,1, 李晴1, 唐文倩1, 王虎虎2, 徐幸莲,2, 邱伟芬1Effects of nuoB on Physiological Properties of Pseudomonas fragi and Its Spoilage Potential in Chilled Chicken
WANG GuangYu,1, LI Qing1, TANG WenQian1, WANG HuHu2, XU XingLian,2, QIU WeiFen1通讯作者:
责任编辑: 赵伶俐
收稿日期:2020-08-14接受日期:2020-10-14网络出版日期:2021-04-16
| 基金资助: |
Received:2020-08-14Accepted:2020-10-14Online:2021-04-16
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王光宇, 李晴, 唐文倩, 王虎虎, 徐幸莲, 邱伟芬. nuoB对莓实假单胞菌生理特性及在冷鲜鸡肉中致腐能力的影响[J]. 中国农业科学, 2021, 54(8): 1761-1771 doi:10.3864/j.issn.0578-1752.2021.08.015
WANG GuangYu, LI Qing, TANG WenQian, WANG HuHu, XU XingLian, QIU WeiFen.
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0 引言
【研究意义】微生物活动引起的肉品腐败导致大量的经济损失和食物浪费[1]。虽然低温可以有效控制部分嗜温性腐败菌的生长,但部分耐冷菌和嗜冷菌仍可以大量繁殖,从而导致肉的腐败[2]。假单胞菌属（Pseudomonas spp.）是有氧条件下储存冷鲜肉中的优势腐败菌属,而莓实假单胞菌（Pseudomonas fragi）是其中最常见的一种,其在肉中的污染比例高达56.7%—79.0%[3,4]。该菌能在2—35℃下的肉中生长,并且进行活跃的代谢活动,分解糖、蛋白和脂肪等物质,导致肉品出现质构软化、变色、产黏和异味等腐败现象。因此,探明P. fragi的潜在作用机制是有效控制冷鲜肉腐败的前提,对保障食品卫生与安全具有重要意义。【前人研究进展】冷鲜肉为P. fragi提供了一个特别合适的生长环境,使该菌成为冷鲜肉中丰度最大的种群。除了与肉品基质的营养成分组成有关外,P. fragi的代谢活动也起到了很大的作用[5]。研究表明,P. fragi可以通过胞外聚合物的作用抑制其他种属细菌的生长,使其成为冷鲜肉中优势腐败菌的同时,在肉表面形成大块菌落和黏液[6]。同时,P. fragi分解肉中的肽和氨基酸还可能会产生多种挥发性化合物[7],进而导致异味的产生。但肉品腐败是一个非常复杂的过程,目前很难完全将腐败特征和腐败菌的代谢活动对应起来。现有的研究多集中在肉中腐败菌的种类数量与肉品质指标之间的关系,关于腐败菌特定基因和腐败相关活动的信息尚不明确。笔者研究团队前期从腐败冷鲜鸡肉中分离到一株具有较强致腐能力的P. fragi NMC25,气调包装环境中该菌的致腐能力受到抑制。转录组测序结果显示细菌呼吸链的nuoB在气调环境中相对于有氧环境显著下调,推测nuoB可能参与了细菌的致腐行为[8]。nuoB编码的NuoB亚基是构成细菌呼吸链中复合体I（complex I,又称为NADH脱氢酶）的重要成员[9]。研究表明细菌体内NADH脱氢酶片段的组装至少需要nuoB的存在[10,11]。而通过插入打断法破坏nuoB后,菌体内无法检测到复合体I活性,且不会出现强烈的极性效应[12]。因此,uoB的功能与复合体I密切相关。复合体I是大多数线粒体和细菌呼吸链中的第一个酶,能够催化电子转移,产生质子驱动力,对保持分解代谢和能量守恒之间的平衡及一些必要的代谢过程起到了重要作用[13]。目前已经有很多研究集中于这种酶在线粒体呼吸链中的核心作用上[14,15,16,17,18]。相对而言,人们对它在细菌能量生活方式中所起到的作用知之甚少。细菌的呼吸链和产能方式不同于真核生物,细菌复合体I由14个不同的蛋白质亚基（NuoA到NuoN）组成。近年来,X-射线晶体学[19]和电镜[20]等技术的使用已经解析了细菌复合体I的结构并扩展了对其机制的理解,同时有证据表明,复合体I在细菌一系列的生长和代谢过程中起重要作用,并且在不同的细菌群体中起到的作用不同[21,22,23,24],这些都有可能影响细菌对冷鲜鸡肉的腐败进程。【本研究切入点】综上所述,受到P. fragi污染的冷鲜鸡肉,在储藏过程中粘附于表面的菌体会迅速生长繁殖,同时细菌的代谢活动会导致肉品质构出现劣变、发黏和异味等现象。根据前期研究推测nuoB可能通过影响复合体I的功能进而影响细菌的生长和代谢过程,但目前尚缺乏该基因如何影响细菌对冷鲜鸡肉的致腐能力的系统和深入研究。【拟解决的关键问题】以前期分离到的强致腐P. fragi NMC25为研究对象,通过构建P. fragi NMC25的nuoB插入失活突变株,分析野生株与突变株的生物学特性差异,并评价原位培养条件下冷鲜鸡肉的腐败特征变化,探讨呼吸链中nuoB对P. fragi致腐效应的影响,为揭示冷鲜鸡肉的腐败规律,开发新型贮藏保鲜技术提供依据。1 材料与方法
试验于2018—2020年在南京农业大学国家肉品质量安全控制工程技术研究中心和南京财经大学食品科学与工程学院国家重点实验室进行。1.1 材料与试剂
NMC25菌株分离自腐败的冷鲜鸡肉,通过16S rRNA测序及种特异性基因carA PCR鉴定为P. fragi[25];胰蛋白胨大豆琼脂（TSA）、胰蛋白胨大豆肉汤（TSB）购于北京陆桥技术有限公司;冷鲜鸡大胸购于南京市苏果超市;本研究引物合成和序列测序均由上海生工生物工程有限公司完成,所用引物如表1所示。Table 1
表1
表1本研究中所用的引物
Table 1
| 引物 Primer | 序列（5′-3′） Sequence (5′-3′) |
|---|---|
| nuoB-F | ATATCTAGACCTGCATAGACATTGATGGAGGTTCTAC AAC |
| nuoB-R | ATATCTAGACTTGATCATCACGTCGCTGTTACGTTC |
| Kpn-Kn-F | ATATATATGGTACCGGAATAGGGAACTTCAAGATCCC CTC |
| Kpn-Kn-R | ATATATATGGTACCAGAGCGCTTTTGAAGCTGG |
| nuoB-outF | GAGGCCTGCGACATAGCGACACAAC |
| nuoB-outR | CAGACTTCGCGCTCGTACCAGTTGG |
| 下划线代表限制性内切酶酶切位点 Underline sequences are cleavage sites of restriction enzyme | |
新窗口打开|下载CSV
1.2 仪器与设备
HVE-50高压灭菌锅（Hirayama,日本）;BCM- 生化培养箱（STIK,美国）;Thermal Cycler PCR仪1600A超净工作台（AIRTECH,苏州）;B1-150a（Applied Biosystems,美国）;DYCP-32B 型琼脂糖水平电泳仪（六一,北京）;Universal Hood II凝胶成像系统（Bio Rad,美国）;Gene Pulser XcellTM电穿孔仪（Bio Rad,美国）;SpectraMax M2多功能酶标仪（Molecular Devices,美国）;Kjeltec 8400全自动凯氏定氮仪（FOSS,丹麦）;PL202-L天平、FE20台式pH计（Mettler Toledo,瑞士）。1.3 方法
1.3.1 ΔnuoB突变株的构建 参考SCHNEIDER等[12]的方法构建ΔnuoB插入失活突变株（图1）。以NMC25基因组为模板,以引物nuoB-F/nuoB-R对包含nuoB在内的同源臂进行PCR扩增,以pKD4质粒为模板,利用引物Kpn-Kn-F/Kpn-Kn-R扩增卡那霉素抗性基因,利用nuoB序列中限制性酶切位点Kpn I将卡那霉素片段插入同源臂扩增片段并连接到载体pCVD442上,获得自杀质粒pCVD442-ΔnuoB::Kn。将其电转化入大肠杆菌β2155获得供体菌β2155/pCVD442- ΔnuoB::Kn。供体菌与P. fragi NMC25通过结合试验,利用卡那霉素抗性筛选得到nuoB插入失活突变株P. fragi NMC25 ΔnuoB::Kn（简称为ΔnuoB）。利用引物nuoB-outF/nuoB-outR对构建突变株进行PCR鉴定,nuoB内部插入打断的阳性克隆片段大小约为3 260 bp,并对扩增产物进行测序鉴定,ΔnuoB突变株构建完成。图1
新窗口打开|下载原图ZIP|生成PPT图1pCVD442质粒图谱（a）和ΔnuoB突变株构建示意图（b）
Fig. 1pCVD442 vector (a) and diagram of the construction of ΔnuoB mutant (b)
1.3.2 生长曲线 将NMC25与ΔnuoB突变株接种于TSB中,在28℃下震摇培养至稳定期,取菌液待用。进行生长曲线测定时,将上述菌液进行梯度稀释,接种至20 mL新鲜TSB培养基中,使菌悬液浓度约为4 lg CFU·mL-1。将菌液在28℃条件下振摇培养16 h,每隔2 h取菌液进行梯度稀释,在合适的稀释度下取0.1 mL菌液涂布于TSA平板上,平板置于28℃恒温培养箱中培养24 h后进行菌落计数,根据菌落计数结果绘制生长曲线。
1.3.3 菌株表面特性 菌株聚集性测定参考XU等[26] 的方法,分别取5 mL NMC25与ΔnuoB稳定期菌液,8 000×g离心5 min后用PBS溶液重悬,测定初始OD600值（A0）。随后将菌悬液置于28℃的恒温培养箱中静置6 h,测定最终OD600值（A1）。菌体的聚集性（%）使用以下公式计算：（A0-A1）/A0×100。
菌株的swarming和swimming泳动性采用软琼脂平板法测定[27,28]。swarming平板的配方为LB 25 g?L-1、葡萄糖0.5 g?L-1和琼脂粉0.5%;swimming平板的配方为胰蛋白胨10 g?L-1、氯化钠5 g?L-1、葡萄糖2.55 g?L-1和琼脂粉0.3%。取两株菌稳定期菌液3 μL分别接种至平板的中心位置,室温下放置20 min使培养基充分吸收菌液。将平板置于28℃的恒温培养箱中培养96 h,每隔24 h取出,使用游标卡尺测定菌株扩散圈的直径大小。
1.3.4 生物被膜生成能力 菌株生物被膜形成能力的测定采用结晶紫染色法[29]。无菌条件下取200 μL稳定期菌液加入96孔细胞培养板中,置于28℃的恒温培养箱中培养,不接种的TSB作为空白对照。分别在12、24、36和48 h取出培养板弃掉孔中菌液,用无菌水洗涤3次,室温下干燥45 min后每个孔中加入200 μL 0.1%的结晶紫溶液染色30 min,弃去染液后用无菌水洗涤3次,随后使用200 μL 95%的乙醇溶液洗脱30 min,最后将培养板置于酶标仪中,在570 nm下测定吸光值。
1.3.5 原位培养条件下致腐能力评价 在原位培养条件下评价NMC25和ΔnuoB对冷鲜鸡肉的致腐能力差异。将冷鲜鸡胸肉切成10 g左右切片并送至南京航宇辐照技术有限公司进行辐照灭菌（6 kGy）。将NMC25和ΔnuoB于TSB中培养至对数期,菌液进行10倍梯度稀释后接种到鸡胸肉切片上,使肉表面细菌数量最终达到3 lg CFU·g-1。未接种的鸡胸肉作为对照。所有样品在8℃下储藏7 d,从第2天开始每天取样进行菌落总数、挥发性盐基氮（TVBN）、pH测定和感官评价。
样品菌落总数测定,将样品无菌条件下转移到含有90 mL生理盐水的均质袋中均质,取均质液进行10倍梯度稀释,选择合适的稀释度取0.1 mL涂布于TSA平板上,平板倒置于28℃恒温培养箱中培养,24 h后进行计数。
样品TVBN含量的测定参照GB 5009.228-2016《食品中挥发性盐基氮的测定》[30]进行并稍做修改。将10 g样品剪碎后加入100 mL去离子水,室温下振荡浸提30 min后过滤,取滤液5 mL加入5 mL氧化镁（10 g?L-1）悬浊液,使用凯氏定氮仪测定。
样品pH的测定参照KANG等[31]的方法,将10 g样品与40 mL预冷的去离子水混合,在15 000 ×g条件下匀浆10 s,使用pH计测定匀浆液的pH。
样品的感官评价参照ZHANG等[32]的方法并稍作修改,由经过培训的5名实验室研究生组成评定小组,依据GB 2707-2016《鲜（冻）畜、禽产品》标准中的感官要求,对样品整体可接受性进行评分。评分等级为1—9分,其中：9分为极好,8分为很好,7分为可接受,6分为可接受与不可接受之间,5分为轻度无法接受,4分为中度无法接受,3分为非常无法接受,2分为极度不能接受,1分为极差。当评分低于6时则视为样品已腐败。
1.3.6 统计分析 除生物被膜生成能力及菌体表面特性试验以外,所有试验均重复3次;生物被膜测定与表面特性试验重复6次,数值以平均值±标准差（SD）的形式表示。所得数据使用SPSS 22进行方差分析和T检验。采用Duncan’s multiple range test进行显著性（P<0.05）分析,用GraphPad Prism 7.0软件作图。
2 结果
2.1 生长曲线
NMC25与ΔnuoB的生长情况如图2所示。在振荡培养条件下,前期菌株生长较慢,4 h后开始迅速生长,并在12 h时细菌数量达到8 lg CFU·mL-1。在整个培养过程中,突变株的生长能力与野生株并无显著差异,说明体外培养时突变株的生长能力并未受到影响。图2
新窗口打开|下载原图ZIP|生成PPT图2P. fragi NMC25与ΔnuoB突变株生长曲线
Fig. 2Growth curves of P. fragi NMC25 and the ΔnuoB mutant
2.2 菌体表面特性
菌体表面特性研究结果发现,经过28℃下6 h孵育,野生株聚集性大小为23.28%,突变株的聚集性大小为23.75%,差异不显著。说明nuoB并未影响NMC25的聚集性。野生株和突变株的swarming泳动能力在24 h分别为5.28和5.32 mm,到96 h时分别达到9.09和8.87 mm（图3-a）,均表现为随着培养时间的延长而逐渐增大,且两者之间差异不显著。两株菌的swimming泳动性同样表现为随培养时间的延长而逐渐增大（图3-b）,24和48 h时两者泳动能力差异并不显著,但在72和96 h,突变株的泳动性分别显著低于野生株。表明nuoB仅影响了细菌的swimming泳动性,并不影响swarming泳动性。图3
新窗口打开|下载原图ZIP|生成PPT图3P. fragi NMC25与ΔnuoB突变株的swarming（a）和swimming（b）泳动性
*和**分别表明在同一培养时间差异显著（P<0.05）和极显著（P<0.01）。下同
Fig. 3Swarming (a) and swimming (b) motilities of P. fragi NMC25 and the ΔnuoB mutant
*, ** indicate significant difference (P<0.05) and extremely significant difference (P<0.01) at the same incubation time, respectively. The same as below
2.3 生物被膜形成
NMC25与ΔnuoB的生物被膜形成能力如图4所示。由图可知,在12 h时两株菌形成的生物被膜最多,随着时间的延长,生成的生物被膜逐渐减少。突变株的生物被膜形成量在12 h时与野生株之间无显著差异,但12 h以后显著低于野生株,说明ΔnuoB突变株的生物被膜形成能力受到了影响。图4
新窗口打开|下载原图ZIP|生成PPT图4P. fragi NMC25与ΔnuoB突变株的生物被膜形成能力
Fig. 4Biofilm formation abilities of P. fragi NMC25 and the ΔnuoB mutant
2.4 原位培养条件下致腐能力评估
经过2 d的培养,两组鸡肉切片中的菌落总数都达到了6 lg CFU·g-1,最终在第7天达到10 lg CFU·g-1。整个培养过程中,两组鸡肉切片的菌落总数均无显著差异,说明突变株在鸡肉上的生长能力没有发生较大变化。对照组的菌落总数在7 d内一直为0,说明辐照能有效杀死肉中存在的微生物（图5）。图5
新窗口打开|下载原图ZIP|生成PPT图5冷藏鸡肉储藏期间的微生物变化
Fig. 5Microbial changes of chilled chicken samples
原位培养条件下,两株菌所污染样品中的TVBN变化如图6所示。所有样品初始的TVBN值约为8 mg/100g。前3 d两组样品的TVBN值增长缓慢,在这期间突变株污染样品TVBN值均未超过15 mg/100g,但野生株污染样品在第2天就已经超过了15 mg/100 g。随后两组样品增长迅速,第7天时,野生株组的TVBN值达到99.68 mg/100g,而突变株组仅为61.04 mg/100g。从第2天开始,突变株组的TVBN值一直显著低于野生株组,说明鸡肉被突变株污染后,产TVBN能力大大降低。在整个培养过程中,对照组的TVBN值较为平稳,始终没有超过15 mg/100g。
图6
新窗口打开|下载原图ZIP|生成PPT图6冷藏鸡肉储藏期间的TVBN变化
***表示在0.001水平上差异显著（P<0.001）。下同
Fig. 6TVBN changes of chilled chicken samples
*** indicate significant difference (P<0.001). The same as below
两株菌所污染样品的pH变化如图7所示。样品的初始pH约为5.8,前2 d所有样品的pH均处于正常范围,2 d之后开始迅速增加。从第5天开始,突变株组的pH显著低于野生株组,在第7天时野生株组pH达到8.2左右,而突变株组pH仅约为7.7。对照组中pH变化较为平稳,一直处于5.7—6.2。
图7
新窗口打开|下载原图ZIP|生成PPT图7冷藏鸡肉储藏期间的pH变化
Fig. 7pH changes of chilled chicken samples
原位培养下两株菌所污染样品的感官变化如图8所示。前2 d所有的处理组样品感官评分都较好,随时间延长,感官评分逐渐降低。从第4天开始,NMC25和ΔnuoB均产生了肉眼可见的黏液和异味（图8-b）,均在4 d时被判定为腐败。但处理组间的外观和气味存在一定差异,突变株组样品产生的黏液和气味均稍弱于野生株组。对照组在整个培养期间的色泽、气味和状态都处于正常水平。
图8
新窗口打开|下载原图ZIP|生成PPT图8冷藏鸡肉储藏期间的感官评分（a）和实物图片（b）
虚线表明样品已达到感官拒绝
Fig. 8Spoilage scores (a) and pictures (b) of chilled chicken samples
The dotted line indicates the samples are sensory rejection
3 讨论
虽然复合体I在线粒体呼吸链中的作用已经进行了深入的研究,但对于它在细菌领域的生理作用却知之甚少。SPERO等[13]研究表明,γ-变形菌纲中的复合体I属于两种类型,对假单胞菌所属分支的生化代谢途径进行分析,结果显示有富马酸盐和硝酸还原酶等厌氧呼吸酶和大量行使细胞功能转运蛋白的富集趋势,表明它们可以转运和代谢一系列营养物质,有利于在不同的环境中生存。这可能是P. fragi能够存在于各种储藏条件如托盘包装、气调包装和真空包装食品中[33,34,35]的一个重要因素。FLEMMING等[36]的研究证实,nuoB的缺失会影响整个复合体I的组装和功能,但不影响呼吸链的其他组成部分。对于复合体I功能丧失的细菌来说,一个主要的生存策略可能是使用其他形式的还原剂（例如还原态铁氧化还原蛋白等）,这些还原剂可以在随后的代谢途径中被重新氧化[13]。因此,nuoB可能与细菌对营养物质的利用和代谢相关,有必要深入探究P. fragi中nuoB的作用方式。ERHARDT等[10]发现在LB培养基中,大肠杆菌nuoF突变株生长速率低于野生株1.5倍,最大生长数量约为野生株的一半。而PRUSS[37]发现在胰蛋白胨培养基培养时,大肠的nuo突变株在稳定期以前生长状况与野生株类似,进入稳定期后会弱于野生株,同时无法有效地将NADH氧化为NAD+,高NADH水平抑制了三羧酸循环中柠檬酸合酶和苹果酸脱氢酶,导致TCA循环及相关的氨基酸利用受到影响。但在碳源充足时,nuo突变株在整个培养周期的生长状况均与野生株类似。综上所述,nuo在碳源缺乏的情况下会影响菌株的生长,而随着碳源的丰富,这种影响会逐渐消失,这与本团队的研究结果一致。在本团队的前期研究中[38],使用Bioscreen C微生物全自动生长曲线分析仪测定400 μL静置培养菌液的生长曲线时发现突变株生长能力弱于野生株;而本试验中,在20 mL的TSB培养基中振荡培养时,nuoB的破坏并未导致突变株的生长缺陷,说明在此过程中,培养环境为P. fragi提供了充足的碳源,不会影响菌株的生长。在原位培养试验中,NMC25与ΔnuoB在冷鲜鸡肉中的生长能力都很强。GB 16869-2005《鲜、冻禽产品》中规定,鲜禽产品菌落总数应不超过106 CFU·g-1,两组鸡肉样品在2 d时就已经不符合标准。笔者课题组前期对冷鲜鸡肉进行宏基因组测序结果发现,腐败鸡肉中P. fragi的丰度最高。同时DOULGERAKI等[39]和LEBERT等[40]的研究也表明,虽然P. fluorescens在鲜肉中初始的数量会比P. fragi多,但随着时间推移,P. fragi会逐渐增多。所以鸡肉也是P. fragi合适的生长基质,并为其提供了足够的碳源,使突变株的生长不受影响。
肉中的腐败菌能够形成生物被膜,使其易粘附在食品接触表面或食品表面导致交叉污染,对食品安全有较大的潜在危害。本研究结果显示,P. fragi的生物被膜形成在12 h时最高,随后随时间的延长逐渐下降。表明12 h内,P. fragi菌体粘附在介质表面后通过进一步的菌体繁殖和彼此之间的相互作用形成了成熟的生物被膜,12 h之后进入主动分散阶段,生物被膜结构逐渐瓦解,菌体脱落,生物被膜形成量下降。细菌生物被膜的形成受到多种因素的影响[41],目前已证实沙门氏菌（Salmonella）和单增李斯特菌（Listeria monocytogenes）的生物被膜形成与表面特性有关[42]。而P. fragi的生物被膜形成能力与表面特性的关系尚不清楚。此外,菌株的扩散及粘附性与其表面特性也密切相关。细菌的表面化合物组成能够调节细菌的聚集能力,并且当聚集的菌体达到一定数量后可行使一定的生理功能[43]。在本研究中,ΔnuoB突变株的聚集性与野生株无显著差异,因此推测nuoB并未影响菌株的表面化合物组成。另外,菌体表面特性试验还发现ΔnuoB突变株的swimming泳动能力相比野生株显著降低。研究发现,大肠杆菌的nuoG经过插入打断后,在0.25%胰蛋白胨琼脂平板中的泳动能力明显下降[37],这与本研究结果一致。这种泳动性变化可能由于nuoB能够影响鞭毛的合成与发育,或者导致细菌无法提供足够的能量供鞭毛或菌毛运动,使得菌体的泳动性下降。菌体的泳动性还是菌体粘附和生物被膜形成过程中必不可少的因素[44],参与的鞭毛数量越多,形成的生物被膜越稳定。因此,试验中发现12 h以后突变株的生物被膜形成量显著低于野生株,也可能与菌体泳动性的下降有关。
TVBN是动物性食品的蛋白质或含氮化合物受细菌和酶的作用分解产生的,是反映肉品新鲜度的重要指标,鲜肉中TVBN含量随其腐败程度的增加而上升[45]。根据GB2707-2016《鲜（冻）畜、禽产品》规定,冷鲜肉中挥发性盐基氮含量不得超过15 mg·100 g-1。在本研究中,虽然两组样品在前3 d的TVBN增长都比较缓慢,但野生株组在2 d时就已经超过标准限量,这与NMC25本身在冷鲜鸡肉中具有较强的分解蛋白能力和较快的生长速度有关。而突变株组在3 d以后TVBN含量才迅速升高,在4 d时超过了标准限量,这表明ΔnuoB分解营养基质产生含氮挥发性化合物的能力下降。前期研究中发现,NMC25主要分解鸡肉肌原纤维蛋白中的肌球蛋白和肌动蛋白[46],nuoB的破坏可能会导致P. fragi分解利用这些蛋白的能力下降。正常鸡胸肉的pH范围是5.7—6.1[47],动物死后肉的pH是由糖酵解过程中糖原产生的乳酸含量决定的,是影响腐败微生物生长的重要因素[2]。pH升高会促进微生物的生长和对营养物质的利用,加速肉的腐败[48,49]。此外,多数假单胞菌的蛋白酶最适pH范围为6.5— 8.0[50],在本研究中,NMC25与ΔnuoB组的pH变化范围为5.8—8.2,处于或接近酶的最适pH,这可能是两株菌均能够快速生长的原因之一。与TVBN变化不同的是,突变株组的pH在第5天时才显著低于野生株组,这表明nuoB能够影响冷鲜鸡肉的腐败进程;同时也说明在腐败前期,细菌分解蛋白质产生的氨以及胺类等碱性含氮挥发性物质不会对肉的pH产生较大影响。
本研究中,野生株和突变株在导致腐败时能够在鸡肉上产生异味和大量的黏液。有研究表明,肉在腐败过程中会改变质构和pH,这些因素会影响挥发性化合物向气态转化,进而形成异味影响感官评价[1]。产黏是肉中非常常见的腐败现象,这些黏液能够保持细菌的水分,保护它们在低温下生长[51]。黏液的产生可能使它们在生存竞争中打败其他微生物,使自己处于优势地位。此外,本研究中ΔnuoB组TVBN含量的超标时间与感官评价时达到感官拒绝的时间一致,但NMC25组不一致,说明仅使用TVBN作为判断鸡肉是否腐败的标志并不准确;JAFFRES等[52]和JOFFRAUD等[53]的研究也认为TVBN并不能很好的指示海产品腐败,这表明对于食品腐败的判断仍需要结合理化指标和感官评价。
4 结论
当呼吸链中nuoB被破坏后,P. fragi的生长能力、聚集性和swarming泳动性未出现显著变化,但swimming泳动性和生物被膜形成量受到抑制,原位培养中鸡肉的TVBN、pH、产黏和异味程度均出现不同幅度的下降,表明nuoB能够影响P. fragi的部分生理特性和对冷鲜鸡肉的致腐能力。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
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DOI:10.1016/j.foodchem.2013.02.034URL [本文引用: 1]
DOI:10.1016/j.fm.2018.07.007URLPMID:30166172 [本文引用: 1]
Modified atmosphere packaging (MAP) is considered an effective method for extending the shelf life of meat. The use of optimal mixture of gases (CO2 and N2) in food packaging containers has been proved to effectively inhibit the growth of microorganisms in poultry meat. In general, a minimum CO2 concentration range of 20%-30% is required for the inhibitory effect. The aim of this study was to investigate the mechanism by which MAP (CO2/N2 30%/70%) inhibits Pseudomonas fragi, a dominant spoilage microorganism in aerobically stored chilled meat. The cell physiological changes were determined by measuring membrane integrity, membrane potential, ATP level, and extracellular proteolytic activity. The results showed that samples stored under MA retained cell membrane integrity, but lost significant membrane potential and ATP synthesis activity. Furthermore, the peptides issued from 2 structural proteins (myosin and actin) were mainly identified in air samples, indicating that these fragments result from bacterial proteolytic activity while MAP inhibited this activity. Overall, the study found that cell metabolism and extracellular protease activity decreased under MAP conditions. This study showed that MAP is an effective food preservation strategy and revealed mechanisms by which MAP inhibits spoilage.
DOI:10.1093/ps/84.5.797URLPMID:15913193 [本文引用: 1]
The effects of chicken breast meat lightness value (L*) on microstructure, protein extraction, and marinating and tumbling was investigated. Pale soft, and exudative (PSE) meat (L* = 57.7, pH 5.72) showed significantly lower salt soluble protein extraction with less heavy myosin chains compared with dark, firm, and dry (DFD) meat (L* = 44.8, pH 6.27). The PSE meat showed larger intercellular spaces among muscle fibers and bundles compared with normal and DFD meat. Marinated and tumbled PSE breast fillets had higher unbound brine compared with the other meats. Further cooking resulted in lower yield and higher shear force values for the PSE meat compared with normal and DFD fillets.
DOI:10.1016/j.meatsci.2016.11.025URLPMID:27918929 [本文引用: 1]
This paper reviews current literature relating to the effects of chilled and frozen storage on the quality characteristics of red meat (lamb and beef). These characteristics include tenderness (shear force), juiciness (fluid losses), flavour (lipid oxidation), colour and microbial loading. Research suggests that chilled storage of red meat can improve certain properties, such as shear force and species-specific flavour, to threshold levels before the effects on flavour and colour become deleterious, and key microbial species proliferate to spoilage levels. For frozen red meat, the negative effects upon quality traits are prevented when stored for shorter durations, although chilled storage conditions prior to freezing and retail display post-thawing can both positively and negatively affect these traits. Here, we review the effects of different chilled, frozen and combined chilled and frozen storage practices (particularly the chilled-then-frozen combination) on meat quality and spoilage traits, in order to contribute to superior management of these traits during product distribution.
DOI:10.1111/1541-4337.12526URLPMID:33325162 [本文引用: 1]
The increasing global population has resulted in increased demand for food. Goods quality and safe food is required for healthy living. However, food spoilage has resulted in food insecurity in different regions of the world. Spoilage of food occurs when the quality of food deteriorates from its original organoleptic properties observed at the time of processing. Food spoilage results in huge economic losses to both producers (farmers) and consumers. Factors such as storage temperature, pH, water availability, presence of spoilage microorganisms including bacteria and fungi, initial microbial load (total viable count-TVC), and processing influence the rate of food spoilage. This article reviews the spoilage microbiota and spoilage mechanisms in meat and dairy products and seafood. Understanding food spoilage mechanisms will assist in the development of robust technologies for the prevention of food spoilage and waste.
DOI:10.1016/j.fm.2016.11.015URLPMID:28040161 [本文引用: 1]
Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management.
DOI:10.1016/S0168-1605(97)00097-4URL [本文引用: 1]
DOI:10.1016/j.ijfoodmicro.2011.04.008URL [本文引用: 1]
The spoilage potential of six bacterial species isolated from cooked and peeled tropical shrimps (Brochothrix thermosphacta, Serratia liquefaciens-like, Carnobacterium maltaromaticum, Carnobacterium divergens, Carnobacterium alterfunditum-like and Vagococcus penaei sp. nov.) was evaluated. The bacteria were inoculated into shrimps, packaged in a modified atmosphere and stored for 27 days at 8 C. Twice a week, microbial growth, as well as chemical and sensory changes, were monitored during the storage period. The bacteria mainly involved in shrimp spoilage were B. thermosphacta, S. liquefaciens-like and C. maltaromaticum whose main characteristic odours were cheese-sour, cabbage-amine and cheese-sour-butter, respectively. The volatile fraction of the inoculated shrimp samples was analysed by solid-phase microextraction (SPME) and gas chromatography coupled to mass spectrometry (GC-MS). This method showed that the characteristic odours were most likely induced by the production of volatile compounds such as 3-methyl-1-butanal, 2,3-butanedione, 2-methyl-1-butanal, 2,3-heptanedione and trimethylamine. (C) 2011 Elsevier B.V.
DOI:10.1016/j.ijfoodmicro.2006.05.014URLPMID:16949172 [本文引用: 1]
Cold-smoked salmon is a lightly preserved fish product in which a mixed microbial flora develops during storage and where the interactive behaviour of micro-organisms may contribute to their growth and spoilage activity. The aim of this study was to assess the effect of the bacterial interactions between the main species contaminating the cold-smoked salmon on bacterial growth, chemical and sensory changes, and spoilage. First, Carnobacterium piscicola, Photobacterium phosphoreum, Lactobacillus sakei, Vibrio sp., Brochothrix thermosphacta and Serratia liquefaciens-like were inoculated as pure cultures on sterile cold-smoked salmon. All bacterial species grew well; Vibrio sp. was the fastest and L. sakei strains developed very rapidly as well with a high maximum cell density on cold-smoked salmon blocks (up to 10(9) cfu g(-1) after 10 days at 8 degrees C). Based on sensory analysis, Vibrio sp. was identified as non-spoilage bacteria, C. piscicola as very lightly and B. thermosphacta as lightly spoiling. L. sakei and S. liquefaciens-like were found to be the most spoiling bacteria. Secondly, C. piscicola and L. sakei, two species frequently occurring in the lactic flora of the product, were inoculated together and each of them in mixed cultures with respectively P. phosphoreum, Vibrio sp., B. thermosphacta, and S. liquefaciens-like. The growth of L. sakei was shown to strongly inhibit most of the co-inoculated strains i.e. P. phosphoreum, B. thermosphacta, S. liquefaciens-like and, to a lesser extent, Vibrio sp. The growth of C. piscicola seemed to be enhanced with B. thermosphacta and to develop earlier with P. phosphoreum and Vibrio sp. Conversely, S. liquefaciens-like and P. phosphoreum were weakly inhibited by C. piscicola. The main observation resulting from the sensory evaluation was the delay in the appearance of the spoilage characteristics in the mixed cultures with L. sakei, in particular L. sakei/ S. liquefaciens-like. On the other hand, the spoilage activity of the non-spoiler strains Vibrio sp. or the moderate spoilage strains B. thermosphacta and C. piscicola was increased when they were associated together. It is concluded that the spoilage behaviour of micro-organisms in mixed culture is significantly different from pure culture and explain the difficulty to find robust quality indices for this product.
