发酵液经抽滤后除去菌丝得到上清液。菌丝体过滤后除去后，留下发酵液，加入磷酸调整pH至2-3，于10-20℃环境内进行静置过夜，过滤收集沉淀，沉淀依次用乙醇、乙酸乙酯、石油醚洗涤后，挥去有机溶液，低温烘干至水分含量小于10％，然后用粉碎机将其粉碎得到产品。
Claims (4)
1.一种血红铆钉菇黑色素生产方法，其主要特征在于以血红铆钉菇为生产菌株，经菌丝体发酵培养后产生胞外黑色素，再通过提取纯化工艺得到天然黑色素产品。
2.根据权利要求1所述的一种血红铆钉菇黑色素生产方法，其特征在于培养基配比为：NaNO3 2gK2HPO4 1g，KCl 0.5g，MgSO4 0.5g，FeSO4 0.01g，蔗糖50g，水1000mL。
3.根据权利要求1及权利要求2所述的一种血红铆钉菇黑色素生产方法，其特征在于发酵条件为发酵温度28℃，初始pH7.0，接种量5％，发酵时间为9天。
4.根据权利要求1、2及3所述的一种血红铆钉菇黑色素生产方法，其主要特征在于血红铆钉菇菌丝体发酵结束后，将菌丝体抽滤除去，留下发酵液，在发酵液中加入磷酸调整pH至2-3，于10-20℃环境内进行静置过夜，过滤收集沉淀，沉淀依次用乙醇、乙酸乙酯、石油醚洗涤后，挥去有机溶液，低温烘干至水分含量小于10％，然后用粉碎机将其粉碎得到天然黑色素产品。
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