Characterization of tannase activity in cell-free extracts of Lactobacillus plantarum CECT 748T

In foods, tannins are considered nutritionally undesirable. Spectrophotometric methods have been used to detect tannin degradation by L. plantarum strains isolated from food substrates. Enzymatic degradation of tannic acid by L. plantarum CECT 748T was examined in liquid cultures and in cell-free extracts by HPLC. Significative reduction of tannic acid was not observed during incubation in the presence of L. plantarum cells after 7 days incubation. However, tannic acid was effectively degraded by cell-free extracts of L. plantarum during 16 h incubation. We have partially characterized L. plantarum tannase activity by measuring its esterase activity on methyl gallate. Tannase activity was optimal at pH 5.0 and 30 degrees C, and showed nearly 75% of the maximal activity at 50 degrees C. The biochemical characteristics showed by L. plantarum tannase are considered favourable for tannin biodegradation in the food-processing industry.     

Assessing biofilm formation by Listeria monocytogenes strains

When a microtitre plate assay was used to quantify biofilm production by Listeria monocytogenes strains following growth in Tryptone Soy Broth (TSB) for 48 h at 20 degrees C, 127 of 138 strains (92.0%) were classified as weak, 9 of 138 strains (6.5%) as moderate and only 2 of 138 strains (1.5%) as strong biofilm formers. The strains included environmental, animal, food (persistent and sporadic strains) and clinical isolates previously typed using esterase electrophoresis (ESE) and multi-locus enzyme electrophoresis (MEE). Strains from different sources produced similar quantities of biofilm, whereas biofilm production by ESE type II strains, irrespective of source, was greater than that observed for other ESE types. No correlation between MEE type and biofilm production was observed. A Petri dish assay which allowed parallel quantification and microscopic examination of biofilms was used to examine biofilm formation by selected L. monocytogenes strains during growth in TSB for 14 days at 20 degrees C. Results from these assays showed that following prolonged incubation, some L. monocytogenes strains categorized as weak biofilm formers by the 48 h microtitre assay, were able to form biofilms similar in terms of quantity and structure to those produced by strains classified as strong or medium biofilm formers. Results from 14-day Petri dish assays confirmed 48 h microtitre assays regarding greater biofilm production by ESE type II strains compared to other ESE types of L. monocytogenes. Biofilm production was similar for ESE type II persistent and sporadic food isolates but reduced for ESE type II clinical strains.     

应用改良的Jameson-effect模型建立植物乳杆菌对单增李斯特菌的抑制模型

本文旨在研究植物乳杆菌对单增李斯特菌的抑制作用,并建立植物乳杆菌对单增李斯特菌的抑制模型。通过稀释法获得植物乳杆菌纯菌培养液及植物乳杆菌/单增李斯特菌的混合培养液,分别置于7℃、14℃、21℃、28℃与35℃恒温培养箱中培养并适时取样计数,单增李斯特菌纯培养数据从Com Base数据库获得。基于两种菌的混合培养数据,应用Baranyi模型为初级模型,拟合得到两种菌的最大比生长速率和迟滞期,进而应用改良的Jameson-effect模型建立植物乳杆菌对单增李斯特菌的抑制模型,并对模型进行数学检验。结果显示,7℃时植物乳杆菌不生长;14℃、21℃、28℃与35℃条件下,植物乳杆菌对单增李斯特菌有显著抑制作用。抑制模型数学检验结果为均方误差值在0.07~0.10之间,偏差因子值在0.98~1.04之间,准确因子值在1附近,表明所建立改良的Jameson-effect模型能够较好地预测植物乳杆菌作为抑菌剂存在条件下单增李斯特菌的生长情况。     

Proteolytic activity of crude cell-free extract of Lactobacillus casei and Lactobacillus plantarum

Crude cell-free extracts of some strains of each L. casei and L. plantarum were assayed for their amino-, imino- and endopeptidase as well as the caseinolytic activity. L-alanine-, L-phenylalanine- and L-leucine-p-nitroanilide but not L-glutamic acid-p-nitroanilide, were hydrolyzed by all the strains indicating an amino-peptidase activity. The activity of proline iminopeptidase was very low compared to that of the aminopeptidase. L. casei could be distinguished from L. plantarum by its high endopeptidase activity against succinyl-phenylalanine- and glutaryl-phenylalanine-p-nitroanilide. The caseinolytic activity of cell-free extract of L. casei ATCC 393 was about one seventh the caseinolytic activity of intact cells, suggesting that the bulk of the cellular proteinase activity is located in the cell wall. It appears that a metallo aminopeptidase and a probable cysteine one are involved in the hydrolysis of amino acid-p-nitroanilide, whereas the endopeptidase activity appears to be related to a cysteine enzyme. Incubation of gels with L-leucine-p-nitroanilide after electrophoresis allowed the revealing of 2 zones of aminopeptidase activity in a strain of L. casei and only one in two others, but in L. plantarum it did not allow the revealing of any. The high proteolytic activities of L. casei compared to those of L. plantarum may explain its more frequent occurence in cheese and its probable role in the ripening of many cheese varities.     

植物乳杆菌SCP53生物膜的形成条件

研究了不同状态下植物乳杆菌SPC53生物膜形成能力的情况。通过改变其生长的环境条件与营养状况,采用结晶紫染色法和扫描电镜(scanning electron microscope,SEM)观察,定量及定性分析其生物膜的形成。结果表明:接种量5×10~7CFU/mL,培养时间36 h,培养温度42℃,pH值5.8是植物乳杆菌SCP53生物膜形成的最适培养条件,在MRS肉汤中依次添加200 mg/mL NaCl、75 mg/mL葡萄糖、7.5 mg/mL果胶、75 mg/mL全脂乳粉,可以促进植物乳杆菌生物膜的形成,形成后的生物膜A_(490)值为4.492 3。对照组A_(490)值为0.136 5,扫描电镜结果显示生物膜与浮游菌体有明显差异。     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Inhibition of Listeria monocytogenes by Lactobacillus sake strains of meat origin

The ability of two Lactobacillus sake strains of meat origin to inhibit the growth of Listeria monocytogenes at 4, 8, 15, 24 and 32°C in a conventional liquid media was investigated. Growth of L. monocytogenes was affected by Lac. sake strains at all temperatures. The inhibition was higher at 15, 24 and 32°C than at refrigeration temperatures. The inhibitory activity of both lactobacilli was similar perhaps due to the fact that Lac. sake 148 produces a bacteriocin inhibitory to L. monocytogenes, while Lac. sake 23 is a strong lactic acid producer. The antagonism exhibited by the lactobacilli on the L. monocytogenes strains seems to display a bacteriostatic rather than a bacteriocidal effect.     

不同方法观察单增李斯特菌生物被膜的比较研究

为探索单增李斯特菌生物被膜较好的定性观测方法,本文分别比较研究了银染法、结晶紫染色法、扫描电镜法等三种不同方法定性观察单增李斯特菌生物被膜的效果。实验结果表明:上述三种方法均可定性观察单增李斯特菌生物被膜;银染法、结晶紫染色法均可快速检出,且均简单实用,容易掌握,便于推广应用;扫描电镜法耗时较长且费用较高,但可清晰地观察到被膜内部结构。      

单增李斯特菌生物膜形成及其调控机制研究进展

单增李斯特菌是一种重要的食源性致病菌,极易在食品接触表面形成难以清除的生物膜,导致食品持续性的污染.细菌生物膜在形成量、活细菌数量、微观结构等方面受到环境因素及菌株自身特性的影响.在单增李斯特菌的生物膜形成过程中,多种调控机制发挥着重要的作用.该文介绍了细菌生物膜的形成过程及影响单增李斯特菌生物膜形成的重要因素,简述了...     

Control of Listeria monocytogenes on turkey frankfurters by generally-recognized-as-safe preservatives

Generally-recognized-as-safe chemicals applied to the surfaces of turkey frankfurters were evaluated for their ability to reduce populations of or inhibit the growth of Listeria monocytogenes. Frankfurters were treated prior to inoculation by dipping for 1 min in a solution of one of four preservatives (sodium benzoate, sodium propionate, potassium sorbate, and sodium diacetate) at three different concentrations (15, 20, and 25% [wt/vol]), with < 0.3% of the preservative being present for each frankfurter. Subsequently, 0.1 ml of a five-strain mixture of L. monocytogenes (10(6) CFU/ml) was used to surface inoculate each frankfurter separately in a sterile stomacher bag. Inoculated frankfurter bags were held at 4, 13, and 22 degrees C, and L. monocytogenes cells were enumerated at 0, 3, 7, 10, and 14 days of storage. The results of this study revealed that at all three concentrations of all four preservatives, the initial populations of L. monocytogenes decreased immediately by 1 to 2 log10 CFU/g. After 14 days of storage at 4 degrees C, L. monocytogenes counts for all treated frankfurters were 3 to 4 log10 CFU/g less than those for the untreated frankfurters. After 14 days of storage at 13 degrees C, L. monocytogenes counts for frankfurters treated with 25% sodium benzoate or 25% sodium diacetate were 3.5 to 4.5 log10 CFU/g less than those for untreated frankfurters, and those for frankfurters treated with 25% sodium propionate or 25% potassium sorbate were 2.5 log10 CFU/g less than those for untreated frankfurters. In all instances, the degree of growth inhibition was directly proportional to the concentration of the preservative. Only frankfurters treated with 25% sodium diacetate or sodium benzoate were significantly inhibitory to L. monocytogenes when held at 22 degrees C for 7 days or longer. Interestingly, the untreated frankfurters held at 22 degrees C were spoiled within 7 days, with copious slime formation, whereas there was no evidence of slime on any treated frankfurters after 14 days of storage.     

Formation of biofilm at different nutrient levels by various genotypes of Listeria monocytogenes

Strains of Listeria monocytogenes differ in their ability to form biofilms. The objectives of this study were to determine whether genetically related strains have similar biofilm-forming capacities and what effect nutrient concentration has on the ability of different strains to produce biofilms. Biofilms of 30 strains of L. monocytogenes, obtained from a variety of sources were grown on stainless steel in tryptic soy broth (TSB) or in a 1:10 dilution of TSB (DTSB) for 24 h at 32 degrees C. The amount of biofilm formed was determined with image analysis after cells were stained with bisBenzimide H 33258 (Hoechst 33258). The strains were genetically subtyped by repetitive element sequence-based PCR (rep-PCR) with the primer set rep-PRODt and rep-PROG5. Data were analyzed with an analysis of variance and Duncan's multiple range test. Eleven strains produced the same amount of biofilm in both media. Fourteen strains produced more biofilm in TSB than in DTSB. Five strains produced more biofilm in DTSB than in TSB. Serotype 4b strains produced more biofilm in TSB than did serotype 1/2a strains, whereas serotype 1/2a strains produced more biofilm in DTSB than did serotype 4b strains. Growth in DTSB resulted in decreased biofilm accumulation for serotype 4b strains. There was no correlation between genetic subtype and the amount of biofilm accumulation. These results indicate that strains of serotype 1/2a and serotype 4b differ in the regulation of their biofilm phenotype. The poor biofilm accumulation of serotype 4b isolates when grown in DTSB could be a factor in the predominance of serogroup 1/2 strains in food processing plants, where nutrients may be limited.     

Efficiency of sanitizing agents for destroying Listeria monocytogenes on contaminated surfaces

In an effort to control the potential hazard of dairy product contamination by contact with processing surfaces, the efficiency of four commercial sanitizing agents was evaluated using the AOAC use-dilution method for their bactericidal activity at 4 and 20 degrees C against Listeria monocytogenes strain Scott-A attached on four types of surfaces (stainless steel, glass, polypropylene, and rubber). Our results indicate that all sanitizers tested were more efficient against L. monocytogenes attached to nonporous surfaces than to porous surfaces. After 10 min of contact time, the limit concentration of disinfectants was at least 5 to 10 times higher for sanitizing rubber than stainless steel or glass surfaces. Concentrations of each sanitizer needed to be higher at sanitation at 4 degrees C than at 20 degrees C to destroy L. monocytogenes attached to stainless steel, glass and rubber when surface contamination was achieved at 4 or 20 degrees C.     

Adhesion to and viability of Listeria monocytogenes on food contact surfaces

Listeria monocytogenes is an important pathogen responsible for major outbreaks associated with food products. Adhesion to surfaces leads to significant modifications in cell physiology. The aim of this work was to determine the adhesion ability of 10 isolates of L. monocytogenes to eight materials commonly used in kitchens and to evaluate the viability of the adhered cells. The materials assayed were stainless steel 304, marble, granite, glass, polypropylene from a bowl and from a cutting board, and two kinds of silestone. All L. monocytogenes strains attached to all surfaces, although to different extents. L. monocytogenes adhered most tightly to granite and marble, followed by stainless steel 304, glass, silestones, and finally polypropylene surfaces. Surfaces at the threshold between hydrophobicity and hydrophilicity, with high electron acceptor capability and a regular pattern of roughness, were more prone to attachment. Polypropylene surfaces displayed the highest percentage of viable bacteria (nearly 100%), whereas marble and granite had a lower percentage of cultivable cells, 69.5 and 78.7%, respectively. The lowest percentage of culturable bacteria was found on white silestone (18.5%). These results indicate that there are differences in adhered cell viability on different materials. Cell viability assays are important to better understand the cross-contamination process because only adhered bacteria that remain viable are responsible for postprocess contamination.     

Behavior of Listeria monocytogenes in a Pseudomonas putida biofilm on a condensate-forming surface

Listeria monocytogenes has been isolated from condensate-forming surfaces in food processing plants. The objective of this research was to observe the behavior of L. monocytogenes on condensate-covered stainless steel with a Pseudomonas putida biofilm. L. monocytogenes-containing biofilms, either with or without added chicken protein, were incubated in a high humidity chamber at 12 degrees C to allow formation of condensate. Samples were analyzed for attached and unattached L. monocytogenes and total plate count periodically for 35 days. Samples were also taken for microscopic observation of Listeria and bacterial extracellular polymeric substances (EPS). L. monocytogenes attached in significantly greater numbers (> 3-log difference) to surfaces with preexisting P. putida biofilms than to Pseudomonas-free surfaces. L. monocytogenes survived in the presence or absence of P. putida with no added nutrients for 35 days, with numbers of survivors in the range of 3 to 4 log CFU/cm2 in the presence of P. putida and less than 2.9 log CFU/cm2 in pure culture. Attached and unattached L. monocytogenes were at similar levels throughout the incubation under all conditions studied. The addition of protein to the biofilms allowed growth of L. monocytogenes in pure culture during the first 7 days of incubation. Numbers of L. monocytogenes were not affected by the presence of P. putida when protein was present. Unattached L. monocytogenes were at levels of 3.6 to 6.7 log CFU/cm2 on the protein-containing surfaces. Microscopic observation of the condensate-covered biofilms indicated that L. monocytogenes formed microcolonies embedded within an EPS matrix over a 28-day period. This research demonstrates that L. monocytogenes can survive on condensate-forming stainless steel in low and high nutrient conditions, with or without the presence of Pseudomonas biofilm. The Listeria can detach and, therefore, have the potential to contaminate product.     

A predictive model for heat inactivation of Listeria monocytogenes biofilm on stainless steel

Heat treatment of potential biofilm-forming sites is sometimes used for control of Listeria monocytogenes in food processing plants. However, little information is available on the heat treatment required to kill L. monocytogenes present in biofilms. The purpose of this study was to develop a predictive model for the heat inactivation of L. monocytogenes in monoculture biofilms (strains Scott A and 3990) and in biofilms with competing bacteria (Pseudomonas sp. and Pantoea agglomerans) formed on stainless steel in the presence of food-derived soil. Biofilms were produced on stainless steel coupons with diluted tryptic soy broth incubated for 48 h at 25 degrees C. Duplicate biofilm samples were heat treated for 1, 3, 5, and 15 min at 70, 72, 75, 77, and 80 degrees C and tested for survivors using enrichment culture. The experiment was repeated six times. A predictive model was developed using logistic regression analysis of the fraction negative data. Plots showing the probability of L. monocytogenes inactivation in biofilms after heat treatment were generated from the predictive equation. The predictive model revealed that hot water sanitation of stainless steel can be effective for inactivating L. monocytogenes in a biofilm on stainless steel if time and temperature are controlled. For example, to obtain a 75% probability of total inactivation of L. monocytogenes 3990 biofilm, a heat treatment of 80 degrees C for 11.7 min is required. The model provides processors with a risk management tool that provides predicted probabilities of L. monocytogenes inactivation and allows a choice of three heat resistance assumptions. The predictive model was validated using a five-strain cocktail of L. monocytogenes in the presence of food soil.     

植物乳杆菌转化生成CLA的研究

从酸菜汁中分离出的一株植物乳杆菌LactobacillusplantarumLT2-6能将亚油酸转化成共轭亚油酸。该菌株在MRS培养基中经0.03%(w/v)的亚油酸诱导培养后,所获得的洗涤细胞具有很强的转化能力。在厌氧环境下利用L.plantarumLT2-6的洗涤细胞进行转化生成CLA的反应。结果表明,适宜反应条件为:温度30℃、pH7.0(0.1mol/L的磷酸盐缓冲系统)、细胞浓度20%(w/v)、游离亚油酸浓度1.5%(w/v)、反应时间64h,获得CLA最高产量为7.87mg/mL,产物为c9、t11/t9、c11-CLA。