amb Advances in Microbiology 2327-0810 2327-0829 beplay体育官网网页版等您来挑战! 10.12677/amb.2024.132011 amb-90128 Articles 生命科学 螺旋藻藻蓝蛋白的提取及应用研究进展
Research Progress on Extraction and Application of Phycocyanin from Spirulina Platensis
吴孟瑶 1 2 邱梦可 1 梅兴国 1 2 湖北科技学院药学院,湖北 咸宁 咸宁市康健医药产业研究院有限公司,湖北 咸宁 26 06 2024 13 02 92 99 10 4 :2024 20 4 :2024 20 6 :2024 Copyright © 2024 beplay安卓登录 All rights reserved. 2024 This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ 藻蓝蛋白既是一种蛋白质,又是一种很好的天然食用色素,越来越多地被应用于医药保健食品、化妆品及荧光探针标记领域。广泛的应用增加了研究人员对藻蓝蛋白成分及其影响的兴趣。本文对以往文献中描述的已有的生产、提取方法,包括主要的物理和化学参数进行全面回顾和比较评估,可以利用这些技术来改进藻蓝蛋白制备工艺,使其更加安全有效。另简单介绍了藻蓝蛋白的广泛应用,使人们对螺旋藻藻蓝蛋白相关知识有更深的了解。
Phycocyanin is not only a kind of protein, but also a good natural edible pigment, which is increasingly used in the fields of medicine, health food, cosmetics and fluorescent probe labeling. The wide application has increased researchers’ interest in phycocyanin composition and its influence. In this paper, the existing production and extraction methods described in previous literature, including the main physical and chemical parameters, are comprehensively reviewed and compared, and these technologies can be used to improve the preparation process of phycocyanin and make it safer and more effective. In addition, the extensive application of phycocyanin is briefly introduced, which makes people have a deeper understanding of phycocyanin related knowledge of spirulina.
螺旋藻,藻蓝蛋白,提取方法,稳定参数,应用
Spirulina
Phycocyanin Extraction Method Stability Parameters Application
1. 引言

螺旋藻是一种广泛应用于食品工业的自然资源,富含多种营养素,包括多种不饱和脂肪酸和色素,尤其是富含藻蓝蛋白 [1] 。据报道,藻蓝蛋白作为一种具有水溶性、无毒、蓝色的光合色素被广泛应用于食品、化妆品和制药工业。从螺旋藻分离纯化藻蓝蛋白已经得到广泛研究。然而,由于螺旋藻中藻蓝蛋白的百分含量、细胞破碎方法的效率以及藻蓝蛋白在储存条件和产品配方中的不稳定性,其应用受到了限制。

本文探讨了生产藻蓝蛋白的提取方法,以及影响该蛋白纯度和稳定性的物理和化学条件。此外,还介绍了其相关应用。所涵盖的知识可为不同行业提供指导,以确定提高藻蓝蛋白产量所需的最佳条件,同时减少时间以降低相关成本。

2. 藻蓝蛋白介绍

藻胆蛋白是螺旋藻中一种荧光蛋白。藻胆体位于类囊体膜上,这是一种超分子蛋白质复合物,是蓝藻主要的光合天线复合体,它们在叶绿素绿光能隙中获取光能,并将高量子效率的激发能转移到光合反应中心,通常是光系统II (PSII) [2] 。根据色素颜色它们被细分为藻红蛋白(PE)、藻蓝蛋白(PC)和别藻蓝蛋白(APC) [3]

藻蓝蛋白是一种高价值蛋白质,由一种由10到19 kDa组成的多肽α单体和一种由14到21 kDa组成的多肽β单体组成异二聚体蛋白,其中α亚基联结了一个藻蓝胆素,β亚基联结了两个藻蓝胆素,这样三个二聚体蛋白首尾相连,就成为一个三聚体的甜甜圈式结构,也存在两个三聚体紧密交叠形成六聚体,也是甜甜圈结构,直径都为11 nm左右 [4] 。藻蓝胆素是一种具有开链四吡咯基团的植物色素的胆碱发色团,它通过硫醚键结合蛋白质,并赋予藻蓝蛋白蓝色 [4]

3. 藻蓝蛋白的提取

为了有效提取藻蓝蛋白,必须考虑几个参数,例如细胞破碎、生物质形式(新鲜或干燥)、温度、光强度、pH、溶剂类型、生物质/溶剂比和防腐剂的使用 [5] 。螺旋藻的细胞膜包括4层,最外层是蛋白质纤维,再是赋予细胞刚性的肽聚糖层,最内层是纤维层 [2] 。破坏细胞膜的方法是获得高提取率藻蓝蛋白和纯度的关键因素。冷冻/融化,混合/均质化,珠磨,超声波及电场,高压均质化及酶提取等方法有最佳条件及参数能得到较高纯度的藻蓝蛋白。

3.1. 细胞破碎方法

冷冻/解冻循环对质膜的持续破坏具有促进作用 [6] 。在这一技术中,溶剂选择是必须考虑的参数。磷酸钠缓冲液(Ph 7.0)在所评价的溶剂中被证明是最合适的 [7] [8] [9] ,从而获得较高的萃取率和纯度(217.18 mg/g, 0.87) [10] 。但重复冷冻、解冻循环耗时长、能耗大,使这种方法只适合实验室规模的研究 [7]

混合与均质化分别为搅拌强度小与大的一种简易提取方法。采用1.71%生物质/溶剂比,均质提取15 min,藻蓝蛋白得率为67.61 mg/g [11] 。浓度为0.02 g/mL的生物质用烘箱70℃干燥4小时,再用0.01 M磷酸盐缓冲液在25℃下提取24小时,藻蓝蛋白得率为103.07 mg/g,纯度0.67 [11] 。混合与均化时温度上升使提取物纯度下降 [8] 。所以该方法并不适用于工业规模。

珠磨就是用高速珠粉碎细胞膜的机械方法 [12] 。此法最佳条件难以确定。已有研究显示低密度珠(例如玻璃)比较适用于低粘度介质 [13] 。该方法工艺简单,时长从5分钟持续到4小时 [14] [15] ,粉碎能力大(60~150 g/L),耗能少,提取率高(217.14 mg/g),非常适合工业化应用 [16]

超声波造成细胞膜减薄、细胞破裂等缺陷 [17] 。极端功率强度可引起剧烈而迅速的膜破裂及高温,但高温造成纯度下降,所以控制好这些参数是超声波提取中非常关键的问题。可利用耦合到水浴的水套提取室进行温度控制。藻蓝蛋白的产率由17.20 mg/g提高到90 mg/g,纯度由0.67提升到了0.93 [18] [19] [20]

电场提取建立在短电脉冲应用的基础上,短电脉冲可以通过电穿孔来促进细胞膜中孔形成和提高细胞膜渗透性,电脉冲可采用脉冲式,也可采用中强度 [21] [22] 。电场强度、脉冲宽度和脉冲形状以及电场持续时间影响藻蓝蛋白的提取。脉冲电场可以选择性提取藻蓝蛋白,这种方法可以在不破坏细胞结构的情况下释放细胞内化合物。在适当的电场作用下,所形成的孔隙尺寸较小,释放时长从15分钟到3小时 [14] [16] [23] 。文献报道脉冲电场提取藻蓝蛋白的最佳条件为40℃、25 kV/cm和150 μs,藻蓝蛋白浓度达到151.94 mg/g,纯度达到0.51 [16]

高压均质是利用高压均质化破坏螺旋藻细胞壁而释放藻蓝蛋白色素的物理方法。近来高压均质已被应用于藻蓝蛋白提取中,所报道产率显着大于常规提取 [24] 。在1400~2000 Pa的压力下,使用磷酸盐溶剂高压均质化藻蓝蛋白的收量在113.50 mg/g~291.90 mg/g之间 [9] [25] 。压力是影响高压均质表达的关键因素,但高压均质提取的优化参数还包括藻类的数量(生物量/溶剂比)、温度和通过高压均质设备的次数 [25] 。相对于采用电场或者超声处理,高压均质更为简便且可扩展,能方便集成于工业生产过程。

酶法提取因其具有比传统提取方法中更高效、环保而成为又一备受关注的手段。利用酶提取可在低温、低压条件下完成,有利于维持藻蓝蛋白稳定性及品质。采用溶菌酶消化干燥螺旋藻细胞壁基质内存在的多糖。收率在20 mg/g~25 mg/g之间,纯度在0.8~0.9之间 [26] 。超声波辅助酶法提取藻蓝蛋白收率提高77.92%,达到92.73 mg/g,纯度1.09 [27] 。此外脉冲电场与酶联合应用也可望成为一种新型藻蓝蛋白的提取技术。

3.2. 藻蓝蛋白提取及稳定性的关键参数

螺旋藻干粉常被用来提取藻蓝蛋白。直接新鲜螺旋藻提取虽然有些研究,没有数据表明新鲜螺旋藻提取能达到较高的提取收率 [16] [23] [28] 。但有研究显示干粉在提取缓冲液中水合两小时会获得较高收率 [29]

温度升高对萃取细胞内化合物有明显意义,但藻蓝蛋白会在高温下丧失稳定性。25~47℃被认为为合适的条件 [30] 。研究表明藻蓝蛋白在45℃以下不降解,47℃~至69℃时降解增强,70℃后降解进一步增强 [30] [31] [32] 。所以最高温度为45℃是维持藻蓝蛋白稳定、避免降解的最佳值 [33]

光线的好坏与强弱会影响藻类生长和光合作用,进而影响藻蓝蛋白产量 [34] 。每一种藻类均生长于特定条件与光。关于光谱对螺旋藻的影响,Walter等人证实了与蓝光相比,用红光操纵的光生物反应器实现了更高的光合效率和藻蓝蛋白 [35] 。Wicaksono等人也证明了红色光谱产生最大量的藻蓝蛋白 [36] 。光和温度的关系也会影响藻蓝蛋白的降解,最好储存在暗处 [31] [36]

pH值对维持藻蓝蛋白稳定,维持光谱特性及独特蓝色至关重要。研究结果表明,在pH 3.0~4.0范围内,藻蓝蛋白构象发生变化,降解率较高 [28] [31] 。藻蓝蛋白稳定的最佳pH在5.5~7.0之间 [30] [31] [33]

溶剂的选择取决于藻蓝蛋白的pH稳定范围。溶剂还会因其离子强度而影响藻蓝蛋白溶解度和蛋白质结构 [4] 。有研究显示磷酸钠缓冲液(pH 7.0)比蒸馏水有更高提取率 [8] [18] [19] [25] 。但也有学者指出,相比较而言,使用蒸馏水 [37] [38] 或磷酸钾缓冲液(pH 6.5) [39] 或氯化钙(Ph 7.4) [11] 时比乙酸盐缓冲液(pH 5.0)得到更低的提取率 [8]

高提取率与生物质/溶剂比呈正相关,但这可能导致藻蓝蛋白含量的降低。磷酸钠缓冲液(pH 6.8)按1:6~1:10配比进行了研究,结果表明1:10配比提取率较高,1:6配比纯度较高 [7] [29] 。磷酸钠缓冲液(pH 7.0)按1:50、1:25和3:50配比进行研究,结果表明1:50配比提取率和藻蓝蛋白纯度最高(提取率80%,纯度1.2) [18] 。为了控制成本,应当优先考虑选择生物质和溶剂的比例。

藻蓝蛋白提取时对环境压力非常敏感。防腐剂主要用于提高藻蓝蛋白稳定性和维持生物活性等方面 [40] 。糖类促进蛋白质与溶剂之间的相互作用,支持蛋白质的折叠 [31] 。糖的种类和浓度对藻蓝蛋白稳定性有影响。加入葡萄糖(20~40% w/w)、果糖(10~15% w/w)和蔗糖(70% w/w)可延长藻蓝蛋白的半衰期 [30] 。Chaiklahan等使用20% w/w的葡萄糖和蔗糖,观察到藻蓝蛋白在6℃下15分钟仍存有62%以上,在不加防腐剂的情况下只有47% [30] 。吴等人论证氯化钠为阻止藻蓝蛋白降解适宜试剂,但当浓度为5% w/w时就会出现浑浊现象 [30] [31]

0.4% w/w柠檬酸浓度使藻蓝蛋白浓度提高至19%,而无柠檬酸情况下只有11% [41] 。另一项研究发现,藻蓝蛋白加入柠檬酸,45天后藻蓝蛋白仍有67%,未加其含量降为3% [42] 。交联剂如甲醛及戊二醛交联剂构成聚合物链与蛋白质间的网络,以防止藻蓝蛋白的降解,但其毒性较高,不适用于食品工业 [43]

4. 藻蓝蛋白的应用 4.1. 食品添加剂色素

藻蓝蛋白是自然界少有的可食用的蓝色色素,清亮可爱,可作为食品着色剂,而且本身是非常丰富的蛋白质,其氨基酸组成齐全,必需氨基酸占总量的37.2%。美国FDA于2013/2014年对藻蓝蛋白在一些食品中的应用做出了许可,包括饮料、奶酪、冰激凌、糖果、口香糖、酸奶、布丁等多种常见食品,我国关于藻蓝蛋白制品《食品添加剂 藻蓝》的国家标准也与2021年3月开始实施(GB 1886.309-2020),因而这些年蓝色的食品也变的越来越多见。

4.2. 化妆品

藻蓝蛋白在化妆品中的应用主要也是以色素的形式体现,常见的产品有润肤霜、增白霜、香皂、眼线膏、唇膏等等。

4.3. 医药保健

藻蓝蛋白的健康功效研究是最近这些年研究最多的内容。目前国外已成功的研制出多种藻蓝蛋白复合药品,认为该蛋白有促进免疫和抗疾病功能。藻蓝蛋白对一些癌细胞的抑制作用明显最新的研究成果显示藻蓝蛋白具有显著抑制肿瘤细胞生长和肿瘤新生血管生成,诱发肿瘤细胞凋亡,能显著地减轻和消除辐照、化疗对血白细胞核骨髓细胞的损伤,并能加速外周血白细胞和骨髓有核细胞的恢复,提高机体免疫力,阻止肿瘤转移复发。

4.4. 生化试剂

藻蓝蛋白是一类能天然发出荧光的物质,具有激发性强、波长偏移宽、摩尔消光系数高、荧光量子产率高等光谱特性 [44] 。经过特别手段制备或修饰的藻蓝蛋白在一些细胞生物学研究手段,如荧光流式细胞术、荧光显微镜、单细胞分析、荧光激发细胞分选及免疫测定法等技术中已经得到应用。根据相关研究报道,以藻蓝蛋白为光敏剂可使激光对肿瘤细胞的杀伤率从12%提高到88%,癌症治愈率提高55%。

5. 结论

本文着重介绍了影响藻蓝蛋白化学降解的主要因素,并提出了提高其稳定性的策略。特别需要注意的是,在细胞破碎的过程中,必须确保释放总色素含量的同时,维持条件的稳定性。据主要研究表明,藻蓝蛋白的保存应在45℃以下范围,pH值介于5.5~6.0,在暗处存储,并采用防腐剂,例如单糖和二糖、柠檬酸、交联剂以及自然聚合物等。

除了藻蓝蛋白之外,螺旋藻所产生的其他次级代谢产物也具备在食品、生物燃料、生物材料、生物肥料和动物饲料等领域中应用的潜力,从而促进工业共生和循环经济的发展。在某些情况下,它们还可以用作药物或疫苗的载体。因此,必须对这些工艺进行优化,以提高其放大效果。

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