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冯疆蓉, 李春杰. 生鲜乳有害微生物污染与危害分析. 草业科学, 2016,33(9):1875-1892
Feng Jiang-rong, Li Chun-jie. Pollution and hazard analysis of harmful microorganisms in raw milk. Pratacultural Science,2016,33(9): 1875-1892  
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《草业科学》编辑部
生鲜乳有害微生物污染与危害分析
冯疆蓉, 李春杰
草地农业生态系统国家重点实验室 兰州大学草地农业科技学院,甘肃 兰州 730020
通信作者:李春杰(1968-),男,甘肃镇远人,教授,博导,博士,研究方向为禾草-内生真菌共生体及草类植物病理学。E-mail:chunjie@lzu.edu.cn

第一作者:冯疆蓉(1991-),女,甘肃白银人,在读硕士生,研究方向为生鲜乳微生物研究。E-mail:fengjr14@lzu.edu.cn

收稿日期: 2016-04-20
基金: 公益性行业(农业)科研专项课题(201403071-6)
摘要

近年来乳制品质量安全是社会关注的焦点,而作为乳制品供应链最上游的生鲜乳的质量安全是乳业持续健康发展的根本保证,其可能含有的乳源性病原体和腐败菌等有害微生物严重影响乳品质量和安全。本文综述了生鲜乳中有害微生物的来源、生鲜乳种类和冷藏、巴氏杀菌对有害微生物的影响,以及主要致病菌的发生、危害和研究现状,以期为监督管理乳制品生产、加工和食物安全提供基础依据。

关键词: 生鲜乳; 乳源性病原体; 巴氏杀菌; 李斯特菌; 金黄色葡萄球菌
中图分类号:TS252.2 文献标志码:A 文章编号:1001-0629(2016)9-1875-18 doi: 10.11829/j.issn.1001-0629.2015-0705
Pollution and hazard analysis of harmful microorganisms in raw milk
Feng Jiang-rong, Li Chun-jie
State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China
Corresponding author: Li Chun-jie E-mail:chunjie@lzu.edu.cn
Abstract

The quality and safety of dairy products is the focus of society in recent years. Raw milk, as the upstream of the dairy supply chain, is the fundamental guarantee for sustainable development of dairy industry, which might be contaminated a variety of microorganisms, in which some of microorganisms are associated with human illness and milk spoilage. This paper stated the source and varieties of harmful microorganisms, and their refrigeration. And then this paper discussed the effect of pasteurization on harmful microorganisms, and the occurrence, hazards and current research of several milkborne pathogens. These would provide basis for dairy production.

Keyword: raw milk; milkborne pathogens; pasteurization; Listeria monocytogenes; Staphylococcus aureus

牛奶被公认为是自然界营养最为均衡的全价食品, 它几乎含有人体所需的所有大量营养元素(如蛋白质、脂质和碳水化合物)以及微量营养元素(如维生素和酶), 不仅是哺乳动物幼崽的营养来源, 也为人类提供营养物质, 在人类饮食结构中具有不可替代的作用和地位[1, 2]。因此, 生鲜乳作为乳制品供应链最上游的初始产物, 其质量安全将直接关系到乳与乳制品的质量和安全。

生鲜乳是微生物生长的理想培养基, 因此含有复杂而多样化的微生物种群, 现检测到的微生物已超过100个属, 400多种, 包括革兰氏阴性细菌(≥ 90种)、革兰氏阳性细菌(≥ 90种)、过氧化氢酶阳性菌(≥ 90种)、乳酸菌(≥ 60种)、酵母菌(≥ 70种)和霉菌(≥ 40种)[3], 包括降低牛奶品质、影响消费者身体健康甚至导致死亡的有害菌, 其以细菌为主, 如结核菌(Mycobacterium tuberculosis)、沙门氏菌(Salmonella)、大肠杆菌(Escherichia coli)、李斯特菌(Listeria monocy-togenes)、弯曲杆菌(Campylobacter)、金黄色葡萄球菌(Staphylococcus aureus)和空肠弯曲菌(Campylobacter jejuni)等曾引起人类多次疾病暴发(表1)。而嗜冷菌、噬菌体和梭菌(Clostridium)等均可导致奶酪、液体奶和一些新鲜乳制品的腐败[28], 此外, 酵母菌和霉菌也与生鲜乳及其乳制品的腐败有关, 近年来在生鲜乳中检测到金黄杆菌属(Chryseobacterium)[29]和Epilithonimonas[30], 但其是否会引起牛奶的变质还有待进一步研究(表1)。因此, 明确生鲜乳中有害微生物的来源及其冷藏和巴氏杀菌对微生物的影响以及重要腐败菌和致病菌的危害与防治, 将有助于帮助企业和农户提升生鲜乳生产质量。

表1 历史上牛奶源性疾病暴发的统计情况 Table 1 Reported human cases and outbreaks of milk-borne diseases in history
1 生鲜乳有害微生物的来源及影响因子

健康的泌乳牛所产的生鲜乳可能由于前期乳房挤奶准备不足、牛奶处理程序的不当以及与挤奶和存储设备相关的一些不好的卫生习惯而引起污染[31], 生鲜乳中的大多数菌在牛棚和挤奶厅中都可以检测到[32], 泌乳牛、牛舍(垫料及管理)、挤奶和存储设备、乳头与乳管、挤奶工、饲料、清洗水、空气和土壤等其它环境因子均可能成为生鲜乳微生物的来源[32, 33, 34]。

1.1 牛场设备及卫生管理

挤奶厅的卫生环境状况可直接影响到肠杆菌(Enterobacter iaceae)污染生鲜乳的程度[35], 如蜡状芽孢杆菌(Bacillus cereus)和假单胞菌属分别可从土壤、饲料、挤奶设备、乳房、牛奶罐和水中进入生鲜乳[36], 大肠杆菌可随清洗设备和牛体的水进入生鲜乳[37, 38], 而且相关设备和泌乳牛体清洗得不彻底或者挤奶工卫生习惯不良好也可能引起金黄色葡萄球菌的严重污染[39]。

粪便和牛床是大肠菌群和环境链球菌的主要来源[40], 生存于牛床材料上的葡萄球菌和一些革兰氏阴性菌可通过乳头和乳管污染生鲜乳[41], 以双歧杆菌(Bifidobacterium)为指标检测牛的粪便是否为生鲜乳污染的主要来源时发现, 88%的生鲜乳样品和95%的粪便样品中均检测到双歧杆菌[42], 此外, 弯曲杆菌也可通过粪便进入生鲜乳[43]。

1.2 空气和饲料

畜舍及挤奶厅内空气及飘浮的灰尘中常常含有许多微生物, 通常包括球菌、细菌芽孢和真菌孢子, 呈气溶胶状态分散在空气中, 与生鲜乳有接触时会引起污染[44]。青贮饲料中也包含了大量对生鲜乳质量和安全存在潜在的危害因子, 是牛奶中李斯特菌、大肠杆菌、霉菌毒素、厌氧孢子形成体(梭菌属)和好氧孢子形成体[主要是芽孢杆菌属(Bacillus)和类芽孢杆菌属(Paenibacillus)]的来源[45]。

1.3 患病乳牛

患病乳牛也是生鲜乳微生物的重要来源, 其中乳房炎是最主要的污染源, 患乳房炎奶牛的牛乳中会有金黄色葡萄球菌和病原性大肠杆菌等, 泌乳牛其它部位患病时, 病原菌也会通过血液循环进入乳汁, 如患结核或布氏杆菌病的牛分泌的乳中会有结核杆菌或布氏杆菌[46]。泌乳牛乳头顶点微生物被视为是生鲜乳微生物的潜在来源, 不同牛场其乳头顶点微生物也有一定的差异[47], 这可能是由于不同牛场牛床材料存在的潜在危害因子不同造成的[32]。Braem等[48] 在患有乳腺炎的泌乳牛乳头顶点检测到细菌17个属, 分属于放线菌(32%)、拟杆菌(1%)、变形菌(42%)和厚壁菌门(25%), 调查还发现环境因子是乳腺炎发生的主要危害因子[48, 49], 最常观察到的乳腺炎病原体乳房链球菌[50]及停乳链球菌主要来源于奶牛所处的环境[51], 金黄色葡萄球菌可能来自牛乳房或人的皮肤[35], 而无乳链球菌是一种专乳房病原体, 在挤奶过程中可以直接从一头牛传输到其它牛[52]。

1.4 温度和时间

储存温度和时间也是导致细菌污染加重的重要原因[41], 温度大于4 ℃时, 微生物生长繁殖较快, 而储存时间超过24 h, 嗜冷菌会大量繁殖, 如蜡样芽胞杆菌(Bacillus cereus)的繁殖和肠毒素的产生均与牛奶储存温度有关[53], 许多致病菌在开始挤奶时数量不足够引起疾病, 但受贮存温度和时间等的影响而逐渐积累增多。

1.5 季节和地区

生鲜乳微生物菌落组成也受季节和地区的影响[54], 研究发现γ -变形菌在春季和冬季, 杆菌在夏季, 放线菌在秋季分别拥有很高的物种丰富度(达到18~21种)[55]。

近年来关于牛场管理卫生情况与生鲜乳质量安全的研究很多[56, 57, 58, 59], 牛场管理规范化对于从源头上消除微生物污染具有很重要的意义。

2 冷藏和巴氏杀菌对生鲜乳有害微生物的影响

微生物可以通过自身产生的毒素、蛋白酶和脂肪酶影响生鲜乳质量、货架期甚至危害人类健康, 通过冷藏和巴氏杀菌可以减少生鲜乳中的大多数微生物, 确保生鲜乳质量与安全, 并延长保质期。

2.1 冷藏

冷链控制在乳制品供应链中扮演很重要的角色, 在生鲜乳的生产、贮存和运输过程中, 为减少生鲜乳中细菌的生长, 全程要求温度控制在1~4 ℃, 这可以抑制绝大多数细菌生长, 但也为嗜冷菌的生长创造了有利条件。用DGGE和克隆两种方法检测发现在冷藏期间细菌的遗传多样性下降, 生鲜乳中的细菌主要有革兰氏阳性菌杆菌属和梭菌属[60], 而冷藏后90%以上是革兰氏阴性菌, 包括假单胞菌属(Pseudomonas)、柠檬酸杆菌属(Citrobacter)、无色杆菌属(Achromobacter)、不动杆菌属(Acinetobacter)、气单胞菌属(Aeromonas)、沙雷氏菌属(Serratia)、产碱杆菌属(Alcaligenes )、色杆菌属(Chromobacerium)、克雷伯氏菌属(Klebsiella)、黄杆菌属(Flavobacterium)和肠杆菌属(Enterobacter)[61, 62, 63]。其中假单胞菌属占据主导地位, 是主要的嗜冷菌[64], 荧光假单胞菌是主要的菌种[65]。研究发现一些嗜冷菌产生的耐热胞外蛋白酶和脂肪酶可导致经过巴氏杀菌和超高温处理的牛奶及其奶制品的结构损坏[66], 使10%的乳脂肪和乳蛋白损失[55], 引起牛奶及其奶制品异味、胀包等质量和安全问题, 且发现这些酶在冬季比夏季更易产生[67]。有研究通过测定蛋白水解和脂肪水解选择性培养基试验发现, 嗜冷菌Pseudomonas gessardii, Pseudomonas lundensis和Pseudomonas fragi也极有可能引起牛奶变质[66]。嗜冷菌可在生存环境中营养物质相对较频发的条件下依然可以存活, 主要是由于它可通过产酶量来弥补酶催化反应速度较低的不足, 而残留在挤奶设备里面的牛奶可以为嗜冷菌的生长提供充足养分[68], 所以去除牛奶运输贮存容器中残留的固体奶对减少嗜冷菌的数量有重要的意义[69], 此外, 在牛奶中充入CO2也可控制嗜冷菌的数量[70]。

2.2 巴氏杀菌

一般来说生鲜乳经过冷藏, 大多数微生物可得到抑制, 但防止微生物增殖还不足以确保生鲜乳质量安全, 极低数量的致病菌就可能引起人类疾病[71], 生鲜乳被污染与大部分和奶酪相关的疾病暴发有关, 在美国[23, 24]、法国[7]、英国[10]和德国[8]等很多国家都有发生(表1)。

巴氏杀菌是减少生鲜乳微生物污染和疾病传播的最有效方法[26], 可杀死生鲜乳中的致病菌、中温菌和低温菌等对人体有害的微生物, 还可以钝化酶类并激活芽孢[72, 73], 延长产品保质期。大多数嗜冷菌在巴氏杀菌处理过程中会被杀死, 但一些嗜热菌、孢子、酶和毒素依然可以存活[55, 74, 75], 其中莓实假单胞菌(Pseudomonas fragi)和Pseudomonas lundensis是生鲜乳中耐热蛋白酶的主要来源[76], 禽分枝杆菌副结核亚种经过巴氏杀菌后也有少量存在[77]。通常情况下芽孢杆菌是巴氏杀菌乳保质期早期(< 7 d)主要的腐败菌, 而对于贮藏在冷藏条件下的巴氏杀菌乳, 类芽孢杆菌是保质期后期(> 10 d)主要的腐败菌[78]。

近年来的一项调查发现巴氏杀菌奶微生物多样性要比预期大[64], 由饮用巴氏杀菌奶引起的疾病暴发在历史上也有发生, 但远少于由生鲜乳引起的疾病暴发(表1)。通过对100份巴氏杀菌前后的奶样进行大肠菌群、大肠杆菌和金黄色葡萄球菌的污染率进行测定, 发现这3种菌的污染率分别由42%、36%和22%减少到9%、2%和2%[79]。1993-2006年对被污染的非巴氏杀菌奶及巴氏杀菌奶致病菌进行测定时发现, 除了在非巴氏杀菌奶中检测到的沙门氏菌、弯曲杆菌、志贺毒素大肠杆菌、布鲁氏菌、李斯特菌和志贺氏菌, 巴氏杀菌奶中还检测到金黄色葡萄球菌、产气荚膜梭状芽胞杆菌(Clostridium perfringens)和芽孢杆菌, 这可能是后期加工过程操作不当引起的污染[80]。

在美国、加拿大和日本等国家, 乳业市场几乎全是巴氏杀菌奶, 我国在2000年之前, 巴氏杀菌奶在液态奶市场占据绝对优势, 而之后至今超高温灭菌奶(常温奶)成为消费的主流。对两者营养价值进行了比较, 得出巴氏杀菌奶相对来说口味更加纯正, 营养更加全面[71, 81], 也有研究者对巴氏杀菌乳发展前景做了论述[82], 并指出它依然是我国未来乳业可持续发展的必由之路, 且将牛奶在HTST巴氏杀菌后立即经过脉冲电场(PEF)处理可以使牛奶保质期延长两周以上[83]。因此, 减少生鲜乳中有害微生物的污染是延长乳制品保质期的根本, 合理的巴氏杀菌操作也必不可少。

3 生鲜乳主要致病菌对牛奶质量和人体健康的影响和危害

生鲜乳可以携带多种病原菌, 是李斯特菌、沙门氏菌、肠杆菌、弯曲杆菌、结核杆菌、金黄色葡萄球菌和沙门氏菌等致病菌及抗菌剂药物残留、毒素的传播载体[35, 84, 85, 86], 是食源性致病菌的重要来源(表1), 调查显示沙门氏菌、李斯特菌、志贺毒素大肠杆菌、空肠弯曲杆菌(Campylobacter jejuni)和小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)在美国农场混合奶中检测概率分别为0~8.9%、2.7%~6.5%、0~3.8%、0~12.3%和1.2%~6.1%[87, 88, 89, 90], 另外来自7个国家食源性疾病的年度报告概要显示, 由牛奶和奶制品所含细菌引起的疾病暴发占到食源性疾病总暴发的1%~5%。

3.1 大肠杆菌

大肠杆菌是一种革兰氏阴性兼性厌氧菌, 是与人体肠内宿主形成互利共生的条件性致病菌[90], 它最早被确定为人类病原体是在1982年[91], 是一种食源性高毒、高侵染和低剂量(少于10~100个细胞)的致病菌[92]。该菌可引起贫血、胃痉挛、腹泻和溶血性尿毒综合征等疾病[93, 94], 不仅是奶牛乳房炎的主要致病菌之一, 也是环境中重要的致病微生物[95], 是评定生鲜乳、乳制品及粪便污染的重要卫生指标。根据不同的生物学特性, 将致病性大肠杆菌分为5类, 肠侵袭性大肠杆菌(EIEC)、致病性大肠杆菌(EPEC)、肠出血性大肠杆菌(EHEC)、弥散粘附性大肠杆菌(DAEC)、肠产毒性大肠杆菌(ETEC)和肠黏附性大肠杆菌(EAEC)[96]。

大肠杆菌人畜共患体的特性归因于其Vero毒素的产生和多药抗药性[97], 由表1可以看出, 未经高温消毒的牛奶及其乳制品很可能引起大肠杆菌血清型为O157和其它一些非O157(O145和O22)的暴发。志贺毒素大肠杆菌(STEC或VTEC)是一组能够产生志贺毒素的大肠杆菌[96], 但并不是所有的STEC都有致病性, STEC导致人类严重疾病主要是因为可以产生Stx基因, 基于抗原性差异将Stx基因分为两类:Stx1(Stx1a、Stx1c和Stx1d)和Stx2(Stx2a、Stx2b、 Stx2c、Stx2d、Stx2e、Stx2f和Stx2g), 其中Stx2a、Stx2c和Stx2d是与人类严重疾病相关最常见的亚型[98, 99], 现已报道的STEC 菌株血清型已超过400种, 但许多并不常见[100], 在生鲜乳及其乳制品中检测到的概率大概在0%~13%[96]。

大肠杆菌O157:H7是肠出血性大肠杆菌(EHEC)中与生鲜乳最相关的致病性大肠杆菌[96], 引起的疾病临床症状有轻度腹泻、溶血性尿毒综合征、出血性紫癜及血栓性血小板减少性紫癜, 严重威胁乳品行业的安全[101, 102], STEC在世界不同地区引起多起食源性疾病的暴发[96], 而由大肠杆菌O157:H7引起的疾病事件占STEC引发的91.4%[103], 此外, 另有研究表明由生鲜乳直接生产的奶酪和巴氏杀菌不彻底以及后期处理不规范均可能导致大肠杆菌O157:H7和大肠杆菌O26:H1的污染[96]。贮存温度对生鲜乳及乳制品中大肠杆菌O157:H7的存活和活性的影响较大, 菌数量及其代谢活性在4 ℃时降到最低[104], 巴氏灭菌(72 ℃下进行15 s)可以使大肠杆菌O157:H7失活[105]。

肠致病型大肠杆菌(EPEC)也是一种很重要的病原菌, 它可使人类患胃肠炎和引起食物中毒[106], 大多数肠致病型大肠杆菌(EPEC)菌株也可以产生Vero毒素(VT), 但其致病性目前并清楚[107]。在埃及, 乳制品中发现最多的血清型是O111、O126、O128、O26、O25 和O125[108, 109], 而在伊拉克, 血清型为O111、O86、O125和O119的菌株经常可以在奶酪中分离到[110]。调查发现在生鲜乳奶酪中检测到携带独立基因大肠杆菌概率为0~55.3%[96], 虽然许多大肠杆菌检测都含有vtx基因, 但并不是所有的都有致病性[92, 111]。在大肠杆菌的防治方面, 表面活性肽不仅具有抑制大肠杆菌O157 的作用, 还具有明显的杀菌效果[112], 添加10%的新鲜大蒜液也能够很好的抑制生鲜乳中大肠杆菌的增殖[95], 有很好的应用前景。

3.2 李斯特菌

李斯特菌(Listeria monocytogenes)是一种革兰氏阳性嗜冷菌, 在pH4.6~9.5、低温、高盐、及水活性低至0.92时均可以生长[113, 114, 115], 具有很强的环境适应能力, 主要通过食品污染传播给人类, 未经高温消毒的生鲜乳及其奶酪是一种很重要的传播途径[116]。目前, 生鲜乳中检测到的李斯特菌有单核细胞增生李斯特菌(L. monocytohenes)、L. ivanovii、英诺克李斯特菌(L. innocua)、威尔斯李斯特菌(L. welshimeri)、西尔李斯特菌(L. seeligeri)、格氏李斯特菌(L. grayi)、绵羊李斯特菌(L. iuanuii)、L. rocourtiae、L. marthii[117]和L. rocourtiae[118] 10种, 单核细胞增生李斯特菌和绵羊李斯特菌是致病菌, 且单核细胞增生李斯特菌是一种人畜共患体且在所有李斯特菌中检出率最高, 目前未见绵羊李斯特菌感染人类的报道[119, 120]。李斯特菌对人体的影响主要通过侵袭人体中枢神经、咽喉和淋巴结, 经胃肠道感染, 并逐渐扩散到局部淋巴结, 最后感染到内脏器官[121], 对胎儿、新生婴儿、老人和免疫系统较弱的群体影响较大, 从轻度流感腹泻到脑膜炎和败血症均有发生[122]。在一些李斯特菌散发病例研究中发现一半以上的疾病暴发与牛奶及其奶制品有关[123]。而且调查发还现, 饮用从零售商购买的牛奶患李斯特菌病的风险更大, 可能是由于贮藏不规范导致李斯特菌在贮藏期间增长的原因[124]。

李斯特菌在环境中广泛存在, 近年来已被广泛研究[125]。现已发现的李斯特菌的血清型至少有13个(1/2a、1/2b、1/2c、3a、3b、3c、4a、4ab、4b、4c、4d、4e和7)[126, 127], 血清型1/2a、1/2b、1/2c和4b最为常见[128], 在牛场检测李斯特菌时发现血清型为1/2a、1/2b、1/2c和4b的菌株达到78%[129] ; 在单增李斯特菌病例时中, 90%以上与1/2a、1/2b和4b有关[130], 在散发性病例中血清型为4b的菌株占到36%[131]。李斯特菌感染途径非常复杂, 19%的牛场样品包括牛粪、牛奶、青贮饲料、土壤和水中检测到李斯特菌[129]; 另有研究者检测发现李斯特菌在设备、环境和生鲜乳中的发生率分别为18.8%(6.3%为单增李斯特氏菌)、54.7%(40.6%为单增李斯特氏菌)和44.4%(22.2%为单增李斯特氏菌)[132]; 此外13.1%加工环境和12.3%外部环境中也监测到李斯特菌[119], 李斯特菌可在土壤、饲料、水和粪便中长期存在的生物学特性[129], 是其进一步形成生物膜的重要条件, 有助于污染最终产品[133], 相比于其它致病菌像沙门氏菌、弯曲杆菌和致病性大肠杆菌, 李斯特菌引起的食源性病例较少, 但由于高的死亡率(30%)引起广泛关注, 大多数国家对李斯特菌实行零容忍政策[134]。

李斯特菌的生长受到细菌素的影响[135], 近年来在生鲜乳及其乳制品中分离到一些产细菌素乳酸菌菌株, 乳酸链球菌素在许多国家的奶酪生产中已有应用[136], 加入乳酸链球菌素12.5 mg· kg-1可使生鲜乳中的单增李斯特菌迅速减少[137], 类似的细菌素还发现很多[138, 139, 140, 141, 142, 143, 144, 145]。毒力不是一个稳定性能, 生鲜乳所处环境及不同的贮存条件均会影响李斯特菌的毒力[146], 像酸和盐胁迫可以促进毒力基因的表达及提高体外毒性, 温度、pH、氧和渗透胁迫可能影响其毒力[147, 148, 149, 150]。在李斯特菌的检测方面, 脉冲场凝胶电泳(PFGE)被证明在检测李斯特菌的流行病学相关集群及食品的追溯调查是很有效的[151, 152], 也有长期监督检测价值[153], PCR-限制性酶分析、DNA探针技术、自动酶连荧光免疫检测系统以及聚合酶免疫检测方法EIA和国标法均可在不同程度上用来检测李斯特菌, 但由于致病性李斯特菌与许多非致病性李斯特菌有许多相似之处, 以及嗜冷和耐盐的特性, 使得其在生鲜乳中的检测和控制成为一大挑战[137, 154]。

3.3 金黄色葡萄球菌

金黄色葡萄球菌(Staphylococcus aureus)是革兰氏阳性菌, 引起最常见的泌乳牛乳房炎[155], 使泌乳牛患有慢性、临床和隐性乳房炎[156], 该菌是一种人畜共患体, 其中一些金黄色葡萄球菌也是一种重要的食源性致病菌, 可引起人类很多疾病, 像食物中毒、胃肠炎、肺炎、败血症等[157], 由于其高发生率和潜在风险性引起人们的广泛关注[158]。金黄色葡萄球菌可通过生鲜乳及其乳制品传播, 有调查[159]发现, 83.5%的生鲜乳样品中可检测到金黄色葡萄球菌, 另有研究[160]发现, 38%的生鲜乳、11%的巴氏杀菌奶被金黄色葡萄球菌污染。

金黄色葡萄球菌的毒力是由于可以产生过氧化氢酶、脂肪酶、DNA酶和溶血素类等酶和毒素的原因, 其中耐热肠毒素(SEs)是最主要的毒力因子[161], 其症状有恶心、剧烈呕吐和痉挛, 有时还会有腹泻; 研究发现, 大多数金黄色葡萄球菌菌株能够产生肠毒素D(68.8%), 而产生肠毒素A的菌株占到12.8%[162], 且 SEA在美国、日本、英国和法国等国家被认为是引起SEP暴发的主要原因[163], 温度是影响其在生鲜乳中产生的关键因子[164]。目前发现的肠毒素除5个经典型肠毒素(SEA-SEE)之外, 近年通过与经典SE序列的相似性比较发现了16种新型肠毒素 (SEG-SElV)[163, 165], 其中SEG、SHE、SEI、SER、SES和SET已被确定为食物潜在中毒剂[166, 167]。通过猴子饲喂试验发现SelK、SelL、SelM、SelN、SelO、SelP、和SelQ在金黄色葡萄球菌引起的食物中毒中可能发挥一定的作用, 但其作用机理尚不清楚[168]。前人对生鲜乳及乳制品中分离得到的227株金黄色葡萄球菌进行测定得到15种肠毒素(SEs/Sels)的基因, 且发现大多数菌株不止含有一个毒素基因, sec(28.6%)是出现频率最高的基因, sea、sed、ser和selj(20%)次之, seg、sei和seh相对较少[169], 同样的研究发现sea是检测出现频率最高的基因(30.7%), 其次为seb(26.9%)和sed(15.37%)[170]。

由于过度使用抗生素药物, 许多金黄色葡萄球菌也具有耐药性, 有研究对分离得到的219株金黄色葡萄球菌进行抗生素试验时发现 70%以上的具有多种抗生素抗性[171], ermC是最常见的耐药基因, ermA是最少频率检测到的基因, 与此结论相反, 在耐甲氧西林葡萄球菌的红霉素和四环素抗性基因的调查中发现ermA基因的检测率最高[172]。随着多药物抵抗机制的改变和mecA阴性耐甲氧西林金黄色葡萄球菌(MRSA)菌株的出现, 金黄色葡萄球菌的抗性变得越来越复杂, 且不同的多位点序列分析中出现了MRSA-IV菌株[171], MRSA在医学上是一种重要的病原菌, 已有报道显示动物很可能是人类MRSA菌株的潜在来源[173, 174], 而对于泌乳牛来说, 生鲜乳是一条很重要的传播途径, 在生鲜乳及其乳制品中也已分离到MRSA菌株[174, 175], 生鲜乳及其乳制品一起被认为是与金黄色葡萄球菌相关食物中毒的主要来源[176]。

早期的研究表明, 泌乳牛乳头上的金黄色葡萄球菌与泌乳早期的感染相关联, 挤奶设备、空气、牛舍、饲料及其人类都可能是金黄色葡萄球菌的来源[177], 调查显示, 金黄色葡萄球菌在整个农场都有发现, 且传播方向可能是由环境传向挤奶设备再到牛奶及其奶制品[178]。近期研究还表明, 牛奶在加工厂的储存温度及在消费者采用的热处理方式是影响金黄色葡萄球菌含量的主要原因, 其它影响因素还包括在农场牛奶的储存时间和温度, 牛奶热处理温度和处理时间[179], 但目前, 关于生鲜乳及其乳制品中金黄色葡萄球菌尤其是MRSA的检测和控制还有待进一步研究[173]。

3.4 其它有害微生物

芽孢杆菌、沙门氏菌、空肠弯曲菌、小肠结肠炎耶尔森菌、流产布鲁氏菌( Brucella abortus)和生孢梭菌(Clostridium sporogenes)等有害菌对生鲜乳质量和安全有不同程度的影响和危害。引起生鲜乳污染的芽孢杆菌主要有枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、球形芽孢杆菌(Bacillus sphaericus)、蜡样芽孢杆菌(Bacillus cereus)、短小芽孢杆菌(Bacillus pumilus)、环状芽孢杆菌(Bacillus circulans)和解淀粉芽孢杆菌(Bacillus amyloliquefaciens)[180, 181], 其中枯草芽孢杆菌、蜡样芽孢杆菌和地衣芽孢杆菌是巴士杀菌乳和超高温杀菌乳的主要污染因素之一[60], 蜡样芽孢杆菌也是6~7 ℃储存的巴氏杀菌奶和乳酪货架期的限制因子[182], 因为其可以产生一种或者多种肠毒素而引起食物中毒[45, 183, 184]。欧盟在2010年测试奶粉时发现呈阳性杆菌毒素的样品达到3.8%[185], 另有研究表明, 在包装乳的腐败中, 60%是由革兰氏阳性杆菌(如芽孢杆菌)引起的[186]。芽孢杆菌可以形成耐热孢子, 在超高温灭菌(UHT)条件下依然存活且一般处于休眠状态, 但在产品贮藏、运输、销售过程中, 遇到适宜条件就会被激活, 逐渐转化为营养细胞, 不断生长繁殖, 产酸、产气, 导致牛奶变质[187]。空肠弯曲菌是引起急性胃肠炎的主要原因[188], 可导致痉挛、血性腹泻、呕吐、腹痛, 并可能引起神经系统疾病格林巴利综合征, 在欧洲生鲜乳中检出频率为0~6%[189, 190, 191], 在意大利北部、波兰、大不列颠、法国等国生鲜乳中均有检测到, 检出频率大概在1.4%~4.6%[192, 193, 194]。初步研究表明, 牛是主要污染源, 但其具体传播途径目前还不清楚[11], 而对于牛来说, 直接饮用生鲜乳是引起疾病暴发的主要原因[195], 全基因多位点序列分析(wgMLST)可用于检测弯曲杆菌疫情[196]。由沙门氏菌感染引起的人类疾病有伤寒和胃肠道疾病, 在罐牛奶中检测到沙门氏菌的频率很低, 一般低于1%[16, 197, 198]。如巴西自2000年之后由沙门氏菌引起的疾病暴发很少, 仅有15起[199]。小肠结肠炎耶尔森菌可使人患类似阑尾炎, 虽然巴氏灭菌会杀了小肠结肠炎耶尔森菌, 但是如果巴氏杀菌不彻底或发生二次污染, 它也可在冷藏温度下繁殖[188], 是导致小儿肠炎的主要原因[88, 200]。结核分枝杆菌是与奶相关的最耐热病原体[201], 而生孢梭菌则可导致乳制品的腐败[202]。

关于生鲜乳有害微生物的检测方法有很多, 梁国添[203]对此做了论述, 且指出生物技术与各种检测方案结合使用是未来乳品安全控制的发展方向, 另有研究称高通量测序法在未来生鲜乳生产环境中微生物的检测上有很重要的意义[204, 205]。

4 展望

微生物是影响生鲜乳消费的主要风险因子[206], 虽然在很多国家销售生鲜乳是合法的, 但生鲜乳有害微生物对人类健康的威胁不容忽视, 其含量是生鲜乳质量和安全的重要指标[207, 208], 目前, 各个国家采用的生鲜乳微生物指标有菌落总数、 芽孢数、总大肠菌群等。目前, 各个国家采用的生鲜乳微生物指标有菌落总数、总大肠菌群和一些致病菌, 我国将菌落总数作为生鲜乳微生物质量合格的安全控制指标, 生鲜乳微生物菌落总数标准为2× 106 cfu· ml-1, 远远高于美国、日本和加拿大等国家且不检致病菌, 具体见表2[209, 210]。常见高菌落总数主要是由于挤奶系统的清洗不彻底引起的[211, 212], 但低菌落总数的生鲜乳也可能含有病原菌[13], 因此, 仅用菌落总数作为生鲜乳微生物质量合格的安全指标是不够的, 一些人畜共患体致病菌的检测也很有必要。

表2 一些国家生鲜乳收购标准 Table 2 Fresh milk acquisition criteria in some country

防治生鲜乳微生物污染的最好方法是控制或者控制每种有害微生物的来源, 但由于特别的生产方式, 这对于生产者来说是很难达到的[213, 214], 因此, 对生鲜乳微生物风险趋势做到及时、准确预警和建立安全生产规程, 是防患于未然的高效手段, 加拿大、美国和新西兰等国均已基于HACCP设立比较完善的生产规程, 对其乳业的健康发展起了重要保障作用, 使其牛奶品质一直保持较高水平。而我国自2008年才开始制定和实施全国范围的生鲜乳质量安全监测计划, 与产业发展需求相比, 研究的系统性仍十分薄弱。

因此, 提高奶站或牧场整体建设水平和工作人员的素质, 以及建立我国自主特色的评价技术与生产规程对我国乳业的健康发展有重要意义, 此外, 普及生鲜乳消费安全知识, 引起广大消费者足够重视, 消费者尤其是婴幼儿和孕妇不要直接饮用生鲜乳及其乳制品可减少相关食源性疾病的发生[213, 215]。

The authors have declared that no competing interests exist.

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