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.     

Effect of pH on the inhibition of Listeria spp. by vanillin and vanillic acid

The antimicrobial effects of vanillin and vanillic acid were verified against several species and strains of Listeria monocytogenes, Listeria innocua, Listeria grayi, and Listeria seeligeri in a laboratory medium adjusted to pH values ranging from 5.0 to 8.0. Medium pH had little influence on the MIC of vanillin as determined by a broth dilution assay, and growth of all test strains was inhibited by concentrations ranging from 23 to 33 mM. In contrast, none of the strains were inhibited by 100 mM vanillic acid at pH > 6.0, but complete inhibition was achieved at pH 5.0 with 10 mM. The effect of pH was further characterized by incubation of L. monocytogenes, L. innocua, and L. grayi in media containing 30 mM vanillin or 60 mM vanillic acid at pH 5.0, 6.0, and 7.0. Bactericidal effects increased with pH in media supplemented with vanillin. An inverse relationship was found for vanillic acid, and the lethality of the compound increased with declining pH. Mixtures of vanillin and vanillic acid exhibited additive inhibitory effects, particularly at lower pH. These natural antimicrobial compounds could prove useful either alone or in mixtures for the control of Listeria spp. in food products.     

50种植物源化合物对阪崎克罗诺肠杆菌的抑菌活性评价

阪崎克罗诺肠杆菌（Cronobacter sakazkaii）是一种食源性条件致病菌,它能够引起菌血症、坏死性小肠 结肠炎和新生儿脑膜炎等多种疾病。本研究选取50 种植物源化合物,检测其在含C. sakazkaii培养基中抑菌圈直径 及最小抑菌浓度（minimum inhibitory concentration,MIC）,旨在评价植物源化合物对C. sakazakii的抑菌作用并筛 查优选高效抑菌剂。结果表明：使C. sakazakii ATCC 29544培养基产生可见抑菌圈的植物源化合物有40 种,其中 7 种（香芹酚、百里醌、百里酚、肉桂醛、柠檬醛、原儿茶醛和原儿茶酸）的抑菌圈平均直径不小于13 mm;在本 研究选取的50 种植物源化合物中,百里酚和香芹酚对C. sakazakii有着最强的抑制作用,对9 株C. sakazkaii的MIC均 为0.1～0.2 mg/mL;百里醌、肉桂醛、柠檬醛和原儿茶醛对C. sakazkaii有良好的抑菌效果,对C. sakazkaii的MIC为 0.30～1.25 mg/mL;阿魏酸、绿原酸、丁香酸、硫辛酸、原儿茶酸、表儿茶素、咖啡酸、丹皮酚和菊苣酸的MIC为 2.50～5.00 mg/mL。以上结果表明,部分植物源化合物对C. sakazakii有良好的抑菌作用,有潜力作为天然抑菌剂应 用于食品加工、流通、贮藏过程中,从而发挥其控制C. sakazakii的作用。     

原儿茶醛对阪崎克罗诺肠杆菌毒力因子的抑制作用

探究植物源活性物质原儿茶醛对阪崎克罗诺肠杆菌泳动运动能力、生物被膜形成能力、黏附和侵入Caco-2细胞能力及9 种与菌体相关毒力基因转录的影响。结果表明,原儿茶醛对6 株阪崎克罗诺肠杆菌实验菌株的最小抑菌质量浓度（minimal inhibitory concentrations,MIC）和最小杀菌质量浓度均为2 mg/mL,对阪崎肠杆菌ATCC29544亚抑制浓度分别为1/200、1/100 MIC和1/50 MIC。亚抑制浓度的原儿茶醛能够抑制阪崎克罗诺肠杆菌在琼脂软平板中泳动运动能力;减弱菌体在12 ℃和25 ℃下不同时间段（24、48、72 h）生物被膜的形成能力;降低细菌黏附及侵入Caco-2细胞能力;下调9 种与菌体相关毒力基因的转录量。研究结果表明原儿茶醛能够抑制阪崎克罗诺肠杆菌多种毒力因子,其有潜力作为抗生素补充剂或抗毒性物质预防及控制阪崎克罗诺肠杆菌引起的相关感染。     

Guaiacol production from ferulic acid, vanillin and vanillic acid by Alicyclobacillus acidoterrestris

Alicyclobacilli are thermophilic, acidophilic bacteria (TAB) that spoil fruit juice products by producing guaiacol. It is currently believed that guaiacol is formed by Alicyclobacillus in fruit juices as a product of ferulic acid metabolism. The aim of this study was to identify the precursors that can be metabolised by Alicyclobacillus acidoterrestris to produce guaiacol and to evaluate the pathway of guaiacol production. A. acidoterrestris FB2 was incubated at 45°C for 7days in Bacillus acidoterrestris (BAT) broth supplemented with ferulic acid, vanillin or vanillic acid, respectively. The samples were analysed every day to determine the cell concentration, the supplement concentration using high performance liquid chromatography with UV-diode array detection (HPLC-DAD) and the guaiacol concentration, using both the peroxidase enzyme colourimetric assay (PECA) and HPLC-DAD. The cell concentration of A. acidoterrestris FB2 during the 7days in all samples were above the critical cell concentration of 10(5)cfu/mL reportedly required for guaiacol production. The guaiacol produced by A. acidoterrestris FB2 increased with an increase in vanillin or vanillic acid concentration and a metabolic pathway of A. acidoterrestris FB2 directly from vanillin to guaiacol was established. The high concentration of vanillic acid (1000mg/L) resulted in an initial inhibitory effect on the cells, but the cell concentration increased after day 2. Guaiacol production did not occur in the absence of either a precursor or A. acidoterrestris FB2 and guaiacol was not produced by A. acidoterrestris FB2 in the samples supplemented with ferulic acid. The presence of Alicyclobacillus spp. that has the ability to produce guaiacol, as well as the substrates vanillin or vanillic acid is prerequisite for production of guaiacol.     

Efficiency of different sanitation methods on Listeria monocytogenes biofilms formed under various environmental conditions

The resistance of Listeria monocytogenes biofilms formed under food processing conditions, against various sanitizing agents and disinfection procedures was evaluated in the present study. The first sanitation procedure included biofilm formation on stainless steel coupons (SS) placed in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) of various concentrations of NaCl (0.5, 7.5 and 9.5%) at different temperatures (5 and 20 °C). The biofilms formed were exposed to warm (60 °C) water for 20 min, or to peroxyacetic acid (2% PAA) for 1, 2, 3 and 6 min. Treatment with warm water caused no significant (P ≥ 0.05) reductions in the attached populations. Conversely, surviving bacteria on SS coupons decreased as the exposure time to 2% PAA increased and could not be detected by culture after 6 min of exposure. Biofilms formed at 20°C were more resistant to PAA than biofilms formed at 5 °C. Salt concentration in the growth medium had no marked impact on the resistance to PAA. The second sanitation procedure included biofilm formation of nonadapted (NA) and acid-adapted (AA) cells in TSBYE of pH 5.0 and 7.0 (i.e., NA-5.0, NA-7.0 and AA-5.0, AA-7.0) at 4 °C. Coupons bearing attached cells of L. monocytogenes were periodically exposed to chlorine (0.465% Cl(-)), quaternary ammonium compound (1% QAC) and 2% PAA. The resistance of attached cells to QAC, PAA and Cl(-) followed the order: AA-5.0>NA-7.0 ≥ AA-7.0>NA-5.0. The most effective sanitizer was QAC followed by PAA and Cl(-). The results can lead to the development of efficient sanitation strategies in order to eliminate L. monocytogenes from the processing environment. Furthermore, such results may explain the presence of L. monocytogenes after sanitation as a result of cell attachment history.     

Thermal oxidation of vanillin affects its antioxidant and antimicrobial properties

Accelerated oxidation of vanillin was studied by isothermal and non-isothermal differential scanning calorimetry (DSC) in model solutions. Exothermic peaks of DSC thermograms, due to the oxidation of vanillin, were observed. Vanillin oxidation to vanillic acid was confirmed by the detection of vanillic acid in heated vanillin samples using GC–MS. The effect of temperature on vanillin oxidation was studied by conducting DSC experiments with pure vanillin at several different final temperatures and by subsequent determination of vanillin and vanillic acid by GC–MS.     

婴幼儿配方粉中香兰素和乙基香兰素的检测方法比较

目的 建立婴幼儿配方粉中香兰素、乙基香兰素的液相色谱法和同位素稀释液相色谱质谱联用两种测定方法。方法 液相色谱法: 样品经乙酸锌沉淀蛋白、Oasis MAX SPE柱净化, HSS T3色谱柱分离后, 在279 nm波长处进行检测, 外标法定量。同位素稀释液质联用法: 样品采用乙腈沉淀蛋白稀释净化后, 采用Waters BEH C18柱分离, 在质谱检测器多反应监测(UPLC-MS/MS)模式下, 用同位素稀释液质联用法进行检测。结果 液相色谱法: 香兰素、乙基香兰素的定量下限(LOQ)均为0.1 mg/kg。香兰素的方法回收率为88%~90.8 %, 相对标准偏差(RSD)小于5.2 %; 乙基香兰素的方法回收率为84%~93.3 %, RSD小于6.2 %。同位素稀释液质联用法香兰素、乙基香兰素的LOQ分别为香兰素1、0.5 mg/kg。香兰素、乙基香兰素的方法回收率分别为82.1%~91.2 %、90.2%~97.6 %, RSD分别小于6.1 %、5.8 %。结论 本文建立的两种方法对同一样品的检测结果一致, 为婴幼儿配方粉中香兰素、乙基香兰素的定量检测提供了较为理想的方法。     

Antimicrobial activity of vanillin against spoilage microorganisms in stored fresh-cut mangoes

The antimicrobial activity of vanillin against four bacteria (Pantoea agglomerans, Aeromonas enteropelogenes, Micrococcus lylae, and Sphingobacterium spiritovorun), four fungi (Alternaria sp., Aspergillus sp., Penicillium sp., and Fusarium sp.), and three unidentified yeasts isolated from spoiling fresh-cut mango slices was verified in laboratory media adjusted to pH 5.0. MICs of vanillin against the fungi (12.5 to 13.3 mM), bacteria (10 to 13.3 mM), and yeasts (5.0 to 6.7 mM) indicated that all the test species were sensitive to the antimicrobial effects of vanillin. Fresh-cut mango slices were dipped for 1 min in solutions containing 40 and 80 mM vanillin before being packaged in rigid trays and stored at 5 and 10 degrees C to verify the effects of vanillin on the development of the spoilage microflora. Microbiological analysis for up to 14 days of storage revealed that treatment with 80 mM vanillin significantly delayed (P < 0.05) the development of total aerobic bacteria and yeast and mold populations. Vanillin may be a practical preservative for processing fresh-cut mango.     

Monitoring ultraviolet (UV) radiation inactivation of Cronobacter sakazakii in dry infant formula using Fourier transform infrared spectroscopy

Cronobacter sakazakii is an opportunistic pathogen associated with dry infant formula presenting a high risk to low birth weight neonates. The inactivation of C. sakazakii in dry infant formula by ultraviolet (UV) radiation alone and combined with hot water treatment at temperatures of 55, 60, and 65 °C were applied in this study. UV radiation with doses in a range from 12.1 ± 0.30 kJ/m2 to 72.8 ± 1.83 kJ/m2 at room temperature demonstrated significant inactivation of C. sakazakii in dry infant formula (P < 0.05). UV radiation combining 60 °C hot water treatment increased inactivation of C. sakazakii cells significantly (P < 0.05) in reconstituted infant formula. Significant effects of UV radiation on C. sakazakii inactivation kinetics (D value) were not observed in infant formula reconstituted in 55 and 65 °C water (P > 0.05). The inactivation mechanism was investigated using vibrational spectroscopy. Infrared spectroscopy detected significant stretching mode changes of macromolecules on the basis of spectral features, such as DNA, proteins, and lipids. Minor changes on cell membrane composition of C. sakazakii under UV radiation could be accurately and correctly monitored by infrared spectroscopy coupled with 2nd derivative transformation and principal component analysis.     

高效液相色谱法测定软饮料中香兰素和乙基香兰素

目的建立软饮料中香兰素和乙基香兰素的高效液相色谱法检测方法。方法样品中的香兰素和乙基香兰素在酸性条件下经乙腈提取,利用盐析作用分层净化,提取液浓缩复溶解后,供液相色谱检测。检测波长308 nm,外标法定量。结果香兰素和乙基香兰素在0.5~50.0?g/m L浓度范围内线性关系良好,相关系数均大于0.9999。添加水平为5、30、63 mg/kg时,香兰素和乙基香兰素的平均回收率分别为98.4%和100.4%,96.7%和100.2%,97.8%和100.3%,相对标准偏差在3.53%~4.70%,检出限均为0.5 mg/kg。结论方法操作简便、快速,经济实用,适用性强,结果准确可靠,可用于样品批量快速检测。     

Antibacterial Activity of Selected Hydroxycinnamic Acids

The objective of this study was to determine the antibacterial effectiveness of selected hydroxycinnamic acids (caffeic, ferulic, pcoumaric) against Escherichia coli and Staphylococcus aureus at pH 5.0, 6.0 and 7.0 and Bacillus cereus at pH 6.0, 6.5 and 7.0. p-Coumaric acid was generally the most effective inhibitor tested causing <99.9% inhibition of E. coli at 1000 μg/ml (pH 5.0, 48 hr), S. aureus at 500 μg/ml (pH 5.0, 48 hr), and B. cereus at 500 μg/ml (pH 7.0, 9 hr). Inhibition increased as pH decreased with E. coli and S. aureus but not B. cereus. Bacillus cereus appeared to be the most susceptible strain with 1000 μg/ml of the compounds tested causing <99% inhibition at all three pH's.     

市售婴幼儿米粉中克罗诺杆菌的分子分型和耐药分析

为了解婴幼儿配方米粉中克罗诺杆菌的污染情况、分子分型特征及耐药性情况,采集广西区内市售6个厂家和品牌不同配方的婴幼儿米粉进行克罗诺杆菌分离及fusA基因的扩增、测序和MLST数据库比对分析。并应用脉冲场凝胶电泳（PFGE）对菌株进行分型鉴定及微量肉汤稀释法测定分离株对8种常见抗生素的最小抑菌浓度（Minimal Inhibitory Concentrations,MICs）。采集的268份婴幼儿米粉共分离到32株克罗诺杆菌,检出率为11.94%,菌株鉴定结果显示32株分离株包括22株C.sakazakii、6株C.malonaticus、3株C.dublinensis和1株C.muytjensii。不同添加成分的婴幼儿配方米粉污染情况差异明显,其中添加淮莲和果蔬的米粉污染率最高为17.78%和15.38%。PFGE分析显示32个菌株共分成32种不同的分子型,相似度为61.20%~92.30%,所有分离株对环丙沙星、庆大霉素和头孢噻肟都敏感,部分菌株（6.25%~43.75%）对氯霉素、四环素、甲氧苄啶/磺胺甲基异恶唑、头孢西丁和萘啶酸表现为中等敏感,有2个菌株对氯霉素表现为抗性。结果表明市售婴幼儿配方米粉中存在一定的克罗诺杆菌污染,尤其是添加营养成分的米粉。婴幼儿配方米粉中克罗诺杆菌污染来源广泛,部分菌株对一些抗菌药表现为中等敏感甚至是抗性,提示克罗诺杆菌对抗菌药的敏感性呈现减弱的趋势。     

High‐performance liquid chromatography procedure for the determination of flavor enhancers in consumer chocolate products and artificial flavors

BACKGROUND: A number of individual high‐performance liquid chromatography (HPLC) procedures exist for the analysis of maltol, theobromine, ethyl maltol, catechin, vanillic acid, caffeine, vanillin, epicatechin, and ethyl vanillin. A single procedure utilizing simple sample preparation and less sophisticated equipment would be advantageous for the analysis of different sample types containing these compounds. RESULTS: An HPLC procedure has been developed using water as the extract for consumer products and artificial flavors. A methanol–water gradient was used to elute the compounds of interest using a reverse‐phase column. Absorbance detection using a wavelength of 273 nm was used to monitor the eluent. Recoveries for these compounds ranged from 88% to 104%. CONCLUSIONS: Results obtained for theobromine, catechin, caffeine, and epicatechin in Standard Reference Material 2380 Baking Chocolate compare well with those found in its certificate of analysis verifying that the procedure is valid. Vanillic acid and ethyl vanillic acid were found as oxidation products of vanillin and ethyl vanillin in both standards and some consumer products. Copyright © 2008 Society of Chemical Industry     

Determination of vanillin,ethyl vanillin,and coumarin in infant formula by liquid chromatography-quadrupole linear ion trap mass spectrometry

A simple, precise, accurate, and validated liquid chromatography-quadrupole linear ion trap mass spectrometry method was developed for the determination of vanillin, ethyl vanillin, and coumarin in infant formula samples. Following ultrasonic extraction with methanol/water (1:1, vol/vol), and clean-up on an HLB solid-phase extraction cartridge (Waters Corp., Milford, MA), samples were separated on a Waters XSelect HSS T3 column (150 × 2.1-mm i.d., 5-μm film thickness; Waters Corp.), with 0.1% formic acid solution-acetonitrile as mobile phase at a flow rate of 0.25 mL/min. Quanti?cation of the target was performed by the internal standard approach, using isotopically labeled compounds for each chemical group, to correct matrix effects. Data acquisition was carried out in multiple reaction monitoring transitions mode, monitoring 2 multiple reaction monitoring transitions to ensure an accurate identi?cation of target compounds in the samples. Additional identi?cation and con?rmation of target compounds were performed using the enhanced product ion modus of the linear ion trap. The novel liquid chromatography-quadrupole linear ion trap mass spectrometry platform offers the best sensitivity and speci?city for characterization and quantitative determination of vanillin, ethyl vanillin, and coumarin in infant formula and fulfills the quality criteria for routine laboratory application.     

百里醌对阪崎克罗诺肠杆菌的抑制作用

为探究百里醌对阪崎克罗诺肠杆菌的抑制作用及可能的抑制机理,检测了百里醌对阪崎克罗诺肠杆菌的最 小抑菌浓度（minimal inhibitory concentrations,MIC）及对其生长动力学模型的影响,并测定百里醌处理后阪崎肠 杆菌胞内ATP浓度、胞内pH值、膜电位、细胞膜完整性的变化,最后利用场发射扫描电子显微镜观察细胞形态的 改变。结果表明：百里醌对阪崎克罗诺肠杆菌的MIC为0.3～0.6 mg/mL;百里醌使阪崎克罗诺肠杆菌的生长迟滞期 延长,最大生长速率减小;百里醌影响了阪崎克罗诺肠杆菌细胞膜的通透性,表现为：质量浓度为MIC和2×MIC 的百里醌使细胞内ATP浓度由6.52 μmol/L分别降低为0.27 μmol/L和0.17 μmol/L,胞内pH值分别由5.69降低为5.22和 4.99,细胞膜完整菌体比例分别降低至80%和22%,引起细胞膜膜电位超极化;场发射扫描电子显微镜观测表明百 里醌使阪崎克罗诺肠杆菌细胞膜褶皱,菌体干瘪。综上所述,百里醌是通过影响细胞膜通透性和改变细胞形态抑制 阪崎克罗诺肠杆菌。这些结果表明百里醌有潜力作为控制阪崎克罗诺肠杆菌的天然抑菌剂应用于食品中。     

Bacillus sphaericus: the highest bacterial tannase producer with potential for gallic acid synthesis

An indigenously isolated strain of Bacillus sphaericus was found to produce 1.21 IU/ml of tannase under unoptimized conditions. Optimizing the process one variable at a time resulted in the production of 7.6 IU/ml of tannase in 48 h in the presence of 1.5% tannic acid. A 9.26-fold increase in tannase production was achieved upon further optimization using response surface methodology (RSM), a statistical approach. This increase led to a production level of 11.2I U/ml in medium containing 2.0% tannic acid, 2.5% galactose, 0.25% ammonium chloride, and 0.1% MgSO(4) pH 6.0 incubated at 37°C and 100 rpm for 48 h with a 2.0% inoculum level. Scaling up tannase production in a 30-l bioreactor resulted in the production of 16.54 IU/ml after 36 h. Thus far, this tannase production is the highest reported in this bacterial strain. Partially purified tannase exhibited an optimum pH of 5.0 with activity in the pH range of 3 to 8; 50°C was the optimal temperature for activity. Efficient conversion of tannic acid to purified gallic acid (90.80%) was achieved through crystallization.     

两株丝状真菌联合转化生产香草醛的研究

本文对黑曲霉(Aspergillus niger)CGMCC0774和朱红密孔菌(Pycnoporus cinnabarinus)CGMCC1115联合转化来源于米糠油脚的阿魏酸生产香草醛进行研究。通过60Co诱变黑曲霉菌株,得到耐较高底物浓度且不降解产物的突变株产香草酸2.48g/L;其转化液经过滤,调pH至5.0,补加5g/L葡萄糖,灭菌处理后,作为朱红密孔菌离菌体转化的底物溶液进行转化,得到1.07g/L香草醛。     

Analysis of the inhibition of food spoilage yeasts by vanillin

The antimicrobial potential of vanillin, the major component of vanilla flavour, was examined against the growth of three yeasts associated with food spoilage, Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii. Minimum inhibitory concentration (MIC) values of 21, 20 and 13 mM vanillin were determined for the three yeast strains, respectively. The observed inhibition was found to be biostatic. During fermentation, the bioconversion of sub-MIC levels of vanillin in the culture medium was demonstrated. The major bioconversion product was identified as vanillyl alcohol, however low levels of vanillic acid were also detected. Neither the vanillyl alcohol nor the vanillic acid was found to be antagonistic to yeast cell growth. The results indicate the importance of the aldehyde moiety in the vanillin structure regarding its antimicrobial activity and that the bioconversion of vanillin could be advantageous for the yeasts, but only at levels below MIC. These bioconversion activities, presumably catalysed by non-specific dehydrogenases, were shown to be expressed constitutively. It was observed that increased vanillin concentrations inhibited its own bioconversion suggesting that the activity required intact cells with metabolic capacity.