CN116814710A - 原花青素在裂殖壶菌发酵过程中的应用及发酵方法 - Google Patents
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Abstract
本发明属于微生物发酵领域，公开了原花青素在裂殖壶菌发酵过程中的应用及发酵方法，包括菌种活化、种子液制备和外源添加原花青素的裂殖壶菌油脂发酵，发酵培养基中添加终浓度为1‑10mg/L的膜过滤除菌后的原花青素，按照5‑10％(v/v)的接种量将种子液接到装有30mL发酵培养基的三角瓶中，在25℃、200rpm摇床中振荡培养96‑120h。本发明所选用的添加因子原花青素是一种低成本、无副作用的天然膳食多酚类化合物。本发明通过在裂殖壶菌发酵体系中外源添加少量特定比例的原花青素，可有效抑制裂殖壶菌发酵过程中胞内油脂过氧化，提高DHA油脂产量、品质和生产效率。与未添加相比，添加适宜浓度的原花青素，生物量提高了11.89％，油脂产量提高了33.58％，DHA产量提高了53.36％。
Description
技术领域
本发明涉及微生物发酵领域，涉及原花青素在裂殖壶菌发酵过程中的应用及发酵方法，更具体地说，涉及一种原花青素在裂殖壶菌发酵过程中抑制脂质过氧化、提高DHA油脂产量的方法。
背景技术
二十二碳六烯酸(Docosahexaenoic acid，DHA)是一种对人体健康非常有益的ω-3多不饱和脂肪酸。DHA是人体大脑和视觉细胞结构脂肪酸不可或缺的成分，具有促进婴幼儿脑部和视力发育、预防或减轻动脉粥样硬化及冠心病、抗肿瘤和延缓衰老等生理功能。许多流行病学研究表明饮食中摄入DHA会降低阿尔茨海默病、双相情感障碍、抑郁症、情绪障碍和精神分裂症的风险。人类无法自身合成DHA，必须从外源性食物中摄取DHA以满足一些重要生理功能的需求。因此，DHA被广泛应用于食品和医药行业。
DHA的传统来源是深海鱼油，但由于海洋环境污染和鱼类过度捕捞，鱼油来源的DHA不可持续，无法满足日益增长的DHA市场需求。裂殖壶菌是一种异养单细胞海洋真核生物，具有生长周期短、易培养、油脂中DHA含量高和食用安全可靠等优点，目前已成为了DHA最有潜力的生产者。在微生物适宜的生长条件下，产油微生物胞内通常仅产生较低含量的脂质，而压力环境则会促进油脂的生成。目前采用氮源耗竭、磷酸盐饥饿、盐度胁迫或激素添加等应激诱导策略已被用于促进裂殖壶菌产DHA油脂产量。然而在裂殖壶菌发酵培养过程中，上述环境胁迫会引起的细胞氧化损伤，并导致细胞内脂质过氧化，进而降低DHA油脂产量。因此大量研究集中在提高裂殖壶菌生产DHA效率和降低生产成本的策略方面。申请号为201710548502专利中公开了一种在裂殖壶菌发酵培养基中添加终浓度为100mg/L～3g/L对苯甲酸衍生物的方法，油脂产量提高了32.12％。但使用含有化学添加剂的培养基培养裂殖壶菌生产的DHA油脂能否应用于食品医药行业有待进一步研究。因此，找到一种安全、廉价、高效的外源添加剂用于在裂殖壶菌发酵过程中抑制脂质过氧化并提高DHA油脂产量，非常有必要。
原花青素是天然存在的膳食多酚类化合物，广泛存在于植物中，是一种低成本、无副作用的天然抗氧化剂，研究表明其具有强抗氧化活性，包括抑制脂质氧化、DNA修复和清除自由基等功能。但是，目前通过添加原花青素来抑制裂殖壶菌发酵过程中胞内脂质过氧化、提高DHA油脂产量未见报道。
发明内容
为了弥补现有技术的不足，本发明提供一种原花青素在裂殖壶菌发酵过程中的应用及发酵方法，通过以下技术方案实现：
原花青素在裂殖壶菌发酵过程中的应用，外源添加原花青素抑制裂殖壶菌发酵过程中细胞脂质过氧化、提高DHA油脂产量的应用。
利用外源添加原花青素培养裂殖壶菌的发酵方法，包括以下步骤：
(1)菌种活化：取出保藏于-80℃的裂殖壶菌甘油管菌种，转接于斜面培养基，其组成为：葡萄糖30g/L，酵母浸粉10g/L，蛋白胨2g/L，海水晶15g/L，琼脂粉15g/L，pH 6.0，置25℃培养箱中培养48-72h；
(2)种子液制备：用接种环挑取3-4环活化的斜面菌种，接入装有30mL种子培养基的250mL三角瓶中，种子培养基组成为：葡萄糖30g/L，酵母浸粉10g/L，蛋白胨2g/L，海水晶15g/L，pH6.0，在25℃、200rpm摇床中振荡培养48-60h；
(3)外源添加原花青素的裂殖壶菌油脂发酵：配制发酵培养基，灭菌，在发酵培养基中添加终浓度为1-10mg/L的膜过滤除菌后的原花青素，按照5-10％(v/v)的接种量将种子液接到装有30mL发酵培养基的250mL三角瓶中，在25℃、200rpm摇床中振荡培养96-120h，离心，收集细胞沉淀，采用酸热法裂解细胞，利用氯仿-甲醇溶液(2:1，v/v)提取细胞中DHA油脂；将油脂进行甲酯化后采用气质联用测定脂肪酸组成，计算PUFA含量和DHA含量。气质联用测定脂肪酸组成方法：采用SPTM-2560聚二氰丙基硅氧烷强极性毛细管色谱柱，载气为高纯度氦气，流速为1mL/min，进样口温度设置为260℃；进样量1μL，分流比1:10；柱温采取程序升温：初始温度140℃，维持5min，以每分钟4℃升温至240℃，240℃维持10min。
与现有技术相比，本发明的有益效果为：
同目前裂殖壶菌发酵过程中添加的化学调节剂相比，本发明所选用的添加因子原花青素是一种低成本、无副作用的天然膳食多酚类化合物。本发明通过在裂殖壶菌发酵体系中外源添加少量特定比例的原花青素，可有效抑制裂殖壶菌发酵过程中胞内油脂过氧化，提高DHA油脂产量、品质和生产效率。与未添加相比，添加适宜浓度的原花青素，生物量提高了11.89％，油脂产量提高了33.58％，DHA产量提高了53.36％。
本发明通过简单有效的发酵调控，提高底物转化率，操作简单，不增加额外的人工和设备，仅通过较低的投入，就可以提高目标产物合成效率和生产强度，从而降低生产成本。且该方法不危害环境，不增加人力物力，简单方便且具有良好的经济效益。本发明中发酵培养基中添加的原花青素是一种天然膳食多酚类化合物，因此生产的DHA可安全应用于婴幼儿奶粉、功能保健品、医药、化妆品及动物饲料等领域。本发明通过在发酵过程中添加原花青素不仅有效降低了藻油DHA的生产成本，保证了较高的藻油DHA品质，十分有利于促进裂殖壶菌发酵产DHA的产业化发展。
具体实施方式
下面结合具体实施例对本发明做进一步说明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到。
实施例1
1、菌种活化：取出保藏于-80℃的裂殖壶菌甘油管菌种，转接于斜面培养基，其组成为(g/L)：葡萄糖30，酵母浸粉10，蛋白胨2，海水晶15，琼脂粉15，pH6.0，置25℃培养箱中培养72h；
2、种子液制备：用接种环挑取3-4环活化的斜面菌种，接入装有30mL种子培养基的250mL三角瓶中，种子培养基组成为(g/L)：葡萄糖30，酵母浸粉10，蛋白胨2，海水晶15，pH6.0，在25℃、200rpm摇床中振荡培养48h；
3、摇瓶发酵：发酵培养基组成为(g/L)：葡萄糖80，酵母浸粉6，谷氨酸钠6，，海水晶15，KH2PO41，MgSO4·7H2O 7，pH6.0，添加原花青素的浓度分别为0.1、0.5、1、5、10、50、100mg/L，在实验过程中，培养基的成分及加入量均保持不变，仅需在不同的发酵培养基中加入原花青素，并做好对照实验。按照10％的接种量将种子液接到装有30mL发酵培养基的250mL三角瓶中，在25℃、200rpm摇床中振荡培养96h，取样测定细胞生物量、油脂产量、不饱和脂肪酸含量和产量、DHA含量和产量，结果如表1。
表1添加不同浓度原花青素的发酵结果
如表1所示，添加不同浓度的原花青素，细胞的生物量有所增加，加入5mg/L的原花青素可有效提高裂殖壶菌发酵产生DHA油脂的产量，油脂产量提高33.58％，DHA产量提高53.36％。本发明中原花青素添加量十分重要，添加量过低和过高均导致发酵各项指标的显著降低。
本发明实施方式不限于此，根据本发明的上述内容，按照本领域的普通技术知识和通用方法，在不脱离本发明上述基本技术思想前提下，本发明还可以有其它的实施方式。本发明还可以做出其它多种形式的修改、替换或变更，均落在本发明权利保护范围之内。
Claims (4)
1.原花青素在裂殖壶菌发酵过程中的应用，其特征在于，外源添加原花青素抑制裂殖壶菌发酵过程中细胞脂质过氧化、提高DHA油脂产量的应用。
2.利用外源添加原花青素培养裂殖壶菌的发酵方法，其特征在于，包括以下步骤：
(1)菌种活化：取出裂殖壶菌甘油管菌种，转接于斜面培养基，置25℃培养箱中培养48-72h；
(2)种子液制备：用接种环挑取3-4环活化的斜面菌种，接入装有30mL种子培养基的三角瓶中，在25℃、200rpm摇床中振荡培养48-60h；
(3)外源添加原花青素的裂殖壶菌油脂发酵：配制发酵培养基，灭菌，在发酵培养基中添加终浓度为1-10mg/L的膜过滤除菌后的原花青素，按照5-10％(v/v)的接种量将种子液接到装有30mL发酵培养基的三角瓶中，在25℃、200rpm摇床中振荡培养96-120h。
3.如权利要求2所述的利用外源添加原花青素培养裂殖壶菌的发酵方法，其特征在于，步骤(1)中斜面培养基的组成为：葡萄糖30g/L，酵母浸粉10g/L，蛋白胨2g/L，海水晶15g/L，琼脂粉15g/L，pH6.0。
4.如权利要求2所述的利用外源添加原花青素培养裂殖壶菌的发酵方法，其特征在于，步骤(2)中种子培养基组成为：葡萄糖30g/L，酵母浸粉10g/L，蛋白胨2g/L，海水晶15g/L，pH6.0。
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