生鮮乳有害微生物污染與危害分析

馮疆蓉,李春杰

(草地農(nóng)業(yè)生態(tài)系統(tǒng)國家重點實驗室 蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院，甘肅 蘭州 730020)

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生鮮乳有害微生物污染與危害分析

馮疆蓉,李春杰

(草地農(nóng)業(yè)生態(tài)系統(tǒng)國家重點實驗室 蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院，甘肅 蘭州 730020)

近年來乳制品質(zhì)量安全是社會關(guān)注的焦點，而作為乳制品供應(yīng)鏈最上游的生鮮乳的質(zhì)量安全是乳業(yè)持續(xù)健康發(fā)展的根本保證，其可能含有的乳源性病原體和腐敗菌等有害微生物嚴重影響乳品質(zhì)量和安全。本文綜述了生鮮乳中有害微生物的來源、生鮮乳種類和冷藏、巴氏殺菌對有害微生物的影響，以及主要致病菌的發(fā)生、危害和研究現(xiàn)狀，以期為監(jiān)督管理乳制品生產(chǎn)、加工和食物安全提供基礎(chǔ)依據(jù)。

生鮮乳；乳源性病原體；巴氏殺菌；李斯特菌；金黃色葡萄球菌

牛奶被公認為是自然界營養(yǎng)最為均衡的全價食品，它幾乎含有人體所需的所有大量營養(yǎng)元素(如蛋白質(zhì)、脂質(zhì)和碳水化合物)以及微量營養(yǎng)元素(如維生素和酶)，不僅是哺乳動物幼崽的營養(yǎng)來源，也為人類提供營養(yǎng)物質(zhì)，在人類飲食結(jié)構(gòu)中具有不可替代的作用和地位[1-2]。因此，生鮮乳作為乳制品供應(yīng)鏈最上游的初始產(chǎn)物，其質(zhì)量安全將直接關(guān)系到乳與乳制品的質(zhì)量和安全。

生鮮乳是微生物生長的理想培養(yǎng)基，因此含有復(fù)雜而多樣化的微生物種群，現(xiàn)檢測到的微生物已超過100個屬， 400多種，包括革蘭氏陰性細菌(≥90種)、革蘭氏陽性細菌(≥90種)、過氧化氫酶陽性菌(≥90種)、乳酸菌(≥60種)、酵母菌(≥70種)和霉菌(≥40種)[3]，包括降低牛奶品質(zhì)、影響消費者身體健康甚至導(dǎo)致死亡的有害菌，其以細菌為主，如結(jié)核菌(Mycobacterium tuberculosis)、沙門氏菌(Salmonella)、大腸桿菌(Escherichia coli)、李斯特菌(Listeria monocy-togenes)、彎曲桿菌(Campylobacter)、金黃色葡萄球菌(Staphylococcus aureus)和空腸彎曲菌(Campylobacter jejuni)等曾引起人類多次疾病暴發(fā)(表1)。而嗜冷菌、噬菌體和梭菌(Clostridium)等均可導(dǎo)致奶酪、液體奶和一些新鮮乳制品的腐敗[28]，此外，酵母菌和霉菌也與生鮮乳及其乳制品的腐敗有關(guān)，近年來在生鮮乳中檢測到金黃桿菌屬(Chryseobacterium)[29]和Epilithonimonas[30]，但其是否會引起牛奶的變質(zhì)還有待進一步研究(表1)。因此，明確生鮮乳中有害微生物的來源及其冷藏和巴氏殺菌對微生物的影響以及重要腐敗菌和致病菌的危害與防治，將有助于幫助企業(yè)和農(nóng)戶提升生鮮乳生產(chǎn)質(zhì)量。

表1　歷史上牛奶源性疾病暴發(fā)的統(tǒng)計情況Table 1　Reported human cases and outbreaks of milk-borne diseases in history

續(xù)表1

年份Year發(fā)生國家Country事件Case相關(guān)致病菌Pathogenicagent起因Reason文獻Reference2007-2012美國USA共發(fā)生81起疾病事件,979人生病,73人住院81cases,979illness,73hospi-talised彎曲桿菌Campylobacter沙門氏菌Salmonella李斯特菌Listeriamonocytogenes志賀毒素大腸桿菌STEC彎曲桿菌Campylobacter生鮮乳和乳制品Rawmilkanddairy[24]2009中國China119名兒童中毒119poisoned金黃色葡萄球菌Staphylococcusaureus生鮮乳及其未經(jīng)高溫消毒奶制品Rawmilkandunpas-teurisedmilk[25]2012美國USA81人生病,10人住院81illness,10hospitalised空腸彎曲菌Campylobacterjejuni未經(jīng)高溫消毒的奶品Unpasteurisedmilk[26]2014瑞士Switzerland14人生病14illness金黃色葡萄球菌Staphylococcusaureus飲用由生鮮乳直接生產(chǎn)的奶酪Unpasteurisedmilk[27]

1　生鮮乳有害微生物的來源及影響因子

健康的泌乳牛所產(chǎn)的生鮮乳可能由于前期乳房擠奶準備不足、牛奶處理程序的不當以及與擠奶和存儲設(shè)備相關(guān)的一些不好的衛(wèi)生習(xí)慣而引起污染[31]，生鮮乳中的大多數(shù)菌在牛棚和擠奶廳中都可以檢測到[32]，泌乳牛、牛舍(墊料及管理)、擠奶和存儲設(shè)備、乳頭與乳管、擠奶工、飼料、清洗水、空氣和土壤等其它環(huán)境因子均可能成為生鮮乳微生物的來源[32-34]。

1.1牛場設(shè)備及衛(wèi)生管理

擠奶廳的衛(wèi)生環(huán)境狀況可直接影響到腸桿菌(Enterobacter iaceae)污染生鮮乳的程度[35]，如蠟狀芽孢桿菌(Bacillus cereus)和假單胞菌屬分別可從土壤、飼料、擠奶設(shè)備、乳房、牛奶罐和水中進入生鮮乳[36]，大腸桿菌可隨清洗設(shè)備和牛體的水進入生鮮乳[37-38]，而且相關(guān)設(shè)備和泌乳牛體清洗得不徹底或者擠奶工衛(wèi)生習(xí)慣不良好也可能引起金黃色葡萄球菌的嚴重污染[39]。

糞便和牛床是大腸菌群和環(huán)境鏈球菌的主要來源[40]，生存于牛床材料上的葡萄球菌和一些革蘭氏陰性菌可通過乳頭和乳管污染生鮮乳[41]， 以雙歧桿菌(Bifidobacterium)為指標檢測牛的糞便是否為生鮮乳污染的主要來源時發(fā)現(xiàn)，88%的生鮮乳樣品和95%的糞便樣品中均檢測到雙歧桿菌[42]，此外，彎曲桿菌也可通過糞便進入生鮮乳[43]。

1.2空氣和飼料

畜舍及擠奶廳內(nèi)空氣及飄浮的灰塵中常常含有許多微生物，通常包括球菌、細菌芽孢和真菌孢子，呈氣溶膠狀態(tài)分散在空氣中，與生鮮乳有接觸時會引起污染[44]。青貯飼料中也包含了大量對生鮮乳質(zhì)量和安全存在潛在的危害因子，是牛奶中李斯特菌、大腸桿菌、霉菌毒素、厭氧孢子形成體(梭菌屬)和好氧孢子形成體[主要是芽孢桿菌屬(Bacillus)和類芽孢桿菌屬(Paenibacillus)]的來源[45]。

1.3患病乳牛

患病乳牛也是生鮮乳微生物的重要來源，其中乳房炎是最主要的污染源，患乳房炎奶牛的牛乳中會有金黃色葡萄球菌和病原性大腸桿菌等，泌乳牛其它部位患病時，病原菌也會通過血液循環(huán)進入乳汁，如患結(jié)核或布氏桿菌病的牛分泌的乳中會有結(jié)核桿菌或布氏桿菌[46]。泌乳牛乳頭頂點微生物被視為是生鮮乳微生物的潛在來源，不同牛場其乳頭頂點微生物也有一定的差異[47]，這可能是由于不同牛場牛床材料存在的潛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頭頂點檢測到細菌17個屬，分屬于放線菌(32%)、擬桿菌(1%)、變形菌(42%)和厚壁菌門(25%)，調(diào)查還發(fā)現(xiàn)環(huán)境因子是乳腺炎發(fā)生的主要危害因子[48-49]，最常觀察到的乳腺炎病原體乳房鏈球菌[50]及停乳鏈球菌主要來源于奶牛所處的環(huán)境[51]，金黃色葡萄球菌可能來自牛乳房或人的皮膚[35]，而無乳鏈球菌是一種專乳房病原體，在擠奶過程中可以直接從一頭牛傳輸?shù)狡渌52]。

1.4溫度和時間

儲存溫度和時間也是導(dǎo)致細菌污染加重的重要原因[41]，溫度大于4 ℃時，微生物生長繁殖較快，而儲存時間超過24h，嗜冷菌會大量繁殖，如蠟樣芽胞桿菌(Bacillus cereus)的繁殖和腸毒素的產(chǎn)生均與牛奶儲存溫度有關(guān)[53]，許多致病菌在開始擠奶時數(shù)量不足夠引起疾病，但受貯存溫度和時間等的影響而逐漸積累增多。

1.5季節(jié)和地區(qū)

生鮮乳微生物菌落組成也受季節(jié)和地區(qū)的影響[54]，研究發(fā)現(xiàn)γ-變形菌在春季和冬季，桿菌在夏季，放線菌在秋季分別擁有很高的物種豐富度(達到18～21種)[55]。

近年來關(guān)于牛場管理衛(wèi)生情況與生鮮乳質(zhì)量安全的研究很多[56-59]，牛場管理規(guī)范化對于從源頭上消除微生物污染具有很重要的意義。

2　冷藏和巴氏殺菌對生鮮乳有害微生物的影響

微生物可以通過自身產(chǎn)生的毒素、蛋白酶和脂肪酶影響生鮮乳質(zhì)量、貨架期甚至危害人類健康，通過冷藏和巴氏殺菌可以減少生鮮乳中的大多數(shù)微生物，確保生鮮乳質(zhì)量與安全，并延長保質(zhì)期。

2.1冷藏

冷鏈控制在乳制品供應(yīng)鏈中扮演很重要的角色，在生鮮乳的生產(chǎn)、貯存和運輸過程中，為減少生鮮乳中細菌的生長，全程要求溫度控制在1～4 ℃，這可以抑制絕大多數(shù)細菌生長，但也為嗜冷菌的生長創(chuàng)造了有利條件。用DGGE和克隆兩種方法檢測發(fā)現(xiàn)在冷藏期間細菌的遺傳多樣性下降，生鮮乳中的細菌主要有革蘭氏陽性菌桿菌屬和梭菌屬[60]，而冷藏后90%以上是革蘭氏陰性菌，包括假單胞菌屬(Pseudomonas)、檸檬酸桿菌屬(Citrobacter)、無色桿菌屬(Achromobacter)、不動桿菌屬(Acinetobacter)、氣單胞菌屬(Aeromonas)、沙雷氏菌屬(Serratia)、產(chǎn)堿桿菌屬(Alcaligenes )、色桿菌屬(Chromobacerium)、克雷伯氏菌屬(Klebsiella)、黃桿菌屬(Flavobacterium)和腸桿菌屬(Enterobacter)[61-63]。其中假單胞菌屬占據(jù)主導(dǎo)地位，是主要的嗜冷菌[64]，熒光假單胞菌是主要的菌種[65]。研究發(fā)現(xiàn)一些嗜冷菌產(chǎn)生的耐熱胞外蛋白酶和脂肪酶可導(dǎo)致經(jīng)過巴氏殺菌和超高溫處理的牛奶及其奶制品的結(jié)構(gòu)損壞[66]，使10%的乳脂肪和乳蛋白損失[55]，引起牛奶及其奶制品異味、脹包等質(zhì)量和安全問題，且發(fā)現(xiàn)這些酶在冬季比夏季更易產(chǎn)生[67]。有研究通過測定蛋白水解和脂肪水解選擇性培養(yǎng)基試驗發(fā)現(xiàn),嗜冷菌Pseudomonas gessardii, Pseudomonas lundensis和Pseudomonas fragi也極有可能引起牛奶變質(zhì)[66]。嗜冷菌可在生存環(huán)境中營養(yǎng)物質(zhì)相對較頻發(fā)的條件下依然可以存活，主要是由于它可通過產(chǎn)酶量來彌補酶催化反應(yīng)速度較低的不足，而殘留在擠奶設(shè)備里面的牛奶可以為嗜冷菌的生長提供充足養(yǎng)分[68]，所以去除牛奶運輸貯存容器中殘留的固體奶對減少嗜冷菌的數(shù)量有重要的意義[69]，此外，在牛奶中充入CO2也可控制嗜冷菌的數(shù)量[70]。

2.2巴氏殺菌

一般來說生鮮乳經(jīng)過冷藏，大多數(shù)微生物可得到抑制，但防止微生物增殖還不足以確保生鮮乳質(zhì)量安全，極低數(shù)量的致病菌就可能引起人類疾病[71]，生鮮乳被污染與大部分和奶酪相關(guān)的疾病暴發(fā)有關(guān)，在美國[23-24]、法國[7]、英國[10]和德國[8]等很多國家都有發(fā)生(表1)。

巴氏殺菌是減少生鮮乳微生物污染和疾病傳播的最有效方法[26]，可殺死生鮮乳中的致病菌、中溫菌和低溫菌等對人體有害的微生物，還可以鈍化酶類并激活芽孢[72-73]，延長產(chǎn)品保質(zhì)期。大多數(shù)嗜冷菌在巴氏殺菌處理過程中會被殺死，但一些嗜熱菌、孢子、酶和毒素依然可以存活[55,74-75]，其中莓實假單胞菌(Pseudomonas fragi)和Pseudomonas lundensis是生鮮乳中耐熱蛋白酶的主要來源[76]，禽分枝桿菌副結(jié)核亞種經(jīng)過巴氏殺菌后也有少量存在[77]。通常情況下芽孢桿菌是巴氏殺菌乳保質(zhì)期早期(<7d)主要的腐敗菌，而對于貯藏在冷藏條件下的巴氏殺菌乳，類芽孢桿菌是保質(zhì)期后期(>10d)主要的腐敗菌[78]。

近年來的一項調(diào)查發(fā)現(xiàn)巴氏殺菌奶微生物多樣性要比預(yù)期大[64]，由飲用巴氏殺菌奶引起的疾病暴發(fā)在歷史上也有發(fā)生，但遠少于由生鮮乳引起的疾病暴發(fā)(表1)。通過對100份巴氏殺菌前后的奶樣進行大腸菌群、大腸桿菌和金黃色葡萄球菌的污染率進行測定，發(fā)現(xiàn)這3種菌的污染率分別由42%、36%和22%減少到9%、2%和2%[79]。1993-2006年對被污染的非巴氏殺菌奶及巴氏殺菌奶致病菌進行測定時發(fā)現(xiàn)，除了在非巴氏殺菌奶中檢測到的沙門氏菌、彎曲桿菌、志賀毒素大腸桿菌、布魯氏菌、李斯特菌和志賀氏菌，巴氏殺菌奶中還檢測到金黃色葡萄球菌、產(chǎn)氣莢膜梭狀芽胞桿菌(Clostridium perfringens)和芽孢桿菌，這可能是后期加工過程操作不當引起的污染[80]。

在美國、加拿大和日本等國家，乳業(yè)市場幾乎全是巴氏殺菌奶，我國在2000年之前，巴氏殺菌奶在液態(tài)奶市場占據(jù)絕對優(yōu)勢，而之后至今超高溫滅菌奶(常溫奶)成為消費的主流。對兩者營養(yǎng)價值進行了比較，得出巴氏殺菌奶相對來說口味更加純正，營養(yǎng)更加全面[71,81]，也有研究者對巴氏殺菌乳發(fā)展前景做了論述[82]，并指出它依然是我國未來乳業(yè)可持續(xù)發(fā)展的必由之路，且將牛奶在HTST巴氏殺菌后立即經(jīng)過脈沖電場(PEF)處理可以使牛奶保質(zhì)期延長兩周以上[83]。因此，減少生鮮乳中有害微生物的污染是延長乳制品保質(zhì)期的根本，合理的巴氏殺菌操作也必不可少。

3　生鮮乳主要致病菌對牛奶質(zhì)量和人體健康的影響和危害

生鮮乳可以攜帶多種病原菌，是李斯特菌、沙門氏菌、腸桿菌、彎曲桿菌、結(jié)核桿菌、金黃色葡萄球菌和沙門氏菌等致病菌及抗菌劑藥物殘留、毒素的傳播載體[35,84-86]，是食源性致病菌的重要來源(表1)，調(diào)查顯示沙門氏菌、李斯特菌、志賀毒素大腸桿菌、空腸彎曲桿菌(Campylobacter jejuni)和小腸結(jié)腸炎耶爾森氏菌(Yersinia enterocolitica)在美國農(nóng)場混合奶中檢測概率分別為0～8.9%、2.7%～6.5%、0～3.8%、0～12.3%和1.2%～6.1%[87-90]，另外來自7個國家食源性疾病的年度報告概要顯示，由牛奶和奶制品所含細菌引起的疾病暴發(fā)占到食源性疾病總暴發(fā)的1%～5%。

3.1大腸桿菌

大腸桿菌是一種革蘭氏陰性兼性厭氧菌，是與人體腸內(nèi)宿主形成互利共生的條件性致病菌[90]，它最早被確定為人類病原體是在1982年[91]，是一種食源性高毒、高侵染和低劑量(少于10～100個細胞)的致病菌[92]。該菌可引起貧血、胃痙攣、腹瀉和溶血性尿毒綜合征等疾病[93-94]，不僅是奶牛乳房炎的主要致病菌之一，也是環(huán)境中重要的致病微生物[95]，是評定生鮮乳、乳制品及糞便污染的重要衛(wèi)生指標。根據(jù)不同的生物學(xué)特性，將致病性大腸桿菌分為5類，腸侵襲性大腸桿菌(EIEC)、致病性大腸桿菌(EPEC)、腸出血性大腸桿菌(EHEC)、彌散粘附性大腸桿菌(DAEC)、腸產(chǎn)毒性大腸桿菌(ETEC)和腸黏附性大腸桿菌(EAEC)[96]。

大腸桿菌人畜共患體的特性歸因于其Vero毒素的產(chǎn)生和多藥抗藥性[97]，由表1可以看出,未經(jīng)高溫消毒的牛奶及其乳制品很可能引起大腸桿菌血清型為O157和其它一些非O157(O145和O22)的暴發(fā)。志賀毒素大腸桿菌(STEC或VTEC)是一組能夠產(chǎn)生志賀毒素的大腸桿菌[96]，但并不是所有的STEC都有致病性，STEC導(dǎo)致人類嚴重疾病主要是因為可以產(chǎn)生Stx基因，基于抗原性差異將Stx基因分為兩類：Stx1(Stx1a、Stx1c和Stx1d)和Stx2(Stx2a、Stx2b、 Stx2c、Stx2d、Stx2e、Stx2f和Stx2g)，其中Stx2a、Stx2c和Stx2d是與人類嚴重疾病相關(guān)最常見的亞型[98-99]，現(xiàn)已報道的STEC菌株血清型已超過400種，但許多并不常見[100]，在生鮮乳及其乳制品中檢測到的概率大概在0%～13%[96]。

大腸桿菌O157：H7是腸出血性大腸桿菌(EHEC)中與生鮮乳最相關(guān)的致病性大腸桿菌[96]，引起的疾病臨床癥狀有輕度腹瀉、溶血性尿毒綜合征、出血性紫癜及血栓性血小板減少性紫癜，嚴重威脅乳品行業(yè)的安全[101-102]，STEC在世界不同地區(qū)引起多起食源性疾病的暴發(fā)[96]，而由大腸桿菌O157：H7引起的疾病事件占STEC引發(fā)的91.4%[103]，此外，另有研究表明由生鮮乳直接生產(chǎn)的奶酪和巴氏殺菌不徹底以及后期處理不規(guī)范均可能導(dǎo)致大腸桿菌O157：H7和大腸桿菌O26：H1的污染[96]。貯存溫度對生鮮乳及乳制品中大腸桿菌O157：H7的存活和活性的影響較大，菌數(shù)量及其代謝活性在4 ℃時降到最低[104]，巴氏滅菌(72 ℃下進行15s)可以使大腸桿菌O157：H7失活[105]。

腸致病型大腸桿菌(EPEC)也是一種很重要的病原菌，它可使人類患胃腸炎和引起食物中毒[106]，大多數(shù)腸致病型大腸桿菌(EPEC)菌株也可以產(chǎn)生Vero毒素(VT)，但其致病性目前并清楚[107]。在埃及，乳制品中發(fā)現(xiàn)最多的血清型是O111、O126、O128、O26、O25 和O125[108-109]，而在伊拉克，血清型為O111、O86、O125和O119的菌株經(jīng)常可以在奶酪中分離到[110]。調(diào)查發(fā)現(xiàn)在生鮮乳奶酪中檢測到攜帶獨立基因大腸桿菌概率為0～55.3%[96]，雖然許多大腸桿菌檢測都含有vtx基因，但并不是所有的都有致病性[92,111]。在大腸桿菌的防治方面，表面活性肽不僅具有抑制大腸桿菌O157 的作用，還具有明顯的殺菌效果[112]，添加10%的新鮮大蒜液也能夠很好的抑制生鮮乳中大腸桿菌的增殖[95]，有很好的應(yīng)用前景。

3.2李斯特菌

李斯特菌(Listeria monocytogenes)是一種革蘭氏陽性嗜冷菌，在pH4.6～9.5、低溫、高鹽、及水活性低至0.92時均可以生長[113-115]，具有很強的環(huán)境適應(yīng)能力，主要通過食品污染傳播給人類，未經(jīng)高溫消毒的生鮮乳及其奶酪是一種很重要的傳播途徑[116]。目前，生鮮乳中檢測到的李斯特菌有單核細胞增生李斯特菌(L. monocytohenes)、L. ivanovii、英諾克李斯特菌(L. innocua)、威爾斯李斯特菌(L. welshimeri)、西爾李斯特菌(L. seeligeri)、格氏李斯特菌(L. grayi)、綿羊李斯特菌(L. iuanuii)、L. rocourtiae、L. marthii[117]和L. rocourtiae[118]10種，單核細胞增生李斯特菌和綿羊李斯特菌是致病菌，且單核細胞增生李斯特菌是一種人畜共患體且在所有李斯特菌中檢出率最高，目前未見綿羊李斯特菌感染人類的報道[119-120]。李斯特菌對人體的影響主要通過侵襲人體中樞神經(jīng)、咽喉和淋巴結(jié)，經(jīng)胃腸道感染，并逐漸擴散到局部淋巴結(jié),最后感染到內(nèi)臟器官[121]，對胎兒、新生嬰兒、老人和免疫系統(tǒng)較弱的群體影響較大，從輕度流感腹瀉到腦膜炎和敗血癥均有發(fā)生[122]。在一些李斯特菌散發(fā)病例研究中發(fā)現(xiàn)一半以上的疾病暴發(fā)與牛奶及其奶制品有關(guān)[123]。而且調(diào)查發(fā)還現(xiàn)，飲用從零售商購買的牛奶患李斯特菌病的風(fēng)險更大，可能是由于貯藏不規(guī)范導(dǎo)致李斯特菌在貯藏期間增長的原因[124]。

李斯特菌在環(huán)境中廣泛存在，近年來已被廣泛研究[125]?，F(xiàn)已發(fā)現(xiàn)的李斯特菌的血清型至少有13個(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最為常見[128]，在牛場檢測李斯特菌時發(fā)現(xiàn)血清型為1/2a、1/2b、1/2c和4b的菌株達到78%[129]；在單增李斯特菌病例時中，90%以上與1/2a、1/2b和4b有關(guān)[130]，在散發(fā)性病例中血清型為4b的菌株占到36%[131]。李斯特菌感染途徑非常復(fù)雜，19%的牛場樣品包括牛糞、牛奶、青貯飼料、土壤和水中檢測到李斯特菌[129]；另有研究者檢測發(fā)現(xiàn)李斯特菌在設(shè)備、環(huán)境和生鮮乳中的發(fā)生率分別為18.8%(6.3%為單增李斯特氏菌)、54.7%(40.6%為單增李斯特氏菌)和44.4%(22.2%為單增李斯特氏菌)[132]；此外13.1%加工環(huán)境和12.3%外部環(huán)境中也監(jiān)測到李斯特菌[119]，李斯特菌可在土壤、飼料、水和糞便中長期存在的生物學(xué)特性[129]，是其進一步形成生物膜的重要條件，有助于污染最終產(chǎn)品[133]，相比于其它致病菌像沙門氏菌、彎曲桿菌和致病性大腸桿菌，李斯特菌引起的食源性病例較少，但由于高的死亡率(30%)引起廣泛關(guān)注，大多數(shù)國家對李斯特菌實行零容忍政策[134]。

李斯特菌的生長受到細菌素的影響[135]，近年來在生鮮乳及其乳制品中分離到一些產(chǎn)細菌素乳酸菌菌株，乳酸鏈球菌素在許多國家的奶酪生產(chǎn)中已有應(yīng)用[136]，加入乳酸鏈球菌素12.5mg·kg-1可使生鮮乳中的單增李斯特菌迅速減少[137]，類似的細菌素還發(fā)現(xiàn)很多[138-145]。毒力不是一個穩(wěn)定性能，生鮮乳所處環(huán)境及不同的貯存條件均會影響李斯特菌的毒力[146]，像酸和鹽脅迫可以促進毒力基因的表達及提高體外毒性，溫度、pH、氧和滲透脅迫可能影響其毒力[147-150]。在李斯特菌的檢測方面，脈沖場凝膠電泳(PFGE)被證明在檢測李斯特菌的流行病學(xué)相關(guān)集群及食品的追溯調(diào)查是很有效的[151-152]，也有長期監(jiān)督檢測價值[153]，PCR-限制性酶分析、DNA探針技術(shù)、自動酶連熒光免疫檢測系統(tǒng)以及聚合酶免疫檢測方法EIA和國標法均可在不同程度上用來檢測李斯特菌，但由于致病性李斯特菌與許多非致病性李斯特菌有許多相似之處，以及嗜冷和耐鹽的特性，使得其在生鮮乳中的檢測和控制成為一大挑戰(zhàn)[137,154]。

3.3金黃色葡萄球菌

金黃色葡萄球菌(Staphylococcus aureus)是革蘭氏陽性菌，引起最常見的泌乳牛乳房炎[155]，使泌乳?；加新?、臨床和隱性乳房炎[156]，該菌是一種人畜共患體，其中一些金黃色葡萄球菌也是一種重要的食源性致病菌，可引起人類很多疾病，像食物中毒、胃腸炎、肺炎、敗血癥等[157]，由于其高發(fā)生率和潛在風(fēng)險性引起人們的廣泛關(guān)注[158]。金黃色葡萄球菌可通過生鮮乳及其乳制品傳播，有調(diào)查[159]發(fā)現(xiàn)，83.5%的生鮮乳樣品中可檢測到金黃色葡萄球菌，另有研究[160]發(fā)現(xiàn)，38%的生鮮乳、11%的巴氏殺菌奶被金黃色葡萄球菌污染。

金黃色葡萄球菌的毒力是由于可以產(chǎn)生過氧化氫酶、脂肪酶、DNA酶和溶血素類等酶和毒素的原因，其中耐熱腸毒素(SEs)是最主要的毒力因子[161]，其癥狀有惡心、劇烈嘔吐和痙攣，有時還會有腹瀉；研究發(fā)現(xiàn)，大多數(shù)金黃色葡萄球菌菌株能夠產(chǎn)生腸毒素D(68.8%)，而產(chǎn)生腸毒素A的菌株占到12.8%[162]，且SEA在美國、日本、英國和法國等國家被認為是引起SEP暴發(fā)的主要原因[163]，溫度是影響其在生鮮乳中產(chǎn)生的關(guān)鍵因子[164]。目前發(fā)現(xiàn)的腸毒素除5個經(jīng)典型腸毒素(SEA-SEE)之外，近年通過與經(jīng)典SE序列的相似性比較發(fā)現(xiàn)了16種新型腸毒素 (SEG-SElV)[163,165]，其中SEG、SHE、SEI、SER、SES和SET已被確定為食物潛在中毒劑[166-167]。通過猴子飼喂試驗發(fā)現(xiàn)SelK、SelL、SelM、SelN、SelO、SelP、和SelQ在金黃色葡萄球菌引起的食物中毒中可能發(fā)揮一定的作用，但其作用機理尚不清楚[168]。前人對生鮮乳及乳制品中分離得到的227株金黃色葡萄球菌進行測定得到15種腸毒素(SEs/Sels)的基因，且發(fā)現(xiàn)大多數(shù)菌株不止含有一個毒素基因，sec(28.6%)是出現(xiàn)頻率最高的基因，sea、sed、ser和selj(20%)次之，seg、sei和seh相對較少[169]，同樣的研究發(fā)現(xiàn)sea是檢測出現(xiàn)頻率最高的基因(30.7%)，其次為seb(26.9%)和sed(15.37%)[170]。

由于過度使用抗生素藥物，許多金黃色葡萄球菌也具有耐藥性，有研究對分離得到的219株金黃色葡萄球菌進行抗生素試驗時發(fā)現(xiàn) 70%以上的具有多種抗生素抗性[171]，ermC是最常見的耐藥基因，ermA是最少頻率檢測到的基因，與此結(jié)論相反，在耐甲氧西林葡萄球菌的紅霉素和四環(huán)素抗性基因的調(diào)查中發(fā)現(xiàn)ermA基因的檢測率最高[172]。隨著多藥物抵抗機制的改變和mecA陰性耐甲氧西林金黃色葡萄球菌(MRSA)菌株的出現(xiàn)，金黃色葡萄球菌的抗性變得越來越復(fù)雜，且不同的多位點序列分析中出現(xiàn)了MRSA-IV菌株[171]，MRSA在醫(yī)學(xué)上是一種重要的病原菌，已有報道顯示動物很可能是人類MRSA菌株的潛在來源[173-174]，而對于泌乳牛來說，生鮮乳是一條很重要的傳播途徑，在生鮮乳及其乳制品中也已分離到MRSA菌株[174-175]，生鮮乳及其乳制品一起被認為是與金黃色葡萄球菌相關(guān)食物中毒的主要來源[176]。

早期的研究表明，泌乳牛乳頭上的金黃色葡萄球菌與泌乳早期的感染相關(guān)聯(lián)，擠奶設(shè)備、空氣、牛舍、飼料及其人類都可能是金黃色葡萄球菌的來源[177]，調(diào)查顯示，金黃色葡萄球菌在整個農(nóng)場都有發(fā)現(xiàn)，且傳播方向可能是由環(huán)境傳向擠奶設(shè)備再到牛奶及其奶制品[178]。近期研究還表明，牛奶在加工廠的儲存溫度及在消費者采用的熱處理方式是影響金黃色葡萄球菌含量的主要原因，其它影響因素還包括在農(nóng)場牛奶的儲存時間和溫度，牛奶熱處理溫度和處理時間[179]，但目前，關(guān)于生鮮乳及其乳制品中金黃色葡萄球菌尤其是MRSA的檢測和控制還有待進一步研究[173]。

3.4其它有害微生物

芽孢桿菌、沙門氏菌、空腸彎曲菌、小腸結(jié)腸炎耶爾森菌、流產(chǎn)布魯氏菌( Brucella abortus)和生孢梭菌(Clostridium sporogenes)等有害菌對生鮮乳質(zhì)量和安全有不同程度的影響和危害。引起生鮮乳污染的芽孢桿菌主要有枯草芽孢桿菌(Bacillus subtilis)、地衣芽孢桿菌(Bacillus licheniformis)、球形芽孢桿菌(Bacillus sphaericus)、蠟樣芽孢桿菌(Bacillus cereus)、短小芽孢桿菌(Bacillus pumilus)、環(huán)狀芽孢桿菌(Bacillus circulans)和解淀粉芽孢桿菌(Bacillus amyloliquefaciens)[180-181]，其中枯草芽孢桿菌、蠟樣芽孢桿菌和地衣芽孢桿菌是巴士殺菌乳和超高溫殺菌乳的主要污染因素之一[60]，蠟樣芽孢桿菌也是6～7 ℃儲存的巴氏殺菌奶和乳酪貨架期的限制因子[182]，因為其可以產(chǎn)生一種或者多種腸毒素而引起食物中毒[45,183-184]。歐盟在2010年測試奶粉時發(fā)現(xiàn)呈陽性桿菌毒素的樣品達到3.8%[185]，另有研究表明，在包裝乳的腐敗中，60%是由革蘭氏陽性桿菌(如芽孢桿菌)引起的[186]。芽孢桿菌可以形成耐熱孢子，在超高溫滅菌(UHT)條件下依然存活且一般處于休眠狀態(tài)，但在產(chǎn)品貯藏、運輸、銷售過程中，遇到適宜條件就會被激活，逐漸轉(zhuǎn)化為營養(yǎng)細胞，不斷生長繁殖，產(chǎn)酸、產(chǎn)氣，導(dǎo)致牛奶變質(zhì)[187]?？漳c彎曲菌是引起急性胃腸炎的主要原因[188]，可導(dǎo)致痙攣、血性腹瀉、嘔吐、腹痛，并可能引起神經(jīng)系統(tǒng)疾病格林巴利綜合征，在歐洲生鮮乳中檢出頻率為0～6%[189-191]，在意大利北部、波蘭、大不列顛、法國等國生鮮乳中均有檢測到，檢出頻率大概在1.4%～4.6%[192-194]。初步研究表明，牛是主要污染源，但其具體傳播途徑目前還不清楚[11]，而對于牛來說，直接飲用生鮮乳是引起疾病暴發(fā)的主要原因[195]，全基因多位點序列分析(wgMLST)可用于檢測彎曲桿菌疫情[196]。由沙門氏菌感染引起的人類疾病有傷寒和胃腸道疾病，在罐牛奶中檢測到沙門氏菌的頻率很低，一般低于1%[16,197-198]。如巴西自2000年之后由沙門氏菌引起的疾病暴發(fā)很少，僅有15起[199]。小腸結(jié)腸炎耶爾森菌可使人患類似闌尾炎，雖然巴氏滅菌會殺了小腸結(jié)腸炎耶爾森菌，但是如果巴氏殺菌不徹底或發(fā)生二次污染，它也可在冷藏溫度下繁殖[188]，是導(dǎo)致小兒腸炎的主要原因[88,200]。結(jié)核分枝桿菌是與奶相關(guān)的最耐熱病原體[201]，而生孢梭菌則可導(dǎo)致乳制品的腐敗[202]。

關(guān)于生鮮乳有害微生物的檢測方法有很多，梁國添[203]對此做了論述，且指出生物技術(shù)與各種檢測方案結(jié)合使用是未來乳品安全控制的發(fā)展方向，另有研究稱高通量測序法在未來生鮮乳生產(chǎn)環(huán)境中微生物的檢測上有很重要的意義[204-205]。

4　展望

微生物是影響生鮮乳消費的主要風(fēng)險因子[206]，雖然在很多國家銷售生鮮乳是合法的，但生鮮乳有害微生物對人類健康的威脅不容忽視，其含量是生鮮乳質(zhì)量和安全的重要指標[207-208]，目前，各個國家采用的生鮮乳微生物指標有菌落總數(shù)、 芽孢數(shù)、總大腸菌群等。目前，各個國家采用的生鮮乳微生物指標有菌落總數(shù)、總大腸菌群和一些致病菌，我國將菌落總數(shù)作為生鮮乳微生物質(zhì)量合格的安全控制指標，生鮮乳微生物菌落總數(shù)標準為2×106cfu·ml-1，遠遠高于美國、日本和加拿大等國家且不檢致病菌，具體見表2[209-210]。常見高菌落總數(shù)主要是由于擠奶系統(tǒng)的清洗不徹底引起的[211-212]，但低菌落總數(shù)的生鮮乳也可能含有病原菌[13]，因此，僅用菌落總數(shù)作為生鮮乳微生物質(zhì)量合格的安全指標是不夠的，一些人畜共患體致病菌的檢測也很有必要。

表2　一些國家生鮮乳收購標準Table 2　Fresh milk acquisition criteria in some country

防治生鮮乳微生物污染的最好方法是控制或者控制每種有害微生物的來源，但由于特別的生產(chǎn)方式，這對于生產(chǎn)者來說是很難達到的[213-214]，因此，對生鮮乳微生物風(fēng)險趨勢做到及時、準確預(yù)警和建立安全生產(chǎn)規(guī)程，是防患于未然的高效手段，加拿大、美國和新西蘭等國均已基于HACCP設(shè)立比較完善的生產(chǎn)規(guī)程，對其乳業(yè)的健康發(fā)展起了重要保障作用，使其牛奶品質(zhì)一直保持較高水平。而我國自2008年才開始制定和實施全國范圍的生鮮乳質(zhì)量安全監(jiān)測計劃，與產(chǎn)業(yè)發(fā)展需求相比，研究的系統(tǒng)性仍十分薄弱。

因此，提高奶站或牧場整體建設(shè)水平和工作人員的素質(zhì)，以及建立我國自主特色的評價技術(shù)與生產(chǎn)規(guī)程對我國乳業(yè)的健康發(fā)展有重要意義，此外，普及生鮮乳消費安全知識，引起廣大消費者足夠重視，消費者尤其是嬰幼兒和孕婦不要直接飲用生鮮乳及其乳制品可減少相關(guān)食源性疾病的發(fā)生[213,215]。

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(責(zé)任編輯茍燕妮)

Pollutionandhazardanalysisofharmfulmicroorganismsinrawmilk

FengJiang-rong,LiChun-jie

(StateKeyLaboratoryofGrasslandAgro-ecosystems,CollegeofPastoralAgricultureScienceandTechnology,LanzhouUniversity,Lanzhou730020,China)

Thequalityandsafetyofdairyproductsisthefocusofsocietyinrecentyears.Rawmilk,astheupstreamofthedairysupplychain,isthefundamentalguaranteeforsustainabledevelopmentofdairyindustry,whichmightbecontaminatedavarietyofmicroorganisms,inwhichsomeofmicroorganismsareassociatedwithhumanillnessandmilkspoilage.Thispaperstatedthesourceandvarietiesofharmfulmicroorganisms,andtheirrefrigeration.Andthenthispaperdiscussedtheeffectofpasteurizationonharmfulmicroorganisms,andtheoccurrence,hazardsandcurrentresearchofseveralmilkbornepathogens.Thesewouldprovidebasisfordairyproduction.

rawmilk;milkbornepathogens;pasteurization; Listeria monocytogenes; Staphylococcus aureus

LiChun-jieE-mail:chunjie@lzu.edu.cn

10.11829/j.issn.1001-0629.2015-0705

2015-12-12接受日期：2016-04-20

公益性行業(yè)(農(nóng)業(yè))科研專項課題(201403071-6)

馮疆蓉(1991-)，女，甘肅白銀人，在讀碩士生，研究方向為生鮮乳微生物研究。E-mail:fengjr14@lzu.edu.cn

李春杰(1968-)，男，甘肅鎮(zhèn)遠人，教授，博導(dǎo)，博士，研究方向為禾草-內(nèi)生真菌共生體及草類植物病理學(xué)。E-mail：chunjie@lzu.edu.cn

TS252.2

A

1001-0629(2016)9-1875-18*

馮疆蓉,李春杰.生鮮乳有害微生物污染與危害分析.草業(yè)科學(xué),2016,33(9)：1875-1892.

FengJR,LiCJ.Pollutionandhazardanalysisofharmfulmicroorganismsinrawmilk.PrataculturalScience，2016,33(9)：1875-1892.