大自然是人类最重要的食物来源,近年来,酵素已成为一种流行的保健食品,大多数酵素产品以水果为主要原料,如海棠、龙眼、苹果梨、葡萄和蓝莓等[1]。发酵是制作酵素过程中的核心环节,也是人类历史上最常用的古老食品保存方法之一。在没有任何冷藏或其他保存方法的情况下,传统发酵可以作为一种替代方法来制作人们的主食[2]。虽然各种新的加工方法层出不穷,各种各样的新型食品日益涌现,但食品发酵在食品加工中仍然是不可替代的[3],并已发展成为最具特色的食品,如泡菜[4]、面包[5]和葡萄酒[6]。传统的发酵饮料通常是通过自然发酵,一种使用野生微生物菌群,譬如细菌、酵母菌和霉菌单独菌种作用方法,抑或是多种菌种共同作用的方法[7]。现有研究表明,发酵食品可以改善原始未发酵原料的营养价值和功能特性[8],并且发酵在增强抗氧化活性方面起着关键作用,这表明微生物与抗氧化特性之间存在一些特定的关系[9⇓⇓⇓⇓-14]。
食用植物酵素是一种植物功能性食品,由一种或多种新鲜蔬菜、水果、菌菇和药食同源中草药等为原料,经多种微生物长期发酵而制成的。近年来因其对健康存在潜在的益处而受到关注,相关的生产工艺在原料、发酵微生物和发酵条件方面取得了重大进展。研究表明,食用植物酵素含有丰富的营养物质和生物活性物质,如矿物质、氨基酸、多酚、有机酸和多糖。因此,许多研究都集中在食用植物酵素在一些疾病如高血糖、高脂血症、肥胖症和心血管疾病等方面的有益作用[15]。目前现有的研究报道主要针对某一方面进行论述,但是很少有对食用植物酵素进行全面归纳总结。因此,这篇综述阐明了食用植物酵素的主要发酵方式、主要生物活性成分、主要功能作用和安全性方面相关的研究进展,以期为食用植物酵素发酵过程中的主要功能作用研究提供理论依据。
食用植物酵素发酵一般采用自然发酵和人工接种发酵的发酵工艺。
1.1 自然发酵
食用植物酵素自然发酵是指以食用植物为原料,利用原料表面附着的微生物和原料内部的微生物进行发酵。经研究发现,自然发酵中常见的微生物有酵母菌、乳酸菌和醋酸菌3种,三者的共同作用可以形成独特风味的发酵制品[16]。在最近几年中,由于高通量测序技术(HTS)具备高通量、高精确度和快速响应的特点,它在食品加工领域的微生物多样性研究中得到了广泛的应用。它可以快速地检测出样品中存在的所有种类微生物,并且能提供大量有关不同种或同一种内各种微生物间相互作用的信息。众多的科研人员已经采用HTS技术对传统发酵食品中的细菌和真菌多样性进行了深入分析。
张琪等[17]采用Illumina MiSeq高通量测序技术对沙棘酵素自然发酵过程中的细菌进行了多样性分析,揭示了细菌群落随时间的推移而发生的动态演变过程,不但为提高沙棘酵素产品质量提供了理论基础,更为研制高效酵素发酵剂做好准备。牛广财等[18]采用Illumina MiSeq高通量测序技术对自然发酵过程中沙棘酵素真菌的多样性及其结构变化规律进行了研究,为生产出更高品质的沙棘酵素产品同时为未来的沙棘酵素产品提供理论支持。汤灿辉等[19]利用Illumina Miseq高通量测序技术分析发酵过程中细菌群落结构的变化,加深对自然沙棘酵素发酵机制的认识,为筛选潜在价值的益生菌和实现精准人工调控提供科学依据。黄娟等[20]采用Illumina高通量测序技术研究了岭南桑葚果酒在两个发酵周期中的细菌多样性,通过对桑葚果酒样本的菌种多样性分析、优质菌株资源选育和桑葚果酒产业化生产等方面的研究,探讨了新酒发酵转化为陈年酒过程中菌种多样性的变化和菌群结构的变化。
1.2 人工接种发酵
食用植物酵素在自然发酵过程中发酵时间长,易受杂菌污染,发酵过程不易控制,最终致使酵素品质下降。人工接种发酵具有发酵时间短,不易受杂菌污染,发酵过程可控等优点。因此人工接种发酵为食用植物酵素发酵方式的更优选择。在已有的研究中,一般采用单一菌种或混合菌种进行人工接种发酵。
目前发现最常用的发酵菌种有乳酸菌、酵母菌、霉菌以及多菌种混合发酵。李艳杰等[21]采用混合菌种发酵时,要考虑菌种的种类、接种量、接种时期,以及对发酵液的影响。由于酵素发酵周期长、菌种多、组分复杂,所以在自然发酵过程研究中存在一定难度[22]。
在人工接种发酵中,乳酸菌(Lactic acid bacteria, LAB)是应用最广泛的菌种之一,其发酵过程中的主要产物是乳酸。在食品发酵中常见的属一般包括乳杆菌属、乳球菌属、肠球菌属、片球菌属和明串珠菌属等[23]。其中乳酸菌、乳球菌、链球菌和肠球菌等为乳酸菌中最常用的菌种[24]。另一个重要的群体是醋酸菌,它可以利用原料中的糖生产醋产品,如苹果醋和传统醋[25]。芽孢杆菌,如枯草芽孢杆菌是第三种重要的发酵细菌,可以利用豆类或块茎作为底物生产纳豆和酱油[26]。此外,两歧双歧杆菌等放线菌门的细菌也有助于生产酸菜和泡菜等发酵食品[27]。酵母在食品工业中发挥着至关重要的作用,它提供的酶能产生理想的生化反应,有利于面包、啤酒、葡萄酒等产品的形成[28]。此外,霉菌也是重要的微生物,如米曲霉可用于酱油的生产[29]。然而,与细菌和酵母相比,霉菌在发酵食品中的应用相对有限,因为需要更多的控制和监测,以确保没有有害毒素的产生[30]。几种植物果实人工接种发酵制成酵素的成分及最佳工艺条件如表1所示。
表1 几种植物果实酵素制品的成分及最佳工艺条件 酵素在当代社会中,植物性食物的地位举足轻重。随着人们对健康的日益重视,越来越多的人被植物性食品所吸引[43]。植物性食品富含多种生物活性物质,如多糖、多酚、维生素、γ-氨基丁酸和胞外多糖[44]等。发酵不但可以提高食品的安全性还可以延长食品保质期[45]。有研究结果表明,与非发酵食品相比,发酵植物性食品不仅提供令人愉悦的风味和质地,而且在预防慢性疾病方面也显示出更大的潜力[46]。这些益处主要归因于微生物在发酵过程中对营养成分的影响[47]。
2.1 多糖
植物性食物中的多糖是一种重要而广泛的具有对人类健康至关重要的多种结构和生物活性的分布类营养素[48]。此前的研究主要集中在阐明植物多糖的内在结构及其功能特性[49]。然而,近年来随着研究人员深入研究,发酵植物性食品的领域愈发广泛,人们越来越认识到植物生长过程中和微生物发酵过程中探讨多糖变化的意义[50]。在植物中,多糖具有能量储存、支撑结构和细胞壁的形成等方面功能。植物中发现的多糖主要有纤维素、阿拉伯木聚糖、果胶和淀粉等[51],这些多糖可分为贮能多糖和结构多糖[52]。
现有文献表明天然产物资源具有化学结构多样、作用靶点多和毒性低等特点,在延缓衰老和预防衰老相关疾病方面具有巨大潜力;其中,植物多糖的作用尤为突出[53⇓-55]。
2.2 多酚
食用植物酵素中的多酚类化合物不仅来自于植物原料本身,还来自于发酵过程中酵母菌、醋酸菌、乳酸菌等微生物的代谢产物。这些代谢产物包括黄酮类、单宁类、酚酸类和花色苷类等[56]。
在常见的蔬菜、水果和中草药中,多酚类化合物是普遍存在的。这些多酚化合物的分子构造包含了多种酚羟基,如黄酮、类黄酮、酚酸和花色苷等。多酚类化合物具有抗氧化作用、抗菌消炎活性、抗癌活性、抗疲劳作用以及抗突变等生物活性。多项研究证实,人体能够高效地吸收多酚类化合物,其中,肠道和肝脏是这些物质的主要代谢中心。此外,多酚类物质还具有抗氧化、抗突变作用以及抗肿瘤活性。在这些器官里,多酚类化合物会经历甲基化、糖基化和硫酸化等化学反应,然后通过胆汁重新进入肠道。因此,多酚类物质能够被胃肠道直接吸收。然而,不同种类的多酚在人体内的分解速率是有区别的,一旦进入人体血液,其平均半衰期通常超过3.3 h。从这些数据中,可以得出结论:多酚的摄入量与身体的吸收周期在促进人体健康方面有着非常紧密的关联[57]。
2.3 维生素
水果和蔬菜中尽管富含维生素C,但其维生素B的含量相对较低。作为一种辅助酶,维生素B在人体的新陈代谢过程中发挥着不可或缺的角色。因此,人们越来越重视对其进行提取和加工利用。根据目前的科学研究,发酵过程有助于B族维生素的生成[58]。RATCHADAPORN等[59]用嗜酸乳杆菌和干酪杆菌发酵的腰果苹果汁的B族维生素含量高于其他益生菌发酵的腰果苹果汁,其中嗜酸乳杆菌和干酪菌发酵的B族维生素含量分别增加约19.25%和23.11%。SZUTOWSKA等[60]对羽衣甘蓝进行了发酵,过程中维生素C的含量下降的这种情况可能与其不稳定的特性和对氧化分解的高度依赖性有关,或者可能是在发酵阶段被乳酸菌转化和利用。
新鲜植物经过发酵后,具有多种有益于人体健康的功能活性,如在降血脂、抗氧化、降血糖、降血压、改善肠道环境和提高免疫力等方面具有一定的预防治疗效果[61]。
3.1 具有降低血脂的作用
食用植物酵素能够加速身体的新陈代谢过程,这包括脂肪的代谢。当脂肪代谢加快时,血液中的脂质含量就会相应减少,从而改善血脂情况。商曰玲[62]通过对特种沙棘酵素活性成分进行测定分析后发现,这种酵素对沙棘的营养成分和活性功能都能得到有效地保留,尤其在降脂方面有卓越的表现,其作用甚至超出了沙棘本身。在发酵原料浓度相近的条件下,酵素中的生物活性成分含量与发酵原料的特性有关。降脂试验表明,混合沙棘酵素降脂效果显著,特别是加入玫瑰花后的混合沙棘酵素,其降脂效果更为明显。袁斌[63]在实验中发现,利用不同种类的果蔬酵素对营养性肥胖小鼠进行灌胃试验后,发现果蔬酵素对减肥降脂有一定效果,但不同种类果蔬酵素的作用效果有所差异。因此,可以考虑将果蔬酵素作为辅助减肥的一种方式。HUANG等[64]实验结果表明,植物乳杆菌K68和果蔬发酵(FVF)对高脂肪高果糖饮食(HFFD)引起的大鼠体重增加和血脂异常均有预防作用。因此,K68和FVF可能具有预防HFFD诱导的高血糖、高胰岛素血症和高脂血症的特性。
3.2 抗氧化作用
食用植物酵素能够显著增强机体的抗氧化能力,降低自由基的产生和积累,从而减轻氧化应激对机体的损害。王辉[65]经过对青梅酵素的抗氧化活性、酶活力和抑菌作用等方面的研究,发现青梅酵素具有较强的抗氧化活性和酶活力。王虎玄[66]则通过对比苹果酵素和未发酵苹果汁的羟基自由基清除率、还原力以及总酚含量,发现苹果酵素具有良好的抗氧化性能。王迪[67]探究了在芸豆酵素发酵过程中代谢产物含量以及抗氧化能力的动态变化。研究发现,通过代谢产物含量的变化,可以清晰地观察到发酵过程的周期性以及发酵液中生物活性物质的累积情况。
3.3 降低心血管疾病作用
食用植物酵素与心血管疾病之间存在积极的关系。植物酵素中的多种活性成分对心血管健康具有潜在的益处,可以降低心血管疾病风险、预防心脑血管疾病等。赵迪等[68]通过对大鼠进行实验,发现决明子菊花的本草酵素能够显著降低自发性高血压大鼠的血压,而对正常大鼠的血压无明显影响。这表明决明子菊花本草酵素对高血压有良好的治疗作用,能有效辅助降低血压。AHRÉN等[69]通过对动物的实验探究,评估了结合蓝莓和具有产生单宁酶能力的益生菌植物乳杆菌DSM 15313的两种益生菌产品对高血压有一定的预防作用,利用植物乳杆菌DSM 15313进行发酵处理的蓝莓对大鼠显示出有降低高血压的作用,这有助于降低心血管疾病的患病风险。BLANCA等[70]研究表明,饮用发酵橙汁能显著提升谷胱甘肽和尿酸水平、增强抗氧化酶的活性、提高胆红素含量以及血浆的抗氧化能力,从而优化血脂状况,减少氧化形式的低密度脂蛋白,并在大鼠中保持白介素-6(IL-6)和C反应蛋白的水平,进一步表明在健康小鼠体内,发酵橙汁对于心血管疾病的潜在危险因素具有更强的防护效果。
尽管食用植物酵素在营养和健康方面有其优点,但也不可忽视其可能带来的一些安全隐患。制作过程中使用了大量植物原料,这导致由于原料和加工环境的差异,微生物种类和数量各不相同,使得酵素的发酵过程变得复杂且难以精确控制[71]。在含糖量高的条件下,某些微生物如酵母和乳酸菌,会快速增长,成为主导菌群,并为产品赋予独特的风味特性[72]。采用传统方式生产酵素时,产品容易遭受病原体污染,导致安全性低、品质不稳定,且难于规模化生产。深入了解酵素微生物的构造与成分对解决这些问题至关重要[73]。相比之下,采用人工接种技术具有显著优点,它能有效缩减发酵所需时间,提高生产效率。通过选用单一菌种进行发酵,可以有效避免其他微生物的污染,确保了发酵过程的纯净性。此外,人工接种技术还有助于产品质量的标准化和规范化,确保了其稳定性[74]。
人工接种发酵是广泛应用于提高产品质量、精确控制发酵工艺的一项重要技术,但操作不当或管理不当,可能会带来风险,也会带来问题。一方面是发酵工艺要靠具体菌种的选择。如果所选菌种的活性不高或被污染,则会对正常的发酵过程造成干扰,从而对产品质量造成影响,或使发酵作业无法正常进行。所以在选择菌种的时候一定要保证它的来源可靠,纯净度高,而且是适合食物发酵的种类。另一方面,温度、时间、湿度、pH值等关键参数必须在整个发酵过程中严格监测。不适当的环境条件可能会抑制菌种生长或产生代谢物,对产品质量和安全性都不利。发酵条件必须严格控制,才能保证人工接种发酵过程的顺利进行。此外,人工接种发酵过程也面临着环境污染的风险,可能会引入细菌、霉菌等污染源,如无菌环境维持不下去或消毒不到位等。这些外来微生物既可能抢夺营养成分,又可能对产品质量安全造成有害物质的释放和威胁。因此,为了保证人工接种发酵过程的成功,选对菌种、控制发酵条件、防止环境污染是必不可少的,必须以严格的操作规范和管理为基础,才能充分利用其优势,提高产品的质量和稳定性。
作为一种含有特定生物活性成分的产品,食用植物酵素在促进人体健康方面的潜力一直为人们所津津乐道。食用植物酵素的前景从目前的研究和发展趋势来看是十分可观的。对于食用植物酵素的研究,今后需要重点关注以下4个方面:(1)在食用植物酵素的制备过程中,首先要保证原材料的品质,其次应当选用玻璃材质或食品级塑料瓶,确保发酵容器无油无污染,最后发酵环境要避免阳光直射和过于潮湿,以防止酵素变质;(2)在食用植物酵素的发酵过程中,无论是自然发酵还是人工接种发酵,微生物污染是最需要注意的问题,纯化发酵菌种能够避免受到杂菌污染,从而使得纯化菌种能够更有效地应用于食用植物的发酵过程;(3)食用植物酵素目前主要在保鲜、改善口感等方面进行探索,随着加工工艺的不断创新和消费者需求的不断变化,食用植物酵素在食品工业中的应用将更加广泛;(4)在食用植物酵素的机制研究上更多地向人类健康倾斜,通过对食用植物酵素代谢过程及在人体内的作用机理的深入研究,为其健康功效的评估提供理论基础,从而为产品的研发及市场推广提供更为科学的依据。
综上所述,作为前景广阔、潜力巨大的健康食品,食用植物酵素今后将继续扮演其重要角色,期待在未来能够看到更多食用植物酵素产品的创新及研究成果。
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Modern food biotechnology has moved a long way since ancient times of empirical food fermentations. Preservation and safeguarding of food are, however, still major objectives of fermentation. In addition, other aspects, such as wholesomeness, acceptability and overall quality, have become increasingly important and valued features to consumers even in developing countries where old traditions and cultural particularities in food fermentations are generally well maintained. Due to limitations in infrastructure and existing low technologies, rural areas in most developing countries have not been able to keep abreast of global developments toward industrialisation. At the same time, fermented foods play a major role in the diet of numerous regions in Africa and Asia. In many traditional approaches, the advantages of some form of inoculation of a new batch, e.g. by back-slopping or the repeated use of the same container (e.g. a calabash) is appreciated and generally practised. Still, the benefits of small-scale starter culture application as a means of improved hygiene, safety and quality control, in support of HACCP approaches, are not yet realised in small-scale fermentation operations. Approaches and considerations for the selection of pure cultures for small-scale, low-tech applications may differ in some respects from the large-scale industrial approaches practised since 100 years. Selection criteria should take account of the substrate, technical properties of the strain, food safety requirements and quality expectations. Lack of experience in the application of starter cultures in small-scale operations and under rural conditions presents a major obstacle but also an exciting challenge to food microbiologist and technologist. Culture preservation, maintenance and distribution demand special logistic and economic considerations. Quality, safety and acceptability of traditional fermented foods may be significantly improved through the use of starter cultures selected on the basis of multifunctional considerations, also taking into account the probiotic concept and possibilities offered for improved health benefits.
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Fermented whole grain (WG) sorghum food products including WG-ting can be obtained from different sample sources and fermentation conditions, leading subsequently to variations in the molecular composition of the products. There is however, a lack of detailed understanding and description of differential molecular profiles of these food products. Thus, the current study is a nontargeted gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach to descriptively elucidate metabolic profiles of two WG-sorghum types [high tannin (HT) and low tannin (LT)] and their derived WG-ting products obtained via fermentation. Metabolites were extracted with 80% aqueous methanol and analyzed on a gas chromatography high resolution time of flight mass spectrometry (GC-HRTOF-MS) system. Chemometric methods such as principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA) were applied to mine the generated data. Our results showed that tannin contents influenced the composition of the raw sorghum and derived WG-ting samples. Metabolite signatures that differentiated raw HT- and LT-sorghum included cyclic compounds, pesticides, 2,4-di-tert-butylphenol, fatty acid esters, and sugar derivatives. Furthermore, fermentation of the HT- and LT-sorghum into WG-ting led to an increase in the levels of fatty acids, fatty acid esters and some other compounds which are vital from a dietary and health context. Equally observed were reduction of some phenols, cyclic compounds, a pesticide and ketone. Thus, the results demonstrated that the inherent metabolic composition of raw sorghum would lead to differential metabolic changes in the fermented products such as WG-ting, with subsequent dietary and health implications. Fermenting ting with Lactobacillus fermentum FUA 3321 was most desirable as relevant metabolites were observed in both HT- and LT-ting samples. Furthermore, the study highlights the applicability of GC-MS metabolomics in understanding WG-ting fermentation.Copyright © 2019 Elsevier Ltd. All rights reserved.
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Fermented foods are currently experiencing a re-discovery, largely driven by numerous health benefits claims. While fermented dairy, beer, and wine (and other alcoholic fermented beverages) have been the subject of intensive research, other plant-based fermented foods that are in some case widely consumed (kimchi/sauerkraut, pickles, kombucha) have received less scientific attention. In this chapter, the current knowledge on the microbiology and potential health benefits of such plant-based fermented foods are presented. Kimchi is the most studied, characterized by primarily acidic fermentation by lactic acid bacteria. Anti-obesity and anti-hypertension properties have been reported for kimchi and other pickled vegetables. Kombucha is the most popular non-alcoholic fermented drink. Kombucha's microbiology is remarkable as it involves all fermenters described in known fermented foods: lactic acid bacteria, acetic acid bacteria, fungi, and yeasts. While kombucha is often hyped as a "super-food," only antioxidant and antimicrobial properties toward foodborne pathogens are well established; and it is unknown if these properties incur beneficial impact, even in vitro or in animal models. The mode of action that has been studied and demonstrated the most is the probiotic one. However, it can be expected that fermentation metabolites may be prebiotic, or influence host health directly. To conclude, plant-based fermented foods and drinks are usually safe products; few negative reports can be found, but more research, especially human dietary intervention studies, are warranted to substantiate any health claim.© 2019 Elsevier Inc. All rights reserved.
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该研究以蓝莓为主要原料,以酵母菌、植物乳杆菌(Lactobacillus plantarum)、干酪乳杆菌(Lactobacillus casei)为发酵菌种制备蓝莓酵素,以超氧化物歧化酶(SOD)酶活性为考察指标,首先通过均匀设计试验确定复合菌种的最佳接种量;其次通过单因素试验,考察发酵时间、发酵温度、初始总可溶性固形物含量及料液比对SOD活力的影响;最后通过响应面试验获得最佳发酵工艺条件。结果表明,酵母菌、植物乳杆菌、干酪乳杆菌三种菌株的最佳接种量分别为0.1%、2%、0.47%,蓝莓酵素的最佳发酵工艺条件为发酵时间41.5 h,发酵温度31 ℃,初始总可溶性固形物含量12 °Bx,料液比1∶5(g∶mL),在此优化条件下,蓝莓酵素的pH值为3.14,酒精度为0.2%vol,总酚含量为3.14 mg/mL,花色苷含量为26.06 mg/mL,乳酸菌活菌数为1.01×10<sup>7</sup> CFU/mL,SOD酶活性为103.01 U/mL。
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以沙棘(Hippophae rhamnoides L.)为原料,利用酿酒酵母(Saccharomyces cerevisiae)、异常汉逊酵母(Hansenula anomala)和植物乳杆菌(Lactiplantibacillus plantarum)多菌种发酵制备沙棘酵素。以超氧化物歧化酶(SOD)、pH、可溶性固形物含量、总酸含量、感官评分、还原力等为评价指标,采用单因素试验及响应面法优化沙棘酵素多菌种发酵工艺。结果表明,沙棘酵素多菌种最佳发酵工艺为初始pH 5.18、酿酒酵母∶异常汉逊酵母∶植物乳植物杆菌1.0∶1.6∶2.6、接种量10.25%。在此工艺条件下,沙棘酵素产品的SOD活性最高,达到2 206.67 U/mL,pH为2.23,总酸含量为78.60 mg/mL,可溶性固形物含量为2.68 °Bx,乙醇含量为0.05 g/100 mL,总酚含量为18.85 mg/mL,总黄酮含量为12.49 mg/mL,维生素C(VC)含量为6.48 mg/mL,多糖含量为22.49 mg/mL,还原力(OD<sub>700 nm</sub>值)为2.65,产品色泽透亮金黄,香气浓郁,感官评分为9.1分,其理化指标及感官品质均符合相关标准要求。该研究为沙棘的综合利用和多菌种发酵酵素的广泛应用提供理论依据。
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以超氧化物歧化酶(SOD)活力为评价指标,通过单因素试验以及正交试验,对植物乳杆菌(Lactobacillus plantarum)发酵的北五味子麦芽酵素的发酵工艺条件进行优化;并对该酵素产品的抗氧化活性进行了研究。结果表明,植物乳杆菌发酵制备北五味子麦芽酵素的最佳发酵工艺条件为北五味子∶麦芽=2∶1(g∶g),发酵时间为3 d、接种量为1.5%、发酵温度为41 ℃。在此最佳工艺条件下,北五味子麦芽酵素SOD酶活力为3 464.80 U/mL。抗氧化活性结果表明,该酵素对超氧阴离子自由基清除能力、羟自由基清除能力、DPPH自由基清除能力和对ABTS+自由基清除能力分别达到了26.03%、97.25%、89.10%和0.613 7 mmol/L,表明该酵素具有较好的抗氧化活性。
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