Research Progress on the Application of Novel Non-thermal Sterilization Technologies in Fermented Fruit and Vegetable Products
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摘要: 发酵果蔬制品是我国传统发酵食品的重要组成部分。制作时,以新鲜蔬菜、水果为原料,利用其自然携带的乳酸菌、真菌等多种菌群相互作用进行发酵。发酵完成后,蔬菜中微生物组成和数量的失控常引起后酸化、软烂、腐败变质等问题,因而开发适用于发酵果蔬制品的杀菌技术具有重要的产业价值。相较于传统热杀菌的破坏作用,采用非加热方式的新型非热杀菌技术不仅可以针对性地杀灭致腐、致病微生物,还能降低对益生菌的影响,并极大限度地减缓发酵果蔬制品产品劣化,因而逐渐成为发酵果蔬制品杀菌领域的研究热点之一。本文综述了常用新型非热杀菌技术在发酵果蔬制品中的应用研究进展,对新型非热杀菌技术在发酵果蔬制品中的微生物的作用及影响因素进行概述,并探讨其对产品质量属性及安全属性的影响,以期为非热杀菌技术在发酵果蔬制品工业化生产中的规模化应用提供理论依据。Abstract: Fermented fruit and vegetable products are an important part of traditional fermented food in China. During production, fresh vegetables and fruits are used as raw materials. The fermentation is conducted by the interactions between microorganisms including lactic acid bacteria and fungi which are naturally carried by raw materials. After fermentation, the composition and quantity of microorganisms in fruits and vegetables tend to become out of control, which often leads to post-acidification, softening, spoilage and other problems. Therefore, the development of sterilization technology suitable for fermented fruit and vegetable products has important industrial value. Compared with the destructive effect of traditional heat sterilization, the non-thermal sterilization technology without heating can not only kill the spoilage and pathogenic microorganisms, but also reduce the impact on probiotics, and greatly alleviate the deterioration of fermented fruit and vegetable products. Therefore, non-thermal sterilization technologies have gradually become one of the research hotspots in the field of fermented fruit and vegetable products sterilization. In this paper, the application progress of common non-thermal sterilization technologies in fermented fruit and vegetable products is reviewed. The sterile effect and influencing factors of non-thermal sterilization technologies in fermented fruit and vegetable products are summarized. Its influence on quality and safety attributes of products is discussed. The aim of this study is to provide a theoretical basis for the large-scale application of non-thermal sterilization technologies in the industrial production of fermented fruit and vegetable products.
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发酵果蔬制品作为我国传统发酵食品的重要组成部分,主要以新鲜果蔬作为原料,利用果蔬自身携带或加入乳酸菌、真菌等微生物菌群作用发酵,最后制成泡菜、果蔬发酵汁等产品[1−2]。发酵果蔬制品不仅具有独特的风味、爽口的质地、鲜艳的色泽等特殊的感官性质,还保留了果蔬原有的维生素、膳食纤维等营养元素,让其成为了人们餐桌上不可或缺的美食[3]。然而,由于果蔬原料自身携带微生物,在发酵、包装及贮运过程中菌群组成和数量不断变化且难以控制,常出现过酸、胀气等现象,不仅影响产品品质,也存在安全性风险,常需要对果蔬原料及成品进行杀菌。常规热杀菌方法往往因其过高的热强度对果蔬感官和营养品质产生负面影响,因此适用于发酵果蔬的非热杀菌技术的发展创新对产品品质提升、货架期延长以及销售范围扩大具有重要作用[4−6]。
新型非热杀菌技术大部分是在物理方法的基础上衍生发展起来的一类新技术,在稳定产品质量属性和安全属性的前提下有效抑制有害微生物的生长[7]。新型非热杀菌技术大多采用声光电等高能光波或射线对微生物进行杀灭,适用于果蔬固体和液体食品的保鲜、包装减菌延长贮藏期,主要包括超高压杀菌技术、脉冲电场杀菌技术、脉冲强光杀菌技术、低温等离子体杀菌技术、高密度二氧化碳等[8−9]。这些非热杀菌技术单独或组合应用于食品加工时,表现出了优异的杀菌选择性和特异性,其杀菌效果主要受到温度、pH等环境因素及设备参数等技术因素的影响,通过环境因素与技术参数的配合调控,可提升杀菌选择性和特异性以优化杀菌效果[10]。此外,非热杀菌技术具有延缓发酵果蔬汁、泡菜等果蔬制品中维生素、膳食纤维等营养成分损失的作用,在保持发酵莲藕片、泡萝卜等发酵果蔬产品原有风味物质组成、抑制色泽褐变、减缓质地软烂的情况下,显著降低生物胺和亚硝酸盐等食品安全危害因素的积累,因而在发酵果蔬制品原料、成品杀菌方面均表现出了广阔的应用潜力[5,11]。
本文综述了近年来常见新型非热杀菌技术在发酵果蔬制品中的应用研究,从杀灭有害微生物菌群以及对益生菌菌群的影响两个方面对微生物的作用进行阐释,总结了影响杀菌效果的环境因素和技术因素,并概述了新型非热杀菌技术对发酵果蔬制品的感官属性及安全属性的影响,以期为新型非热杀菌技术在发酵果蔬制品产业中的规模化应用提供理论依据。
1. 新型非热杀菌技术概述
不同于传统热杀菌技术的热强度对食品的破坏作用,新型非热杀菌技术大多利用物理手段,采用高压、电场、磁场、射线等非加热形式使生物分子细胞壁、细胞膜及细胞相关生化功能发生改变,起到杀菌、钝酶以及改变食品结构及功能特性的作用,能最大限度维持食品感官和营养成分,延长货架贮藏期[12]。新型非热杀菌技术是工程技术、物理学、化学和微生物学结合的交叉学科,近年来已被广泛应用于肉类[13]、水产品[14]、果蔬[15]、乳类[16]等食品的保鲜、包装成品减菌等方面,是最具潜力的灭菌新技术。表1总结了近年来国内外研究者较为关注的几种新型非热杀菌技术灭菌机理及特点。
表 1 常用新型非热杀菌技术的类型及特点Table 1. Common types and characteristics of novel non-thermal sterilization technologies杀菌技术 灭菌机理 特点 参考文献 超高压杀菌
(high pressure processing,HPP)通常在100~1000 MPa压力下,改变分子间距,破坏微生物结构、酶的构象。 速度快、均匀 [17] 高压脉冲电场杀菌
(pulsed electric field,PEF)强电场脉冲使细胞膜穿孔,导致胞内物质泄漏流出,微生物生理生化终止。 功率消耗低、
强穿透力[4,18] 脉冲强光杀菌
(pulsed intense light,PL)利用紫外线至红外线区域(100~1100 nm)的广谱波长的强脉冲和短脉冲杀灭微生物。 瞬时、强效、节能、易控 [19] 超声波杀菌
(ultrasonic sterilization,US)超声波空化效应导致微生物结构损伤。 易于操作、成本低廉 [20] 电子束辐射杀菌
(radio sterilization)UV、X、γ射线使微生物分子化学键断裂或直接破坏细胞
结构。高效、耗时短 [21] 微波灭菌技术
(microwave,MW)频率在300 MHz~300 GHz的电磁波与微生物蛋白质等物质发生耦合,破坏微生物组成。 速度快、节能、易控 [22] 低温等离子体灭菌技术
(cold plasma technology,CPS)高压电极间放电产生活性粒子、光辐射、冲击波等复合物理化学效应破坏微生物结构。 能耗低、效率高、无污染 [23] 高压/高密度二氧化碳杀菌
(high pressure carbon dioxide,HPCD)加压增加CO2密度,使CO2渗透到细胞中发生酸化效应,降低胞内pH,破坏细胞膜,导致微生物失活。 新型高效、绿色、可持续 [24] 随着消费者对高品质、高营养果蔬食品的多样化需求增加,新型非热杀菌技术逐渐成为了食品加工新技术的焦点。在果蔬中的研究主要集中于如何在保证食品感官、营养的前提下对不同类型果蔬食品的原料保鲜及包装成品上实现高效杀菌,延长保质期。目前国内外研究已证明新型非热杀菌技术在果蔬液体和固体食品已有显著的杀菌效果。研究显示,超高压杀菌、高压脉冲电场杀菌、超声波杀菌、微波杀菌等利用高压力学、电学、磁学作用机理,具有高效、无残留、热效应低、作用均匀等优势适用于果蔬液体制品杀菌中,保障“原汁原味”。如Buitimea-Cantúa等[25]利用了HPP和PEF技术在高压及强电场下作用于覆盆子汁,可明显抑制覆盆子汁中的霉菌和酵母生长,改善极易变质的覆盆子汁的品质;让一峰等[26]利用高压脉冲电场可以短时间、低能耗、高效率地对哈密瓜汁进行强化杀菌,不仅能杀灭引起变质的微生物,还对其理化指标基本无影响;Danshi等[27]利用微波、超声波、超高压灭菌技术几种技术处理发酵苹果汁,不仅能在不同程度上杀灭微生物,还能保证发酵苹果汁的特色风味。脉冲强光杀菌、电子束辐射杀菌、高密度二氧化碳杀菌、低温等离子体的研究大多集中于果蔬固体原料杀菌及产品包装上,如韦雪等[28]探究了脉冲强光联合气调包装对鲜切马铃薯杀菌的影响,在12 J/cm2的脉冲强光下还能维持马铃薯的硬度和原有滋味。孟宪伟等[29]使用低温等离子体技术处理鲜切胡萝卜,对胡萝卜无损伤,并能明显延缓品质劣变。侯新磊等[30]利用低温等离子体技术于四川泡菜中,结果显示,低温等离子体能明显抑制泡菜贮藏过程中腐败微生物的生长。Lee等[31]利用HPP技术处理包装后的韩国泡菜,能明显消除不利微生物的生长,延长泡菜保质期。Zambon等[32]利用二氧化碳作为香菜叶、胡萝卜、椰子三种果蔬的包装填充气体,并高压处理使其达到超临界二氧化碳条件,结果表明高压二氧化碳可有效减缓包装后的微生物生长并能延长新鲜果蔬的货架期。
尽管国内外专家已经研究证明了新型非热杀菌技术在果蔬食品上的潜在用途,但其还存在一些局限性,仍需不断深入研究,使其达到商业化需求。
2. 新型非热杀菌技术对发酵果蔬制品中微生物的作用及影响因素
2.1 新型非热杀菌技术对发酵果蔬制品中微生物的作用
果蔬常因含水量高、营养物质丰富、植物组织易损而容易被微生物污染,在发酵、包装、贮运等加工过程中微生物组成和数量也会不断变化,导致果蔬发酵制品发生腐败变质。冷藏是发酵果蔬制品贮藏的常用方法,其原理是低温可以抑制产品中有害微生物的生长、降低酶活性、减缓生化反应的发生,但冷藏同时也会在一定程度上抑制乳酸菌的发酵,对发酵果蔬的风味、营养等产生一定的负面影响[33]。随着发酵蔬菜的“低盐”、发酵果蔬汁的“健康”越发受到消费者的青睐,新型非热杀菌技术能对有害微生物进行干预并保留有益微生物稳定发酵品质成为了优势。
2.1.1 新型非热杀菌技术对有害微生物的作用
发酵果蔬制品是指以一种或多种新鲜水果、蔬菜为原料,经过酵母菌、乳酸菌等多种有益菌发酵而成的功能性发酵食品,主要有泡菜、发酵果蔬汁等。由于果蔬原料自身携带或发酵加工过程中污染致腐或致病微生物,会引发公共卫生问题,带来巨大经济负担。因此迫切需要通过适当的加工方法来提高发酵果蔬制品的微生物安全性。新型非热杀菌技术可破坏腐败菌和致病菌等有害微生物的结构,从而保障果蔬发酵品质。近期研究表明,新型非热杀菌技术在对发酵果蔬原料进行杀菌时表现出了优越的微生物特异性和针对性,对果蔬中存在的有害微生物进行杀灭或减除,如表2所示。
表 2 新型非热杀菌技术对有害微生物的杀灭效果Table 2. Sterile effect of novel non-thermal sterilization techniques on harmful microorganisms来源 非热杀菌技术及处理参数 有害微生物 杀灭效果 参考文献 莲藕酱 超高压HPP(150~550 MPa)+CO2包装 大肠杆菌Escherichia coli O157:H7 低于检测限度 [34] 芒果 紫外-C辐照 大肠杆菌Escherichia coli
阪崎肠杆菌Cronobacter sakazakii低于检测限度
减少2.4~2.6 lg CFU/g[35] 樱桃汁 超高压(550 MPa 2 min) 霉菌、酵母 低于检测限度 [36] 萝卜 微波低温等离子体(900 W 667 Pa 10 min) 鼠伤寒沙门氏菌Salmonella typhimurium 低于检测限度 [37] 超声波US(25 kHz)+
大气低温等离子体(400~800 Hz 1 min)大肠杆菌Escherichia coli O157:H7 减少0.71 lg CFU/g 鼠伤寒沙门氏菌Salmonella typhimurium 减少0.59 lg CFU/g Feijoa果泥 超高压HPP(400/250/200 MPa;4/2 min)+密集相CO2(0.4% w/w) 大肠杆菌Escherichia coli
枯草芽孢杆菌Bacillus subtilis
酿酒酵母Saccharomyces cerevisiae减少1.7~4.3 lg CFU/g
减少>6.5 lg CFU/g
减少4.7~6.2 lg CFU/g[38] 胡萝卜
青菜超声波US+紫外-C发光二极管UV-C LED 大肠杆菌Escherichia coli O157:H7
大肠杆菌Escherichia. coli O104:H4
产肠毒素a的金黄色葡萄球菌
enterotoxin A-producing S. aureus均低于检测限度 [39] 苹果汁 超高压HPP(600 MPa 7 min)
脉冲电场PEF(0.76 kV/cm 40 ℃ 2 μs/个共21个脉冲)酿酒酵母
Saccharomyces cerevisiae低于检测限度 [40] 蓝莓 脉冲光PL(0.15 J/cm2和 0.3 J/cm2;3脉冲/s)+洗涤水
紫外线(~13和28 mW/cm2)+洗涤水沙门氏菌
Salmonella减少4.5~5.7 lg CFU/g [41] 番茄 超高压HPP(600 MPa 7 min)
脉冲电场PEF(0.76 kV/cm 40 ℃ 2 μs/个共21个脉冲)酿酒酵母
Saccharomyces cerevisiae减少4.4~5.4 lg CFU/g [40] 生菜 脉冲光PL(每脉冲~0.15和0.3 J/cm2;3脉冲/s)+洗涤水
紫外线(~13和28 mW/cm2)+洗涤水沙门氏菌
Salmonella减少1.9~3.1 lg CFU/g [41] 研究显示,新型非热杀菌技术在合理的参数条件下对有害菌的杀菌率最大可降低大约5 lg CFU/g的要求,延长了发酵果蔬制品的保质期并提高了食品安全性,在发酵果蔬制品灭菌上具有巨大潜力。首先,新型非热杀菌技术能明显抑制致腐微生物的生长。如超高压技术可以降低加速胡萝卜汁风味劣化的酿酒酵母的生长[42],低温等离子体可以减少泡菜“生花”的膜醭毕赤酵母的数量[43],超高压技术、高压二氧化碳技术、高压脉冲电场技术、紫外辐照技术独立或相结合处理,可有效减少“顽固”的芽孢杆菌的数量[44]。其次,还可以杀灭果蔬来源的致病性大肠杆菌、金黄色葡萄球菌、单核细胞增生李斯特菌、沙门氏菌等致病微生物[45]。如Lee等[31]研究了超高压灭菌技术在600 MPa下处理韩国叶用芥菜泡菜,处理后大肠杆菌的初始数量低于检测限度(1 lg CFU/g)。Pan等[46]使用低温等离子体处理新鲜果蔬,能完全灭活果蔬表面大肠杆菌。Lee等[39]等研究了超声波和紫外线-C发光二极管(UV-C LED;275 nm)联合处理可减缓胡萝卜、芹菜、辣椒粉和卷心菜等蔬菜中致病性大肠杆菌和金黄色葡萄球菌的污染。Huang等[41]采用脉冲光和紫外线结合水洗涤蓝莓、番茄、生菜新鲜果蔬,能明显降低西红柿洗涤水中沙门氏菌存活率。
相较于传统杀菌而言,新型非热杀菌技术对致腐和致病菌有着优越的特异性和选择性优势,但仍存在着一定的局限性。微生物会因高压、辐射等因子诱发细胞的亚致死损伤和活的非可培养(VBNC)现象的产生[47]。如Nasiłowska等[48]研究了HPP(300~500 MPa)处理胡萝卜汁和甜菜汁,结果发现胡萝卜汁在400 MPa的压力下处理5 min会触发李斯特氏菌的亚致死损伤,大肠杆菌会在500 MPa触发;在甜菜汁中,400 MPa下处理10 min会导致两种菌亚致死损伤现象。因此,有关新型非热杀菌技术诱导有害菌亚致死形态和VBNC状态需要得到业内高度重视,探明不同非热杀菌技术诱导的机制,对规避食品安全风险具有重要意义。
2.1.2 新型非热杀菌技术对有益微生物的作用
益生菌在发酵过程中产生细菌素、胞外多糖、多酚类物质、酶等多种功效成分,使得发酵果蔬制品具有改善营养健康、调节胃肠道功能等作用[49]。因此在使用新型非热杀菌技术杀灭有害微生物的同时仍需保留有益微生物的数量,改善发酵果蔬制品的功能性作用。目前,国内外许多研究已证明新型非热杀菌技术对乳酸菌的影响较小,甚至改善乳酸菌发酵。如Zhang等[50]研究了低温等离子体(700 W/120 s)、脉冲强光杀菌(1.0 Hz/600 J/10次)处理植物乳杆菌(Lactobacillus plantarum)发酵的枣汁益生菌饮料,结果表明,与未杀菌处理相比,L. plantarum在枣汁中的生长能力不受两种杀菌处理的影响,甚至更有利于提高枣汁发酵性能的稳定。刘秋豆[51]使用紫外辅助超声波处理发酵芒果汁,结果显示与水浴灭菌相比,紫外辅助超声波灭菌后植物乳杆菌、嗜热链球菌、鼠李糖乳杆菌、嗜酸乳杆菌的增殖倍数更高,且紫外辅助超声波灭菌处理后,益生菌总数显著高于水浴灭菌中益生菌总数。Ma等[52]比较了HPP和巴氏杀菌后苹果汁发酵中L. plantarum的存活率,经HPP处理后,该菌种的生存能力增加了约2 lg CFU/g,改善了苹果汁发酵特性。
2.2 影响杀菌效果的因素
新型非热杀菌技术涉及的杀菌原理极其复杂,其杀菌效果受到多种因素的影响,主要包括温度、pH等环境因素和电压、功率、时间等处理参数的影响。
2.2.1 环境因素
杀菌时,温度、pH等环境因素对新型非热杀菌技术的杀菌效果有显著的影响。不同温度、不同pH条件下会导致微生物对新型非热杀菌技术的压力、电磁、光辐射以及活性粒子等敏感性会有所不同,呈现出的杀菌效果也会有所差异。例如,朱香澔等[53]使用超高压杀菌技术对西番莲果汁进行杀菌,当果汁环境温度在42.5 ℃时,能将果汁中的微生物全部杀灭。钱静亚等[54]研究了枯草芽孢杆菌介质温度对脉冲磁场杀菌效果的影响,发现低温和高温条件下均可强化磁场的杀菌效果,对细胞膜的破坏性更为严重。Muranyi等[55]研究发现,低温等离子体在pH5的偏酸性条件下可将芽孢杆菌数量减少4.7 lg CFU/g;而中性pH下仅减少2.1 lg CFU/g。洪晨等[56]研究不同pH下脉冲强光对大肠杆菌的影响时,发现当pH4和pH8时,目标菌数量均降低3个以上,而中性pH下仅减少2个。综上所述,在保证食品质量与安全的前提下,科学性地改变食品基质的温度、pH等环境因素可有效提高非热杀菌技术对有害菌的抑制能力。
2.2.2 技术因素
发酵果蔬制品中大多数产品都是多菌共酵体系下的结果,为了达到预期杀菌目的,新型非热杀菌技术需要设置一定的杀菌参数并结合杀菌动力学研究,破坏微生物细胞壁、膜等结构、损伤蛋白质及酶活性等方面达到杀灭不同种类微生物的目的。新型非热杀菌技术大多是处理压力、功率与时间等技术参数会对杀菌效率产生影响。
压力、功率与时间是超高压、高压脉冲电场、高压二氧化碳、超声波灭菌、低温等离子体等新型非热杀菌技术的关键。研究显示,压力参数越大,时间越长,破坏细胞膜结构、钝酶能力更强,杀菌效果越明显。然而,杀菌参数需要根据不同食品基质及微生物特性进行合理设计。如Feng等[57]为了最大程度保证果汁品质并杀灭微生物,选择超高压杀菌技术和超声波技术于草莓-苹果-柠檬汁混合果汁中,通过多次实验后,得出在超高压压力及时间参数设置为500 MPa/15 min及超声功率设置在376 W/10 min时,才能最大限度降低霉菌及大肠杆菌数量。Fan等[58]研究了将微波功率设置为3250 W/30 s可最适合用于制备低盐豆腐的参数。Wu等[59]使用低温等离子体处理樱桃时,与过强的等离子强度参数(80 kV/80 s和60 kV/140 s)相比,在60 kV/30 s或100 s时是杀灭樱桃表面微生物和保证外观平整的最优参数。孙新[60]研究了高密度二氧化碳(HPCD)处理胡萝卜泡菜时菌落总数和乳酸菌总数的减菌效果与压力和时间呈正相关,然而HPCD处理条件为20 MPa、30 min时,在减菌、质构、细胞完整性和营养保留方面起到的促进作用明显优于较高或较低的处理条件。
虽然在大多数情况下,压力、功率、时间等技术参数与杀菌效果成正比,但考虑到时间成本及食品特性等问题,非热杀菌技术的参数需要进行合理设计才能达到最理想的灭菌状态。
3. 新型非热杀菌技术对发酵果蔬制品品质的影响
3.1 质量属性
3.1.1 营养品质
膳食纤维和维生素、酚类化合物、黄酮类化合物等生物活性物质都是发酵果蔬制品的重要营养成分。这些营养成分常在杀菌及贮藏过程中发生流失,非热杀菌技术不仅可以保留维生素、酚类、黄酮类等活性物质,还能改善不溶性膳食纤维等营养成分[23]。
不溶性膳食纤维(IDF)是膳食纤维的一种,具有促进肠道蠕动,改善肠道菌群以等有益的生理作用,但其颗粒尺寸较大会对感官特性和功能有不利影响,需要适度改善尺寸大小促进摄取。研究表明,新型非热杀菌技术不仅可以保留总膳食纤维含量,还能改善不溶性膳食纤维结构。如Yu等[61]采用超高压技术处理泡菜,与空白对照相比,实验组泡菜中IDF结构更松散,晶体强度和热稳定性更低,明显改善了IDF结构及功能。Feng等[62]采用电子辐照处理大豆中不溶性膳食纤维,发现电子辐射技术可以破坏IDF晶体的结构,在6 kGy剂量的电子辐照下可以得到IDF的最小颗粒和最大比表面积,增加表观黏度,改善大豆中IDF功能。Chen等[63]采用高静水压处理卷心菜粉,其总膳食纤维含量(36.06%±1.65%)明显高于未经处理的卷心菜粉。李爽[64]采用超声波处理发酵豆渣,得出经超声波处理后豆渣膳食纤维粒径减小,表面积增加,豆渣纤维表面变得疏松多孔,显著提升了发酵豆渣膳食纤维的持水、持油及膨胀能力。
维生素C作为果蔬中最为主要的营养成分,由于其热稳定性低,易于氧化等特性,在加工中需避免高温、曝气等过程导致维生素C发生不利变化。目前,已有研究证明,部分非热杀菌技术能最大限度保留果蔬中维生素C含量,但对发酵产品中的维生素C含量及影响机制还尚不明确。如Mieszczakowska-Frąc等[65]在分析创新技术对水果和蔬菜产品维生素C含量影响的研究中表示,超高压(HPP)、超声波(US)、脉冲电场(PEF)同巴氏杀菌相比,对新鲜果蔬中维生素C有显著的保护潜力。李树锦等[66]利用辐照技术于苦瓜和圣女果中,结果显示辐照技术对维生素C的影响并不明显。
此外,使用非热菌技术还能保留并增加其他活性物质(如酚类化合物、胡萝卜素、类黄酮等)。例如,Viacava等[67]评估了静态和多脉冲轻强度高静压处理胡萝卜,结果发现,经过处理后的胡萝卜游离酚类和结合酚类分别增加了163.05%、36.95%,类胡萝卜素中的总叶黄素增加了27.61%。还有研究发现低温等离子体处理后有利于发酵红葡萄酒的酚类化合物的保存[68]。Yuan等[69]研究了低温等离子体、超高压、高压二氧化碳作为预处理对真空冻干枣片性能的影响,发现非热杀菌技术对冻干枣片总苯酚含量、总类黄酮、抗坏血酸含量和抗氧化能力均显示提升。
以上研究结果表明,新型非热杀菌技术不仅能保留还能改善果蔬原料及发酵产品的营养品质,还能有效避免或减少传统热处理对生物活性化合物的不良影响。
3.1.2 感官品质
发酵果蔬制品的感官品质对于消费者来说尤为重要,其风味(包括滋味、气味)、色泽、质地等都会直接影响消费者对发酵果蔬制品好坏的判定。发酵果蔬制品在贮藏过程中通常会因微生物组成结构的不稳定导致质构软化、色泽褐变、风味过酸或出现异味等现象。研究表明,新型非热杀菌技术是最大限度保持并能改善发酵果蔬制品的风味、色泽、质地等感官品质的加工杀菌技术。
风味是发酵产品品质的核心和灵魂,其特定的挥发性风味物质可为发酵果蔬制品的独特的气味做出巨大贡献;非挥发性物质(糖类、有机酸、氨基酸等)可为发酵果蔬制品的滋味提供物质基础[70]。杜喜玲[71]检测了泡菜在不同压力参数下的超高压技术处理后的风味物质,结果发现在一定的压力时间下,超高压技术处理大大增加了风味物质的种类与数量(醇类、酯类、酚类)。Yuan等[72]研究了超高压技术生产腌制的中国莲藕片的风味物质,与空白组的腌莲藕片产生18种挥发性化合物相比,HPP处理后的莲藕片产生了32种更利于风味的挥发性化合物。高苏敏等[73]研究了超声波协同低盐处理萝卜泡菜,与传统自然腌制泡菜相比,相同盐含量下,超声波组泡菜水中有机酸和游离氨基酸含量更高,挥发性风味物质种类更丰富,并在超声波组样品中检测出的二甲基三硫阈值小,对风味贡献更大。综上,新型非热杀菌技术在改善发酵果蔬风味物质的方面起到了积极作用。
色泽也是发酵果蔬制品重要的品质属性之一[74]。在果蔬中,花青素发挥着重要作用,是最大、最多样化的植物色素组,负责各种水果和蔬菜的红色、紫色和蓝色等色泽。Marszalek等[75]研究对比了超高压处理、高压二氧化碳、高压均质化三种高压技术对果蔬中花青素的影响,指出三种技术均能在一定程度上减少花青素的流失,而高压二氧化碳对花青素在加工和储存过程中流失更少。Ma等[76]研究巴氏杀菌、臭氧、超声波、高压静水压力处理后李子汁的褐变程度及颜色变化,结果表明高压静水压力灭菌处理后的李子汁褐变程度最小。
质地也是腌制工艺类发酵果蔬制品的重要感官指标。对于泡菜而言,爽脆的质地更受到人们的喜爱,而温度、酸度、微生物是导致泡菜质地变软的主要因素。新型非热杀菌技术不仅可避免因加热导致果蔬软化,同时也能杀灭引起质构软烂的微生物。例如微波杀菌可作为莴苣泡菜的杀菌方式应用于生产,与巴氏杀菌泡菜相比,微波杀菌可明显提升泡菜硬度及弹性[77]。赵楠等[78]使用微波辅助低温等离子体处理榨菜,与巴氏杀菌相比,榨菜硬度和质地明显提升34%和44%,改善了榨菜贮藏期质构软化问题。
目前,大量实验室研究已证实新型非热杀菌对发酵果蔬感官品质的影响较小,甚至起到积极作用,但由于扩大化生产技术及生产设备的限制,仍需逐步探索适用于发酵果蔬制品规模化生产的非热杀菌技术,维持产品品质。
3.2 安全属性
果蔬在发酵过程中,含氮物质在微生物的作用下产生亚硝酸盐和生物胺,过量的摄入会严重影响人类身体健康,致使中毒、癌症甚至死亡,危害产品安全品质。与传统杀菌技术相比,新型非热杀菌技术的应用具有压低亚硝峰、减缓生物胺积累的效果。
研究证明新型非热杀菌技术可以减缓发酵果蔬制品中亚硝酸盐的产生,使其稳定在国家安全食品标准(<20 mg/kg)范围内。李昌宝等[79]研究了巴氏杀菌、微波杀菌、臭氧杀菌三种杀菌方式对低盐腌渍黄瓜的亚硝酸盐含量的影响,发现微波杀菌和臭氧杀菌更可将亚硝酸盐含量降低致0.31 mg/kg。王媛[80]研究了低温等离子体于泡菜发酵过程中亚硝酸盐的消长机制,与未经过处理的空白组发酵液(发酵3 d达28.51 mg/L,>20 mg/kg的国家标准)对比,经等离子体处理后的泡菜发酵液亚硝酸盐含量一直保持在国家标准范围内,且在整个发酵过程中未出现“亚硝峰”。
生物胺是一种对人类健康产生负面影响的含氮化合物,其含量主要是由于酵母或乳酸菌等微生物代谢产生氨基酸脱羧后的结果。新型非热杀菌技术在减缓生物胺的积累呈现优势。Niedźwiedź等[81]研究了低温等离子体技术对红葡萄酒酚醛成分和生物胺含量的影响,与传统保存方法(添加30和100 mg/L的亚硫酸钾)和联合方法(30 mg/L亚硫酸钾组合低温等离子体)进行比较,结果表明低温等离子体处理后,在贮藏期生物胺的含量呈现出下降的结果。
4. 结论与展望
新型非热杀菌技术大多采用物理方法进行杀菌,具有低温、高效、穿透力强、安全无残留等特点,因而被广泛应用于果蔬原料灭菌中。新型非热杀菌技术在果蔬制品杀菌时表现出了优越的微生物特异性、选择性,可对致病、致腐微生物进行选择性杀灭或减除,并保留益生乳酸菌。在合适的温度、pH等环境条件下,合理设置参数或协同其他杀菌技术可以明显提高杀菌效果。还可以达到保留蔬菜营养品质(维生素C、膳食纤维等)风味、色泽、质地等感官品质的目的,同时保证产品安全性,因而为发酵果蔬制品的健康化、安全化生产提供了技术保障。
然而,由于扩大化生产技术及配套设备的限制,新型非热杀菌技术在发酵果蔬制品工业中的规模化应用仍较少,其中辐射灭菌、微波灭菌、臭氧灭菌技术等的应用普遍成熟,低温等离子体灭菌技术、超高压灭菌技术、高压二氧化碳灭菌技术等技术尚未实现工业化,亟待开发适用于规模化、工业化发酵果蔬制品生产的高通量非热杀菌技术。此外,目前新型非热杀菌技术在发酵果蔬制品领域的应用多集中于对蔬菜原料或包装材料的杀菌或减菌,其在发酵果蔬成熟产品中的应用潜力仍待挖掘。发酵果蔬制品作为多菌共酵的复杂体系,非热杀菌工艺在发酵果蔬成品中的适应性改造将有利于其扩大杀菌范围的优势。同时,由于非热杀菌技术原理复杂,往往涉及多种因素,非热杀菌技术对于特定微生物的杀菌作用靶点、决定杀菌选择性的影响因素还认识不清,对其杀菌选择性与适用性的作用机制阐释将有助于其杀菌效果的进一步优化。因此,通过扩展新型非热杀菌技术的适用性范围,挖掘选择性杀菌的作用机制,突破杀菌效率与通量的技术瓶颈,非热杀菌技术将在发酵果蔬制品领域展现更大的应用潜力,助力发酵果蔬制品产业的高质量发展。
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表 1 常用新型非热杀菌技术的类型及特点
Table 1 Common types and characteristics of novel non-thermal sterilization technologies
杀菌技术 灭菌机理 特点 参考文献 超高压杀菌
(high pressure processing,HPP)通常在100~1000 MPa压力下,改变分子间距,破坏微生物结构、酶的构象。 速度快、均匀 [17] 高压脉冲电场杀菌
(pulsed electric field,PEF)强电场脉冲使细胞膜穿孔,导致胞内物质泄漏流出,微生物生理生化终止。 功率消耗低、
强穿透力[4,18] 脉冲强光杀菌
(pulsed intense light,PL)利用紫外线至红外线区域(100~1100 nm)的广谱波长的强脉冲和短脉冲杀灭微生物。 瞬时、强效、节能、易控 [19] 超声波杀菌
(ultrasonic sterilization,US)超声波空化效应导致微生物结构损伤。 易于操作、成本低廉 [20] 电子束辐射杀菌
(radio sterilization)UV、X、γ射线使微生物分子化学键断裂或直接破坏细胞
结构。高效、耗时短 [21] 微波灭菌技术
(microwave,MW)频率在300 MHz~300 GHz的电磁波与微生物蛋白质等物质发生耦合,破坏微生物组成。 速度快、节能、易控 [22] 低温等离子体灭菌技术
(cold plasma technology,CPS)高压电极间放电产生活性粒子、光辐射、冲击波等复合物理化学效应破坏微生物结构。 能耗低、效率高、无污染 [23] 高压/高密度二氧化碳杀菌
(high pressure carbon dioxide,HPCD)加压增加CO2密度,使CO2渗透到细胞中发生酸化效应,降低胞内pH,破坏细胞膜,导致微生物失活。 新型高效、绿色、可持续 [24] 表 2 新型非热杀菌技术对有害微生物的杀灭效果
Table 2 Sterile effect of novel non-thermal sterilization techniques on harmful microorganisms
来源 非热杀菌技术及处理参数 有害微生物 杀灭效果 参考文献 莲藕酱 超高压HPP(150~550 MPa)+CO2包装 大肠杆菌Escherichia coli O157:H7 低于检测限度 [34] 芒果 紫外-C辐照 大肠杆菌Escherichia coli
阪崎肠杆菌Cronobacter sakazakii低于检测限度
减少2.4~2.6 lg CFU/g[35] 樱桃汁 超高压(550 MPa 2 min) 霉菌、酵母 低于检测限度 [36] 萝卜 微波低温等离子体(900 W 667 Pa 10 min) 鼠伤寒沙门氏菌Salmonella typhimurium 低于检测限度 [37] 超声波US(25 kHz)+
大气低温等离子体(400~800 Hz 1 min)大肠杆菌Escherichia coli O157:H7 减少0.71 lg CFU/g 鼠伤寒沙门氏菌Salmonella typhimurium 减少0.59 lg CFU/g Feijoa果泥 超高压HPP(400/250/200 MPa;4/2 min)+密集相CO2(0.4% w/w) 大肠杆菌Escherichia coli
枯草芽孢杆菌Bacillus subtilis
酿酒酵母Saccharomyces cerevisiae减少1.7~4.3 lg CFU/g
减少>6.5 lg CFU/g
减少4.7~6.2 lg CFU/g[38] 胡萝卜
青菜超声波US+紫外-C发光二极管UV-C LED 大肠杆菌Escherichia coli O157:H7
大肠杆菌Escherichia. coli O104:H4
产肠毒素a的金黄色葡萄球菌
enterotoxin A-producing S. aureus均低于检测限度 [39] 苹果汁 超高压HPP(600 MPa 7 min)
脉冲电场PEF(0.76 kV/cm 40 ℃ 2 μs/个共21个脉冲)酿酒酵母
Saccharomyces cerevisiae低于检测限度 [40] 蓝莓 脉冲光PL(0.15 J/cm2和 0.3 J/cm2;3脉冲/s)+洗涤水
紫外线(~13和28 mW/cm2)+洗涤水沙门氏菌
Salmonella减少4.5~5.7 lg CFU/g [41] 番茄 超高压HPP(600 MPa 7 min)
脉冲电场PEF(0.76 kV/cm 40 ℃ 2 μs/个共21个脉冲)酿酒酵母
Saccharomyces cerevisiae减少4.4~5.4 lg CFU/g [40] 生菜 脉冲光PL(每脉冲~0.15和0.3 J/cm2;3脉冲/s)+洗涤水
紫外线(~13和28 mW/cm2)+洗涤水沙门氏菌
Salmonella减少1.9~3.1 lg CFU/g [41] -
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