Growth Kinetics and Model Comparison of Cronobacter sakazakii in Reconstituted Powdered Infant Formula

Cronobacter sakazakii is a life-threatening bacterium, infrequently implicated in illnesses associated with the consumption of powdered infant formula (PIF). It can cause rare but invasive infections in neonatal infants who consume contaminated PIF. The objective of this research was to investigate the growth kinetics and develop mathematical models to predict the growth of heat-injured C. sakazakii in reconstituted PIF (RPIF). RPIF, inoculated with a 6-strain cocktail of non-heat-treated (uninjured) or heat-injured C. sakazakii, was incubated at different temperatures to develop growth models. Except for storage at 6 °C, C. sakazakii grew well at all test temperatures (10 to 48 °C). Uninjured C. sakazakii exhibited no observable lag phase, while a lag phase was apparent in heat-treated cells. A simple 3-parameter logistic equation was used to fit growth curves for non-heat-treated cells, while both Baranyi and Huang models were suitable for heat-treated C. sakazakii. Calculated minimum and maximum growth temperatures were 6.5 and 51.4 °C for non-heat-treated cells, and 6.9 and 50.1 °C for heat-treated cells of C. sakazakii in RPIF, respectively. There was no significant difference between growth rates of non-heat-treated and heat-injured cells in RPIF. For heat-treated cells of C. sakazakii, the lag phase was temperature-dependent and very short (between 25 °C and 48 °C). These results suggest that both non-heat-treated and heat-injured C. sakazakii cells may present a risk to infants if the pathogens are not completely destroyed by heat in RPIF and then exposed to subsequent temperature abuse. Practical Application: C. sakazakii is a life-threatening bacterium found in powdered infant formula (PIF). This study shows that the uninjured bacterium exhibits very short or no lag phase if not refrigerated and can grow well in reconstituted PIF (RPIF), while the heat-injured cells can multiply at an equivalent rate following metabolic recovery. Temperature abuse may allow C. sakazakii to grow and endanger infants fed with RPIF. Predictive models developed in this study can be used to estimate the growth and conduct risk assessments of this pathogen.     

婴幼儿奶粉中阪崎克罗诺杆菌隐性污染状况分析及针对性检测技术研究进展

婴幼儿奶粉中存在的阪崎克罗诺杆菌对婴幼儿危害性极强,卫生标准中对该微生物有严格限制,但检出现象仍然时有发生。存在许多因素导致阪崎克罗诺杆菌在婴幼儿奶粉生产线中的潜伏,本文阐述了其在生产空间中的暴露特征,重点论述逆境胁迫下该菌耐受表型和活的非可培养表型形成的隐性污染对监测和清除污染残留的不利影响。讨论了各类检测技术的优缺点,强调检测灵敏度、回收率和区分活的非可培养状态菌对于控制隐性污染的重要性。以期为阪崎克罗诺杆菌的监测和防控提供新思路,为保障婴幼儿奶粉生产安全提供参考和建议。     

LAMP法检测乳粉中的阪崎肠杆菌

据WHO最新报告显示,乳粉中的阪崎肠杆菌是导致婴幼儿感染和死亡的主要原因之一。FDA于2002年公布了婴幼儿乳粉中阪崎肠杆菌检测的推荐方法,但检测周期长达1周左右,而且操作复杂,难以满足控制该疾病暴发和传播的需要。DNA环介导等恒扩增(loop-mediated isothermal amplification,LAMP)技术是一种特异性、灵敏度高、检测时间短的新型基因扩增技术。本文以阪崎肠杆菌为研究对象,建立了一套应用于婴幼儿乳粉中阪崎肠杆菌快速检测方法。针对阪崎肠杆菌16s RNA基因设计4条引物特异性识别靶基因的六个特殊区域,并在63℃进行恒温扩增。实验结果表明,使用该方法能够在5h之内检测出复原乳样品中101CFU/mL的阪崎肠杆菌。灵敏度可以达到100fg阪崎肠杆菌基因组DNA。应用环介导等温扩增法,克服了传统检测周期长、操作复杂的缺点,不仅能够用于婴幼儿乳粉中阪崎肠杆菌的检测,而且满足了对于婴幼儿乳粉中阪崎肠杆菌快速检测的迫切需要。     

基于PAMAM树枝状大分子纳米免疫传感器快速检验婴幼儿奶粉中的阪崎肠杆菌

以聚酰胺胺树状大分子材料结合还原性石墨烯纳米复合物为电活性修饰膜层,固定于玻碳电极表面,采用物理吸附法固定化辣根过氧化物酶标记阪崎肠杆菌抗体制备成纳米免疫传感器,建立一种快速检测婴幼儿配方奶粉中阪崎肠杆菌的方法。优化的试验条件:以K3[Fe(CN)_6]为探针,过氧化氢浓度为0.5 mmol/L,pH 7.0的PBS溶液为检测底液,孵育温度37℃、孵育时间20 min。采用微分脉冲伏安法研究免疫传感器响应电流与阪崎肠杆菌浓度之间的关系。结果表明,免疫响应电流与阪崎肠杆菌浓度之间呈线性关系(ΔIpc(μA)=-1.1817 lg C+1.723,R~2=0.9989),其检出限为5.8×10~1 CFU/mL(S/N=3)。该法用于检测婴幼儿配方奶粉中的阪崎肠杆菌,其结果与国标方法(实时荧光PCR法)的数据一致,而且具有样品预处理简单、灵敏度高、快速、费用低等优点,可用于婴幼儿食品中阪崎肠杆菌的快速检测。     

市售配方粉中阪崎肠杆菌检测方法的比较

目的比较和分析两种不同的阪崎肠杆菌定性检测方法的区别。方法修改采用美国FDA和ISO阪崎肠杆菌检测方法,同时对194份市售配方粉进行定性检测。结果经过选择性增菌和分离,第二法中待鉴定的阪崎肠杆菌疑似菌落只有8个,明显少于第一法,而且其特征性菌落易于辨认。运用两种方法分别检测到4个和5个阳性样品,其中第二法检出的1个阳性样品在第一法中未检出。结论第二法中的选择性增菌液肉汤mIST—Vm和阪崎肠杆菌显色培养基的选择性明显优于第一法中相应的培养基,可以大大减少工作量,简单省时。     

奶粉中阪崎肠杆菌的污染及快速检测方法的研究

目的：了解福建省市售婴幼儿配方奶粉中阪崎肠杆菌的污染状况、污染途径并寻找快速准确的检测方法。方法：应用分离鉴定、PCR 和荧光PCR 等方法检测,分离鉴定采用阪崎肠杆菌显色培养基和全自动微生物生化仪。结果：阪崎肠杆菌在192 份市售婴幼儿配方奶粉中的检出率为1.56%,在60 份原料奶粉中的检出率为13.33%,30 份生产车间环境样本均未检出。结论：福建省市售婴幼儿配方奶粉中存在阪崎肠杆菌的安全隐患,某工厂奶粉中阪崎肠杆菌的污染主要来自原料奶粉。荧光PCR 和显色培养基可用于阪崎肠杆菌的快速筛选和分离。     

免疫磁珠结合显色平板快速检测婴儿配方奶粉中阪崎克罗诺杆菌

该研究利用羧基化磁珠偶联兔多克隆抗体,制备特异性免疫磁珠,并优化制备条件,然后与显色培养基联用,建立免疫磁珠-显色平板检测方法,用于富集分离婴儿奶粉中的阪崎克罗诺杆菌。结果表明,磁珠与抗体的最佳偶联温度37℃、偶联时间2.0 h,1 mg磁珠的最佳抗体添加量为200μg,饱和偶联量为182.77μg。制得的免疫磁珠,捕获目标菌的浓度范围为10²～10⁷ CFU/mL,捕获率为68.4%～82.4%,与非目标菌反应率低于10%。检测方法应用于模拟污染样品,在目标菌浓度10²～10⁵ CFU/mL时,捕获率为66.2%～71.5%。相比传统方法,该方法的检测周期缩短了2～3 d。     

奶粉中阪崎肠杆菌PCR和荧光PCR检测方法的研究

建立了奶粉中可致婴幼儿高死亡率的阪崎肠杆菌的PCR和荧光PCR检测方法。利用细菌16S和23SrDNA的保守区设计通用引物,对6株阪崎肠杆菌16S-23SrDNA间区序列(ITS)进行扩增和测序,在比对阪崎肠杆菌ITS序列的基础上,设计了11条PCR和荧光PCR检测引物,组合成30对PCR引物,并筛选出一对种特异性引物,建立了奶粉中阪崎肠杆菌PCR和荧光PCR检测方法。用10株阪崎肠杆菌,18株近源菌株验证实验表明,本文所建立的PCR和荧光PCR方法特异性强;加菌实验表明,奶粉样品中阪崎肠杆菌检测低限为(2.2～5.4)CFU/100g,灵敏度高;新建的PCR和荧光PCR方法与FDABAM(美国食品及药品管理局微生物分析手册)方法比对实验表明,三种方法的检测结果完全一致。由于PCR和荧光PCR检测方法快速、可靠,因此可替代传统检验方法。     

免疫磁分离和可视化金纳米杂交探针法快速检测婴幼儿配方乳粉中的阪崎克罗诺杆菌

利用免疫磁分离和金纳米杂交探针策略,开发一种快速和灵敏的可视化检测婴幼儿配方乳粉（powdered infant formula,PIF）中阪崎克罗诺杆菌（Cronobacter sakazakii）方法。抗体功能化的磁颗粒捕获C. sakazakii,金纳米探针对聚合酶链式反应的产物进行分析,这种探针法可以替代传统的电泳。免疫磁颗粒是由100?μL磁颗粒、120?μL碳化二亚胺盐酸盐、80?μL?C.?sakazakii单克隆抗体和体积分数0.02%的吐温-20制备。特异性检测中仅有C.?sakazakii是阳性结果。在纯培养基和不经预富集的人工污染PIF中,分别能检测到102?CFU/mL和103?CFU/g的C.?sakazakii。经过3?h的预富集,此方法可以在PIF中检测到低至4.5×101 CFU/g的C. sakazakii。可视化检测、电泳和紫外扫描光谱结果一致。因此,免疫磁分离和金纳米探针的方法可以替代凝胶电泳,对于快速检测C. sakazakii具有重要的意义。此方法将有助于检测PIF样本的C. sakazakii。     

奶粉中阪崎克罗诺杆菌生长与失活建模的研究进展

奶粉中含有丰富的营养物质,极易被微生物污染,而阪崎克罗诺杆菌是污染奶粉的主要病原菌之一。本文对国内外阪崎克罗诺杆菌生长、失活建模的研究进行综述,生长模型的建立为病原微生物的监测提供有效信息,失活模型的建立能反映传统、新型微生物控制技术和历史应激对阪崎克罗诺杆菌的失活作用效果。最后,针对目前阪崎克罗诺杆菌在预测建模研究中存在的问题进行探讨,并对阪崎克罗诺杆菌的研究方向提出建议,以期为今后的研究提供参考。     

Direct Real-Time PCR with Ethidium Monoazide: A Method for the Rapid Detection of Viable Cronobacter sakazakii in Powdered Infant Formula

The goal of this study was to establish a rapid assay for the specific detection of viable Cronobacter sakazakii in powdered infant formula (PIF). Samples were subjected to treatment multiple times with ethidium monoazide with a concentration gradient (gEMA) prior to PCR to discriminate viable from dead C. sakazakii cells. To improve the current detection limits, we developed a new buffer for direct quantitative real-time PCR (DqPCR) without DNA isolation. Using 17 PIF samples, our rapid assay was compared with the new U.S. Food and Drug Administration (FDA) method published in the Bacteriological Analytical Manual in 2012. Although both the new FDA method and our rapid assay, which consists of DqPCR combined with gEMA (gEMA-DqPCR), produced negative results for all 17 PIF samples, 5 of the 17 PIFs were positive by DqPCR when they were not treated with EMA. Furthermore, for PIF samples artificially contaminated with viable C. sakazakii, gEMA-DqPCR successfully detected between 1 and 9 CFU of viable C. sakazakii in 300 g of PIF within 9 h, including a 6-h preincubation. Our results indicate that multiple EMA treatments are required to avoid false-positive results due to the contamination of commercial PIF with dead or injured C. sakazakii cells. Our rapid assay may also improve the sensitivity of the screening portion required by the new FDA method published in the Bacteriological Analytical Manual in 2012.     

阪崎氏肠道杆菌与婴儿配方奶粉

阪崎氏肠道杆菌(E.Sakazakii),是一种罕见但经常引起致命性新生儿脑膜炎的致病菌(致命率达40%～80%)。感染这种高致命性的病毒,与食用污染有阪崎氏肠道杆菌的婴儿配方奶粉有关。一、食用被阪崎氏肠道杆菌污染的婴儿配方奶粉有关的新生儿死亡病历1.美国Himelright(2002)报道,2001年4月在田纳西州,一个出生时只有33.5周,1.27kg的早产男婴,由于呼吸困难而在Cesearian地区入院,出生11d后,婴儿发烧、心动过速、血流减弱,神经紊乱,脊髓中有阪崎氏肠道杆菌生长。静脉注射抗生素也不能阻止神经损坏,此婴儿发病9d后夭折,被诊断为致命性的脑膜炎。为…     

Thermal Tolerance and Survival of Cronobacter sakazakii in Powdered Infant Formula Supplemented with Vanillin, Ethyl Vanillin, and Vanillic Acid

The thermal tolerance Cronobacter sakazakii was examined in sterile powdered infant formula (PIF) rehydrated at 58 °C in water or apple juice supplemented with vanillin, ethyl vanillin, or vanillic acid. All three compounds decreased thermal tolerance during-rehydration and the lowest decimal reduction time (D-value, 0.19 ± 0.01 min) was measured in PIF rehydrated in apple juice supplemented with 20 mM vanillic acid. At this level of supplementation no C. sakazakii were detected in PIF stored for 48 h at 10 and 24 h at 21 °C subsequent to a sublethal heat treatment. Thermal tolerance during rehydration and survival in reconstituted PIF were influenced by compound type, concentration, and temperature. Supplementation of PIF with vanillin, ethyl vanillin, or vanillic acid could enhance the safety of PIF or other dehydrated foods contaminated with C. sakazakii.     

豆基婴儿配方粉化学与营养特性研究

有机大豆粉富含优质蛋白、矿物质和微量元素,是一类非乳配方粉的主要原材料。本研究以有机大豆粉及大豆分离蛋白为蛋白源,开发出不含乳糖的豆基婴儿配方粉。通过配方粉的化学成分、氨基酸、微观结构、渗透压等指标的测定与分析对配方粉的品质进行了研究,使用蛋白质功效比值法评价配方粉的营养,结果表明该豆基婴儿配方粉营养成分与GB 10765—2021《食品安全国家标准 婴儿配方食品》设计值相符合,必需氨基酸组成优于市售豆基婴儿配方粉,氨基酸评分（amino acid score,AAS）更平衡,且强化组氨酸等必需氨基酸可以满足婴儿需求,起到代替母乳的作用;透射电子显微镜观察发现该配方粉与市售豆基婴儿配方粉相比,颗粒分布更加均匀,具有更好的体系稳定性,且该配方粉渗透压（（281.00±1.20）（mOsm/kg H2O））符合婴儿体液平衡的需求。蛋白质功效比值（protein efficiency ratio,PER）分析结果表明该配方粉的大鼠实验组体质量总增长率和PER均优于酪蛋白参比组,具有较好的促进生长发育功效,营养价值较高,本研究可为研制豆基婴儿配方粉提供基础数据。     

配方奶粉生产过程中阪崎肠杆菌的风险与控制

阪崎肠杆菌是20世纪发现的一种对婴幼儿具有严重健康威胁的致病微生物,在婴幼儿配方奶粉的原料和生产过程中都有污染阪崎肠杆菌的风险。准确对配方奶粉生产过程中阪崎肠杆菌的污染风险进行评价,并相应制定科学有效的风险控制措施,对于降低风险、提高产品品质、保障食品安全具有重要意义。本文综述了配方奶粉生产过程中阪崎肠杆菌的存活规律、配方奶粉原料中可能存在的被阪崎肠杆菌污染的风险,以及通过环境、空气、设备以及制度操作等方面对阪崎肠杆菌风险进行控制的各种措施。     

Nonthermal Inactivation of Cronobacter sakazakii in Infant Formula Milk: A Review

Up-to-date, nonthermal technologies and combinations of them, in accordance with the “hurdle technology” concept, are being applied by different research groups in response to calls by the International Food and Human Health Organizations (ESPGHAN, 2004; FAO/WHO, 2006, 2008) for alternatives to thermal control of Cronobacter sakazakii in reconstituted powdered infant formula milk. This review highlights (i) current knowledge on the application of nonthermal technologies to control C. sakazakii in infant formula milk and (ii) the importance of the application of nonthermal technologies for the control of C. sakazakii as part of the development of strategies in the context of improving food safety and quality of this product.     

中国市售配方粉中阪崎肠杆菌和其它肠杆菌的污染状况

目的:调查和研究我国市售配方粉中阪崎肠杆菌和其它肠杆菌的污染状况。方法:采用传统的微生物学分离和鉴定方法检测212份市售配方粉中阪崎肠杆菌和其它肠杆菌。试验采用无菌水增菌、EE肉汤选择性增菌、VRBGA平板选择性分离、TSA平板分离培养和生化鉴定等。结果:从11份配方粉中分离到阪崎肠杆菌(占检测样品总数的5.19%),从103份样品中检出肠杆菌(占检测样品总数的48.58%)。检出的肠杆菌包括:泛菌属某些种、肺炎克雷伯氏菌、阴沟肠杆菌、醋酸钙-鲍曼复合不动杆菌、阪崎肠杆菌、非脱羧勒克氏菌、伤口埃希氏菌和大肠埃希氏菌等。结论:配方粉中肠杆菌的污染可能会导致婴幼儿,特别是婴儿的严重感染。本研究结果将有助于针对我国市售配方粉的卫生预防和控制。     

阪崎肠杆菌的研究进展

在概述了历年来对阪崎肠杆菌的研究进展，对其分类、来源、微生物学特性（包括菌落形态、生长特性等）、致病性、以及对失活特性（温度、水分活度和抗生素）进行了介绍，提出通过对不同污染来源的控制，从临床、环境、生产加工等环节减少阪崎肠杆菌的措施。     

奶粉中沙门氏菌和阪崎肠杆菌的测定能力验证结果分析

目的 提高实验室用传统生化方法检测沙门氏菌和克罗诺杆菌属(阪崎肠杆菌)的能力和水平。方法 用空白添加法和交叉试验检查培养基性能后,以奶粉样品为材料,用传统生化培养方法进行检测。结果 发现在传统生化检测技术中,对于排除非沙门氏菌来说,BS板作用突出,XLD和沙门氏菌显色平板容易受干扰菌影响。W样品检出沙门氏菌,Q样品未检出沙门氏菌;V样品检出阪崎肠杆菌,G样品未检出阪崎肠杆菌。结论 通过该研究,本实验室的沙门氏菌和克罗诺杆菌属(阪崎肠杆菌)的传统生化检测技术水平和能力得到了提高。