Carbon dioxide stimulates the production of amylovorin L by Lactobacillus amylovorus DCE 471, while enhanced aeration causes biphasic kinetics of growth and bacteriocin production

The effects of both oxygen and carbon dioxide on growth of and product formation by Lactobacillus amylovorus DCE 471, a promising new sourdough starter culture, were assessed through controlled, in vitro fermentation experiments, using a temperature of 37 degrees C and a constant pH of 5.4. It was seen that aeration affected both cell growth and amylovorin L production. At aeration rates of 1 l min(-1) and more, the bacterial population was subjected to oxidative stress as reflected by biphasic growth patterns. During the first growth phase, the maximum specific growth rate increased with increasing aeration rates stabilizing at the highest oxygen concentrations. The maximum obtainable cell yields decreased. During the second growth phase, the amylovorin L production was stimulated at the highest aeration. However, amylovorin titers were never higher in the presence of oxygen compared with the anaerobic fermentations. Carbon dioxide did not influence cell growth of L. amylovorus DCE 471. The maximum specific growth rate and the biomass concentrations were merely affected. On the other hand, the maximum soluble bacteriocin titers coincided with the highest carbon dioxide flow rates. These results indicate that mild aeration of type II sourdoughs might enhance both cell yield and amylovorin L production by L. amylovorus DCE 471, thereby contributing to the competitiveness of the strain. Growth in an ecosystem together with yeasts producing carbon dioxide might exert a positive effect on the production of amylovorin L as well.     

Analysing the lag-growth rate relationship of Yersinia enterocolitica

A generalised z-value concept has been applied to analyse the relationship between the lag and the growth rate of Yersinia enterocolitica at a range of temperature, atmospheric carbon dioxide and oxygen percentages. The product of the specific growth rate and the lag (the "work to be done" during the lag phase) is found to be independent of temperature. However, it does depend on the CO2 and O2 concentrations, though the effect of oxygen was less noticeable than the effect of carbon dioxide.     

Inhibition of Listeria innocua in hummus by a combination of nisin and citric acid

The effect of nisin or citric acid or combinations of these two inhibitors on the inactivation of a cocktail of three Listeria innocua strains was investigated in a model brain heart infusion (BHI) broth and hummus (chickpea dip). In BHI broth, citric acid had a limited ability to inhibit L. innocua growth. Nisin initially reduced L. innocua concentrations by about 3 log cycles; however, L. innocua reached concentrations similar to those of the control after 5 days at 22 degrees C. In combination, the effects of 500 IU/ml nisin and 0.2% citric acid were synergistic and resulted in complete elimination of L. innocua in the BHI broth. The inhibition of L. innocua by nisin (500 or 1,000 IU/g), citric acid (0.1, 0.2, or 0.3%), or their combinations also was evaluated in hummus. Citric acid alone did not affect L. innocua growth or the aerobic bacterial plate count. A combination of 1,000 IU/g nisin and 0.3% citric acid was somewhat effective (approximately 1.5-log reduction) in controlling the concentration of L. innocua and the aerobic plate count for up to 6 days. This combination also may be useful, in addition to proper hygienic practices, for minimizing the growth of the pathogen Listeria monocytogenes in hummus.     

Effects of the indigenous microflora of minimally processed lettuce on the survival and growth of Listeria innocua

Interactions between the natural background microflora of shredded lettuce and Listeria innocua (in lieu of Listeria monocytogenes ) were studied. The effect of increasing the initial size of indigenous populations (from 10³ to 10⁶–10⁷ CFU g⁻¹) was tested for its ability to reduce L. innocua growth on shredded lettuce. Co-culture experiments were performed in model media, where bacterial isolates from the indigenous microflora were tested for possible inhibitory effects. Varying the size of the indigenous populations had no effect on L. innocua survival or growth. However, interactions with individual species and mixed populations from lettuce did affect the survival and growth of L. innocua in model media. In general, mixed populations diminished L. innocua growth. In the undiluted lettuce medium, the various species tested individually either reduced or did not affect the growth of L. innocua . However, when the medium was diluted, some species extended the survival of L. innocua . Competition between the indigenous microflora and L. innocua resided mostly with the Enterobacter spp. and not with the pseudomonads. Enterobacter cloacae was particularly effective in reducing L. innocua growth. Lactic acid bacteria also reduced L. innocua growth in undiluted media. It is concluded that interactions with the natural background microflora may play an important role in determining the dynamics of Listeria populations on shredded lettuce.     

Combined physico-chemical and water transfer modelling to predict bacterial growth during food processes

The quality and safety of food products depend on the microorganisms, the food characteristics and the process. The prediction of conditions that prevent growth in complex situations due to the characteristics of the process and of the food cannot be obtained by predictive models of bacterial growth only. Thus, a combined modelling approach was developed by integrating three models, which were selected in a first step: (1) a bacterial model that predicts the bacterial growth from the physico-chemical properties of the media; (2) a water transfer model that predicts the effects of the drying process variables on the medium characteristics; and (3) a thermodynamic model that predicts the water activity aw and the pH of the media from its composition. A second step consisted in separately validating each selected model in which all of the physical, chemical or biological parameters appearing in the equations were previously measured. The third step combined the three knowledge models. The global model was validated on the basis of experimental results concerning the growth of Listeria innocua on the surface of a gelatine gel, the surface of which was submitted to a drying process (changes in relative humidity and air velocity). It was shown that bacterial growth models had to be modified: a specific model was set up to predict the maximum growth rate and another for the lag. Additionally, growth models set up in broth could not be applied in gelatine, leading to the development of a specific growth model on a solid surface. The thermodynamic model accurately predicted the pH and aw of bacterial broth in which high concentrations of solutes were added, and those of the solid media, the gelatine. The water transfer model was applied on gelatine data to predict the evolution of its surface aw during the drying process. The three models-bacterial, water transfer and thermodynamic, separately validated-were combined according to an integrated modelling strategy. The water transfer model coupled with the thermodynamic model predicted the aw on the gel surface. The predicted surface aw explained why growth inhibition was observed. Indeed, growth stopped at a predicted surface aw <0.94, corresponding to L. innocua minimum aw during the drying process. The global model satisfactorily predicted L. innocua growth on the surface of the gel. This study proves the validity of the approach and shows that the combination of the water transfer and thermodynamic models compensates for the lack of aw measurement techniques.     

食品中3种致病李氏菌MPCR-DHPLC检测方法的建立

应用复合PCR(multiplex PCR,MPCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的快速高通量检测方法。根据单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的特异基因序列分别设计引物,MPCR扩增的产物经DHPLC技术进行快速检测。以94株参考菌株做特异性实验,并开展了重现性检测实验。MPCR-DHPLC方法同步检测到单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的特异性阳性吸收峰,未检测到李斯特菌属其他近源种和非近源种参考菌株的阳性吸收峰,且重现性良好。该方法具有很好的特异性,可以快速、准确、高通量地检测食品中单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌,是食品微生物快速检测的新技术。     

基于MALDI-TOF MS技术对李斯特氏菌属两种细菌的快速鉴定

为提高基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF MS)在李斯特氏菌属鉴定中的分辨能力,建立快速准确鉴定单增和英诺克李斯特氏菌的质谱学方法。通过采集79株单增和57株英诺克李斯特氏菌的指纹图谱,利用Clin Pro tools软件对数据进行统计学分析,建立数学判别模型并验证其准确性。峰统计结果显示,两组数据峰强度差异显著的特征峰有16个,推测出单增李斯特氏菌生物标志物6个,英诺克李斯特氏菌10个,发现在单增李斯特氏菌中质量峰3985/7970 u和3972/7942 u是独立且连锁存在。基于遗传算法的判别模型交叉验证率和检测识别能力最强,分别为99.44%和100.00%,经验证准确率达到96%以上,可实现对单增和英诺克李斯特氏菌的快速准确鉴定。同时,利用Bruker Biotyper软件将以上菌株建库形成了实验室内部李斯特氏菌谱库,对8株未测李斯特氏菌进行搜库鉴定,匹配分数均高于商品化数据库,提升了MALDI-TOF MS对李斯特菌属的自动鉴定能力。     

The effect of reuterin on the lag time of single cells of Listeria innocua grown on a solid agar surface at different pH and NaCl concentrations

The lag time of single cells of Listeria innocua grown on the surface of Brain Heart Infusion Agar was studied by microscopy and image analysis. An experimental set-up that enabled relocation of the cells on the agar surface was developed and used to collect data from 50 to 100 individual cells at a time. Reuterin was added at different concentrations (0-10 AU/ml) and it was observed that it increased both the lag time of the cells and its variance. Furthermore, for a large proportion of cells, reuterin completely prevented the cell division within the time of observation. Reuterin in combination with low pH inhibited the cell division even more efficiently. A similar effect was observed for the combination of reuterin and sodium chloride. Our experimental set-up provides a good model system for generating data on the lag time of single cells on solid surfaces, which can improve the predictions of microbial growth on solid food matrices.     

Reduced detectability of Listeria monocytogenes in the presence of Listeria innocua

Recent foodborne crises have demonstrated the importance of monitoring food safety. In terms of microbiological criteria, food safety requires the reliable detection of pathogens such as Listeria monocytogenes along the food chain by appropriate analytical methods. However, indications exist that accompanying Listeria innocua strains suppress the growth of L. monocytogenes during selective enrichment, which may cause reduced or even inhibited detection. To study these effects, the limit of detection of L. monocytogenes was investigated in the presence of L. innocua using the International Organization for Standardization standard method ISO 11290-1 and the VIDAS LDUO system, an automated method based on enzyme-linked fluorescence technology. The challenge was to provide low initial Listeria concentrations at sufficient precision to quantify the influence on the probability of detection of L. monocytogenes. The application of reference materials appropriate for quantitative test methods and a standardized dilution procedure were necessary to ensure accurate CFU levels of defined proportions of mixtures of both Listeria species. During selective enrichment, overgrowth of L. monocytogenes by L. innocua could be confirmed, leading to high rates of false-negative results. Moreover, with both methods, a significant decrease in the detectability of L. monocytogenes could be quantified at ratios of 2:1 at very low concentrations representative of natural contamination levels often found in foods and environments. It is concluded that there is a need to improve existing procedures with respect to selective enrichment, as well as the detection techniques.     

Physiological state of single cells of Listeria innocua in organic acids

The growth of Listeria innocua in three organic acids was monitored by optical density measurements in a Bioscreen C automatic plate reader (Labsystems, Finland). The method described by Metris et al. [Metris, A., George, S.M., Baranyi, J., 2006. Use of optical density detection times to assess the effect of acetic acid on single-cell kinetics. Applied and Environmental Microbiology 72, 6674-6679.], was used to estimate both the growth rate and the lag time of single cells. It was found that the logarithm of both the growth rates and the population lag times increased linearly with sorbic acid concentration in the same way as previously described with acetic acid but the relationship was not linear with lactic acid. Of the three acids tested, sorbic was the most inhibitory for an equivalent undissociated acid concentration. The effect of lactic acid was dependent on both the growth phase of the inoculum and the inoculum concentration.     

Effects of atmospheric conditions, NaCl and pH on growth and interactions between moulds and yeasts related to blue cheese production

The starter culture Penicillium roqueforti, undesired cultures Penicillium caseifulvum and Geotrichum candidum and the potential starter culture Debaryomyces hansenii were examined for their growth and interactions at environmental conditions similar to the Danish blue cheese Danablu. The combined effect of low oxygen (0.3%) and high level of carbon dioxide (25%) at 4% NaCl w/v (a(w) 0.97) and pH 4.5 and 6.5 on radial growth was examined on a cheese medium at 10 degrees C. P. roqueforti and G. candidum were well adapted to growth at low levels of oxygen and high levels of carbon dioxide but G. candidum was not able to grow in the presence of 4% NaCl (w/v). Growth of P. caseifulvum was strongly inhibited at atmospheric conditions comprising 25% carbon dioxide, especially in combination with 0.3% oxygen. Generally D. hansenii showed strong growth at all environmental conditions examined, except at 0.3% oxygen combined with 25% carbon dioxide and 4% NaCl (w/v). Growth and sporulation of P. roqueforti was highly affected in the presence of G. candidum at 25% carbon dioxide irrespective of levels of oxygen and NaCl in the cheese media. P. caseifulvum caused a pronounced inhibitory effect towards growth of P. roqueforti and D. hansenii at 21% oxygen. D. hansenii caused weak inhibition of P. roqueforti at 21% oxygen, while positive interactions between the two species were indicated at 25% carbon dioxide and 0.3% oxygen.     

Modelling the competitive growth of Listeria monocytogenes and Listeria innocua in enrichment broths

The overgrowth of Listeria innocua in enrichment broths designed for the isolation of Listeria monocytogenes is believed to result from two factors: a selective growth advantage of L. innocua, and/or an inhibitory interspecies interaction. The generation times of 13 isolates of L. innocua and L. monocytogenes were determined in Brain Heart Infusion (BHI) and a variety of enrichment media. No significant differences were found in growth characteristics between either species in the various media, suggesting that the growth advantage of L. innocua in enrichment media was not as significant as previously described. Kinetic analysis of mixed cultures of L. monocytogenes and isolates of L. innocua producing a variety of inhibitory activities demonstrated the possibility of an inhibitory interaction between these two species resulting in the overgrowth of the enrichment culture with L. innocua. Modelling the evolution of the ratio between two populations in an enrichment process was used to analyze the impact of a selective growth advantage in L. innocua in an enrichment process for growth of L. monocytogenes. These findings support the widely held view that an overgrowth of L. innocua in the enrichment process can result from both a selective growth advantage as well as the production of inhibitory compounds. From a practical perspective, these interactions can result in an increase in false negatives.     

Effect of a(w), controlled by the addition of solutes or by water content, on the growth of Listeria innocua in broth and in a gelatine model

The effect of a(w) on the growth of Listeria innocua was investigated in broth and on the surface of a gelatine food model. In broth, a(w) was controlled from 0.91 to 0.99 by the addition of solutes such as NaCl, KCl, glucose, sucrose and glycerol. In the gelatine food model, a(w) was controlled by removal of water. In the a(w) range, 0.92-0.99, the generation times observed in broth in the presence of NaCl, KCl, sucrose and glucose were similar but were longer than those in glycerol. For lag times, the inhibition of L. innocua growth followed the order: NaCl = KCl = sucrose>glucose>glycerol. When comparing growth at a(w) 0.95 for the three media--broth + NaCl, gelatine gel (a(w) controlled by removal of water) and gelatine gel with NaCl (gel + NaCl, a(w) controlled by NaCl)--the shortest generation time was observed in broth + NaCl, followed by gel + NaCl and, finally, on gel with a larger gap between the last two. The generation time on gel was five times greater than the generation time in broth + NaCl and 2.5 times greater on gel + NaCl. It was concluded that not only the structure of the media (solid or liquid) had an effect on Listeria inhibition but also and mainly the way the a(w) was adjusted. Removal of water was more stressful to Listeria than the addition of NaCl.     

INHIBITION OF LISTERIA INNOCUA AND L. MONOCYTOGENES IN A LABORATORY MEDIUM AND COLD-SMOKED SALMON CONTAINING LIQUID SMOKE

Five commercial liquid smokes were tested in vitro and the most inhibitory to Listeria monocytogenes ATCC 19115 and L. innocua ATCC 33090 was Charsol Supreme. Chum salmon samples (100-g each) were brined, dipped for 15 s at varying concentrations of liquid smoke, inoculated with L. innocua, cold-processed and analyzed. Liquid smoke concentrations of 60–100% reduced L. innocua by 3-log₁₀/g in the final product. Dwell times of 15 s to 5 min using 60% liquid smoke gradually decreased Listeria survival with an optimum 5-min dip. Isoeugenol was antilisterial in vitro but lacked synergism with liquid smoke in cold-smoked salmon. An immunoassay kit detected low inoculum levels (< 100 CFU/g) of L. innocua in one of three samples that were treated with liquid smoke for two and four minutes. Charsol Supreme was antilisterial but could not be relied on to totally eliminate Listeria in cold-smoked salmon. Panelists found the 0 to 2-min dipped sockeye salmon slightly desirable with no significant (p < 0.05) differences. The 5-min treatment was significantly (p < 0.05) darker, scored lower in desirability and flavor and contained 93 ppm of phenolic compounds.     

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.     

Enumeration and growth of naturally occurring Listeria spp. in unpackaged ham

A total of 301 unpackaged retail ham samples were tested for the presence and number of Listeria spp. after 7 days at 5 degrees C to simulate domestic storage. Thirteen samples (4.3%) contained Listeria monocytogenes, with the highest count being 1.6 x 10(3)cfu g(-1). Thirteen samples contained other Listeria spp. Genotyping showed that only one L. monocytogenes isolate from the 14 tested was of a type previously identified in New Zealand human cases. Listeria-contaminated batches were incubated at 5 degrees C over approximately 3 weeks to assess the growth rate of natural contaminants. None contained L. monocytogenes, but growth occurred in one sample containing Listeria welshimeri and four containing Listeria innocua. Growth was usually slow at 0.002-0.004 log h(-1). In one sample, L. innocua grew at 0.02 log h(-1) although the maximum number reached was only 4.0-5.0 x 10(3)cfu g(-1). In five other samples little growth, if any, occurred. Growth of naturally occurring Listeria spp. at 5 degrees C was therefore generally slower than predicted by the Pathogen Modelling Programme (PMP) or did not occur.     

Effects of acid adaptation on the survival of Listeria monocytogenes on modified atmosphere packaged vegetables

The survival and growth of Listeria monocytogenes and L. innocua on ready-to-use (RTU) packaged vegetables (lettuce, swedes, dry coleslaw mix, bean-sprouts) were studied. The effects of acid adaptation of Listeria spp. on their survival during subsequent storage were also investigated. Listeria innocua behaviour was similar to that of L. monocytogenes on all vegetables examined. The survival and growth patterns of Listeria varied with the packaged product. Populations on packaged lettuce and swedes significantly increased ( P  < 0.05, by 1–1.5 log cycles) during a 14-day storage period. During the same period, Listeria counts gradually decreased (by 1–1.5 log cycles) on coleslaw mix. Acid adaptation enhanced survival of Listeria spp. during storage in packages of vegetables which had relatively high in-pack CO₂ levels (i.e. 25% in packaged coleslaw, bean-sprouts). It is concluded that adapting listerial cells to mildly acidic conditions rendered cultures more resistant to relatively high (25–30%) CO₂ atmospheres.     

Preincubation time and the use of oxygen indicators in determining the microbiological quality of aseptically packed pea and tomato soup

In order to get accurate information about the preincubation time needed for viscous aseptic products, the growth characteristics of three food poisoning organisms Staphylococcus aureus, Clostridium perfringens and Bacillus cereus in pea soup, and one spoilage organism, Lactobacillus plantarum, in tomato soup, were followed during a preincubation period at 30 degrees C for 14 days. The sterile food packs were sealed into polyamidepolyethylene laminate bags containing different headspace gas concentrations (21% oxygen + 80% nitrogen, 5% oxygen + 95% nitrogen and 100% nitrogen). Bacterial growth was followed every second day by counting the number of colony forming units and following the headspace oxygen and carbon dioxide concentrations. The bacterial strains differed in their growth characteristics, especially in their ability to consume oxygen from the headspace, and also in their ability to produce carbon dioxide. The headspace oxygen concentration was also followed by oxygen indicators in order to determine the correlation with the oxygen concentration of the headspace and the growth of bacteria. The bacterial growth followed more closely the concentration of carbon dioxide than the concentration of oxygen. It was concluded that further studies are needed to determine the preincubation period for viscous aseptic products in order to furnish accurate data on the growth characteristics of low numbers of bacteria in aseptic foods at different headspace gas compositions.