Evaluation of the effect of Lactobacillus rhamnosus probiotic culture added to yogurt over Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis populations

The effect of different types of probiotics present in yogurt over known populations of Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis was evaluated. The three types of yogurt used were: without added probiotics, with added probiotics (Lactobacillus casei CRL_431 and L. acidophilus CRL_730 CHR HANSEN) and another one with the same probiotics mentioned above and Lactobacillus rhamnosus (LR-35) culture. About 10(9) CFU/ mL of each potentially pathogenic bacteria was added to each type of yogurt tested, and kept in refrigeration at 4 degrees C during its shelf life, about 30 days. Bacterial count was done the initial day and every four days. Results obtained show that there is a difference in the inhibition between yogurts without added probiotics and the commercial yogurt with added probiotics; there is a clear inhibitory effect of the last one over S. aureus, E. coli O157:H7 and Listeria monocytogenes. The yogurt with added probiotics and L. rhamnosus did not show any additional inhibitory effect over the bacteria tested when compared with the yogurt with added probiotics. S. enteritidis could not be evaluated because it was not detectable in any yogurt samples evaluated four days after its inoculation. This study confirms the antagonic effect of probiotic cultures over potentially pathogenic bacteria for human beings and animals that may be present in food. Nevertheless, the use of L. rhamnosus did not produce any additional inhibitory effect.     

Evaluation of the effect of probiotic cultures on two different yogurt brands over a known population of Staphylococcus aureus and the production of thermonuclease

The effect of probiotic cultures over known populations of Staphylococcus aureus inoculated in yogurt was studied; also the production and stability of its thermonuclease during yogurt storage was evaluated. In three different occasions, two different yogurt brands, one with additional probiotic cultures (Lactobacillus casei and L. acidophilus), were inoculated with known populations of S. aureus in high and low concentration (10(9) CFU/g and 10(7) CFU/g), respectively. These samples were stored for 28 days at 5 degrees C. Every four days the count of lactic bacteria, S. aureus and pH were evaluated, according to the methodology described in the Compendium of Methods for the Microbiological Examination of Foods, Vanderzant & Splittstoesser. The presence of thermonuclease was determined using petrifilm for S. aureus from 3M company. The pH and lactic bacteria population were constant during the testing period. Yogurt with additional probiotic cultures (high and low concentration) lowered the population of S. aureus to non detectable levels in 8 days; but, S. aureus could be cultured from yogurt without probiotics even after 24 days of incubation. Same time, the presence of thermonuclease was positive in all tests; it was not affected by probiotics. The presence of thermonuclease is related to the production of S. aureus enterotoxin. This work emphasizes again the beneficial effects of probiotic cultures in yogurt over bacteria and the importance of keeping hygienic practices in order to avoid the contamination of food with S. aureus and the eventual production of its enterotoxin, since it is not affected by probiotics.     

Evaluation of the activity of probiotic cultures over Listeria monocytogenes during the production and storage of yogurt

The effect of probiotic cultures over Listeria monocytogenes during the production and storage of yogurt was evaluated. A yogurt mixture (10.6% non-fat solid liquids, 3% fat and 0.3% gelatin) was prepared, homogenized and pasteurized. Yogurt was inoculated with 0, 10(2), 10(4) and 10(6) CFU/mL of L. monocytogenes and 0.02% of traditional lactic culture YC 180 (Streptococcus thermophilus and Lactobacillus bulgaricus) and probiotic culture ABY-1 (Bifidobacterium longum, B. bifidum, B, infantis, Lactobacillus acidophilus, Streptococcus thermophilus y Lactobacillus delbrueckii subsp. bulgaricus). It was incubated for 3 h at 43 degrees C until pH reached an approximate value of 4.8, followed by refrigeration at 5 degrees C for 21 days. During fermentation, samples were taken every hour, and during storage every 3 days, analyzing pH and lactic, bifidobacteria and pathogen count for each time. It was demonstrated that there was no significant simple effect for the type of culture used (ABY-1 and YC 180) (p = 0.684) over the amount of L. monocytogenes present in yogurt during the fermentation and storage periods. The presence of bifidobacteria in the ABY-1 culture did not present a significant effect over L. monocytogenes. Neither the effect of time presented a significant effect over L. monocytogenes (p = 0.448). In this case, the ABY-1 and YC 180 cultures present a bacteriostatic effect over the pathogen. The probiotic cultures had a bacteriostatic but not bactericidal effect over L. monocytogenes. This is not related to the protective effect of these cultures in bowel, since in-vivo conditions favor the production of antimicrobial substances, such as bacteriocins that act over pathogens.     

Presence of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. in food from animal origin in Costa Rica

The presence of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp was analyzed in two kinds of food from animal origin in Costa Rica. 100 samples of non pasteurized milk, from the principal producing zones of the country, and 100 samples of chicken giblets, purchased in retail markets, were analyzed according to the methodology described by Food and Drug Administration, 1995. Escherichia coli O157:H7 was analyzed in both kinds of food, while L. monocytogenes was evaluated in raw milk and Salmonella spp. in chicken giblets. Five strains of E. coli O157:H7 were isolated, three of them coming from chicken giblets and the other two from raw milk. 15% positivity for Salmonella spp. was found in chicken giblet samples and 3% positivity for L. monocytogenes in raw milk samples. The results obtained show the importance of the adequate processing of food from animal origin in order to decrease the potential transmission of pathogenic agents. The introduction of Hazard Analysis Critical Control Points system (HACCP) and Good Manufacturing practices in food industry keep on being the principal control measures and inocuity warranty.     

Effect of a probiotic mixed culture on texture profile and sensory performance of Minas fresh cheese in comparison with the traditional products

The effect of a mixed probiotic culture on instrumental texture, and on sensorial and related properties of Minas fresh cheese during refrigerated storage was investigated. Three cheese-making trials were prepared: T1, with the traditional type O starter culture (Lactococcus lactis subsp. lactis + L. lactis subsp. cremoris), T2 with only lactic acid and T3, with lactic acid and the probiotic ABT culture (Lactobacillus acidophilus La-5 + Bifidobacterium animalis Bb-12 + Streptococcus thermophilus). Instrumental texture profile analysis and related properties were monitored during storage for up to 21 days. Lb. acidophilus and B. animalis were present in high levels throughout storage of cheeses T3, above 6 log cfu.g(-1), threshold required for probiotic activity, and stimulation of the La-5 growth was observed. Cheeses with added probiotic ABT culture, as well as those made adding lactic acid only, showed to be less brittle and with more favorable sensorial features, due to higher pH values. Results indicated that the use of probiotic ABT culture complementary to lactic acid for the purpose of substituting the type O (Lc. lactis subsp. lactis + Lc. lactis subsp. cremoris) culture, traditionally employed for Minas cheese production, is advantageous.     

Effect of microwave oven over the growth and survival of Escherichia coli O157:H7 inoculated in bovine minced meat samples

The use of microwave ovens in food industry is a growing trend. It is used for thawing, drying and cooking food, but the microorganism's inactivation that this treatment may exert or not is still a subject of worldwide discussion. At the same time. Escherichia coli O157:H7 now presents itself as an emerging pathogen, distributed worldwide and associated with food. Its resistance to adverse environments has been widely discussed. The purpose of this work was to determine the effect of different times of exposure and cooking intensities of microwave oven on the survival of this bacterium inoculated into minced meat samples. These were inoculated with a high (10(7)-10(9) CFU/mL) or low (10(7)-10(7) CFU/mL) population of E. coli O157:H7, frozen for 3 days at-4 degrees C and thawed in a Whirlpool microwave oven according to their weight. They were radiated at levels of 70%, 80%, 90% and 100% for periods of 30, 60, 90 and 120 seconds. In each sample the rate of survival of the bacteria was determined according to the methodology described by Vanderzant & Splittstoesser. The results obtained showed that the rate of destruction of the bacteria analyzed was significant (p < 0.005). The number of bacteria present in the meat samples diminished dramatically as the exposure time and temperature increased, even though, for the complete elimination of this microorganism, a prolonged exposure was necessary, even though it did cause undesirable organoleptic characteristics in the food samples.     

Surface modification of poly(styrene) by the attachment of an antimicrobial peptide

Polymers that directly inhibit the growth of microorganisms at their surface are potentially useful. To investigate the feasibility of such materials, poly(styrene) (PS) resin beads that had had poly(ethyleneglycol) (PEG) grafted onto the surface were further derivatized by covalently linking an antimicrobial peptide onto the surface. The antimicrobial peptide was composed of 8 lysine and 7 leucine (6K8L) residues. The resulting surface‐modified polystyrene (SMPS) was microcidal in a concentration and time‐dependent manner against several micro‐organisms including E. coli O157 : H7, L. monocytogenes, S. aureus, P. fluorescens, and K. marxianus when suspended in phosphate buffer. The SMPS inhibited the growth of pathogenic E. coli O157 : H7 in trypticase soy broth. SMPS was bactericidal at pH 3.5 to 7, retained activity after heating to 200°C for 30 min, and could be extensively washed without loss of antimicrobial activity. Bioassays and HPLC analyses of buffer that had been preincubated with SMPS indicated that antimicrobial activity may have been due, at least in part, to the slow release of a peptide–PEG ligand from the PS to the buffer. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 609–616, 2001     

Characterization and Genomic Analysis of Escherichia coli O157:H7 Phage UAE_MI-01 Isolated from Birds

Verotoxin-producing Escherichia coli O157:H7 is responsible for the majority of foodborne outbreaks worldwide and may lead to death. Bacteriophages are natural killers of bacteria. All previously reported E. coli O157:H7 phages were isolated from ruminants or swine. Here, we report for the first time a phage isolated from bird feces in the United Arab Emirates (UAE), designated as UAE_MI-01, indicating birds as a good source of phages. Thus, phages could be a tool for predicting the presence of the host bacteria in an animal or the environment. UAE_MI-01 was found to be a lytic phage that was stable at wide ranges of pH, temperature, and chemical disinfectants, and with a burst size of almost 100 plaque-forming units per host cell after a latent period of 20 min and an adsorption rate constant (K) of 1.25 × 10⁻⁷ mL min⁻¹. The phage genome was found to be 44,281 bp long with an average GC content of 54.7%. The presence of the phage indicates the presence of the host cell E. coli O157:H7 in wild birds. Therefore, other birds, mainly poultry, could be also investigated for the presence of this pathogenic bacterium. To the best of our knowledge, this is the first report of an E. coli O157:H7 bacteriophage isolated from a bird.     

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties.     

用于分离大肠杆菌O157∶H7的免疫磁珠的制备与表征

以Fe2+、Fe3+、羧基化葡聚糖(CMD)为原料,采用共沉淀法,一步合成了CMD-Fe3O4磁性纳米粒子,并对其进行表征。用EDC和NHS将纳米粒子表面的羧基活化,再进一步与标记有荧光基团的anti-E.coli结合,合成了表面结有大肠杆菌O157∶H7抗体的新型免疫磁珠anti-E.coli-CMD-Fe3O4,可以用于分离大肠杆菌O157∶H7。     

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.     

Production of a Potentially Probiotic Product for Animal Feed and Evaluation of Some of Its Probiotic Properties

Nowadays, probiotics have been proposed for substituting antibiotics in animal feed since the European Union banned the latter compounds in 2006 to avoid serious side effects on human health. Therefore, this work aimed to produce a probiotic product for use in animal feed by fed-batch fermentation of whey with a combination of kefir grains, AGK1, and the fermented whole milk used to activate these kefir grains. The probiotic culture obtained was characterized by high levels of biomass (8.03 g/L), total viability (3.6 × 10⁸ CFU/mL) and antibacterial activity (28.26 Activity Units/mL). Some probiotic properties of the probiotic culture were investigated in vitro, including its survival at low pH values, under simulated gastrointestinal conditions, after freezing in skim milk at −20 °C, and in the commercial feed during storage at room temperature. The viable cells of lactic and acetic acid bacteria and yeasts exhibited higher tolerance to acidic pH and simulated gastrointestinal conditions when the cells were protected with skim milk and piglet feed, compared with washed cells. The results indicated the feasibility of producing a probiotic product at a low cost with a potential application in animal feed.     

鼠源性抗EHEC O157:H7 Stx2噬菌体Fab抗体库的构建及筛选

目的构建鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,并从中筛选特异性的抗体。方法用EHEC O157∶H7 Stx2类毒素免疫BALB/c小鼠,取脾分离淋巴细胞,提取总RNA,RT-PCR分别扩增抗体轻、重链(к和Fd)基因,经双酶切依次克隆入噬粒载体pComb3X中,电转化大肠杆菌XL1-Blue,以辅助噬菌体M13K07进行超感染,构建抗EHEC O157∶H7 Stx2的Fab噬菌体抗体库。以纯化的Stx2为抗原进行筛选,获得抗EHECO157∶H7Stx2的特异性Fab抗体,Western blot法检测噬菌体抗体与毒素抗原的结合活性,并对所得阳性克隆进行基因序列分析。结果构建了一个库容为1.56×107的Fab抗体库,筛选出3株特异性较强的阳性克隆,其中2个可与Stx2A1亚单位抗原反应,1个可与Stx2B亚单位抗原反应。基因序列分析显示,轻、重链可变区氨基酸序列与GenBank中已注册的鼠免疫球蛋白可变区氨基酸序列同源性分别为98.5%和99.6%。结论已成功构建了鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,为进一步制备抗EHECO157∶H7Stx2的治疗性人源化抗体奠定了基础。     

E. coli O157:H7 Desorption by Rhamnolipid Biosurfactant in Water-Unsaturated Porous Media

Animal waste is a valuable resource, which can add organic matter, nitrogen, phosphorous and potassium as well as other nutrients needed for plant growth when it is used in the agricultural field. When applied to an agricultural field, infectious agents or pathogenic organisms in the animal waste are usually retained in the soil. At the same time, surfactants are popularly utilized in pesticide applications to aid pesticide solubility and mobility. Consequently, the retained pathogenic strains might be flushed out of the soil matrices with a possibility of contaminating the groundwater. In this research, we investigated desorption of E. coli O157:H7 pre-deposited in silica sand and its subsequent release and transport in the presence of rhamnolipid biosurfactant in unsaturated laboratory columns. Based on the experimental observations, it was discovered that retained E. coli O157:H7 was released during flushing with rhamnolipid biosurfactant solutions. E. coli O157:H7 release rate coefficient increased both with the increase of rhamnolipid biosurfactant concentration and the increase of water saturation. With the increase of rhamnolipid biosurfactant concentration, the air–water surface tension decreased. With the increase of water saturation, the air–water interfacial area decreased. Both the decreased air–water surface tension and air–water interfacial area favored E. coli O157:H7 release. Increase of E. coli O157:H7 release rate coefficient with the decrease of air–water surface tension was more pronounced for low air–water interfacial area (i.e., high water saturation). Similarly, increase of E. coli O157:H7 release rate coefficient with the decrease of air–water interfacial area was more pronounced for low air–water surface tension (i.e., high rhamnolipid biosurfactant concentration). E. coli O157:H7 retention in unsaturated systems was found to be controlled by the capillary force, which was greatly impacted by water saturation and air–water surface tension. Increase of water saturation and rhamnolipid biosurfactant concentration both contributed to the decrease of the capillary force, resulting in increased E. coli O157:H7 release rate.     

Design of a Hazard Analysis and Critical Control Points (HACCP) plan to assure the safety of a bologna product produced by a meat processing plant

The Hazard Analysis and Critical Control Point (HACCP) is a systematic integral program used to identify and estimate the hazards (microbiological, chemical and physical) and the risks generated during the primary production, processing, storage, distribution, expense and consumption of foods. To establish a program of HACCP has advantages, being some of them: to emphasize more in the prevention than in the detection, to diminish the costs, to minimize the risk of manufacturing faulty products, to allow bigger trust to the management, to strengthen the national and international competitiveness, among others. The present work is a proposal based on the design of an HACCP program to guarantee the safety of the Bologna Special Type elaborated by a meat products industry, through the determination of hazards (microbiological, chemical or physical), the identification of critical control points (CCP), the establishment of critical limits, plan corrective actions and the establishment of documentation and verification procedures. The used methodology was based in the application of the seven basic principles settled down by the Codex Alimentarius, obtaining the design of this program. In view of the fact that recently the meat products are linked with pathogens like E. coli O157:H7 and Listeria monocytogenes, these were contemplated as microbiological hazard for the establishment of the HACCP plan whose application will guarantee the obtaining of a safe product.     

Use of date (Phoenix dactylifera L.) blanching water for reconstituting milk powder: Yogurt manufacture

The processing of dates yields high volumes of blanching water. The use of such blanching water for reconstituting skim milk powder to produce low fat yogurt was studied. Physicochemical properties and antioxidant activity of two date cultivars (Medjoul and Confitera) blanching water were determined. Quality characteristics of yogurts (control, Medjoul and Confitera) were evaluated during 28 days of refrigerated storage. Results showed that Confitera blanching water is considered a good source of natural antioxidants and organic acids, and has a promising future as a functional ingredient, whereas Medjoul blanching water had a high content of sugars. Regarding yogurt characteristics: Confitera yogurts presented highest populations of lactic acid bacteria, and gave soft gels of weak structure, Medjoul yogurts presented higher firmness and sensory scores than Confitera.     

Assimilation of cholesterol in broth,cream, and butter by probiotic bacteria

High serum cholesterol concentrations are associated with the development of coronary heart disease. It has been reported that some cultures of Lactobacillus spp. actively take up cholesterol from laboratory media. In the present study, the abilities of ten probiotic lactic acid bacteria to assimilate cholesterol in broth medium, cream, and butter were tested and compared. The cholesterol reduction ratios of these human‐origin bacteria were determined in MRS‐THIO broth supplemented with 150 μg/mL cholesterol. The amount of cholesterol assimilated by the bacteria was measured by gas chromatography. Cholesterol assimilation of these ten bacteria in broth was found to be similar to assimilation in cream. Two of these ten bacteria (Lactobacillus maltaramicus AC 3–16 and L. casei subsp. casei AB16–65) were chosen for making soured butter. The results indicate that the probiotic bacteria applied to cream and butter, as well as to the broth medium, caused a reduction of the cholesterol level of the product's fat content. This study provides some evidence that probiotic cultures have a cholesterol level‐reducing effect, and soon we will be able to produce butter without cholesterol via microorganisms.     

Q235钢上中温化学镀镍磷合金工艺

采用正交试验考察了镀液中配位剂柠檬酸钠和乳酸钠含量及pH对Q235碳钢上中温化学镀层沉积速率的影响,研究了稳定剂苯并三氮唑、硫代硫酸钠及其复配对镀液稳定性和沉积速率的影响.得到较理想的工艺配方及操作条件为:NiS046H2O30 g/L,NaH2PO2·H2O 30 g/L,乳酸50 g/L,柠檬酸钠2 g/L,CH3...     

A sandwich-type assay based on quantum dot/aptamer bioconjugates for analysis of E. Coli O157:H7 in microtiter plate format

An optical assay based on CdSe/ZnS quantum dots (QD)/NH₂-Apt bioconjugates for the pathogen detection was presented. QDs with carboxyl functional groups and 72-mer aptamer (against E. coli outer membrane proteins) were used as probes and sensing element. E. coli O157:H7 was selected as a model pathogen and 96-well plate assay in the sandwich hybridization format was constructed. Poly-L-lysine-coated 96-well plate surfaces were used as support material where thiol functionalized aptamers were immobilized by 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide (sulfo-SMCC). After incubation with the bacteria, CdSe/ZnS QDs/aptamer bioconjugates were added. The fluorescence signals were followed before and after addition of bioconjugates. Probe concentrations, incubation time with E. coli O157:H7 were also optimized. The bioassay could detect the pathogen down to 10² CFU/mL with high selectivity. The detection system was successfully employed in samples, in the presence of interfering compounds.     

A Novel Nano-Antimicrobial Polymer Engineered with Chitosan Nanoparticles and Bioactive Peptides as Promising Food Biopreservative Effective against Foodborne Pathogen E. coli O157-Caused Epithelial Barrier Dysfunction and Inflammatory Responses

For food quality and safety issues, the emergence of foodborne pathogenic bacteria has further accelerated the spread of antibiotic residues and drug resistance genes. To alleviate the harm caused by bacterial infections, it is necessary to seek novel antimicrobial agents as biopreservatives to prevent microbial spoilage. Nanoantimicrobials have been widely used in the direct treatment of bacterial infections. CNMs, formed by chitosan nanoparticles and peptides, are promising antibiotic alternatives for use as excellent new antibacterial drugs against pathogenic bacteria. Herein, the current study evaluated the function of CNMs in the protection of foodborne pathogen Escherichia coli (E. coli) O157 infection using an intestinal epithelial cell model. Antibacterial activity assays indicated that CNMs exerted excellent bactericidal activity against E. coli O157. Assessment of the cytotoxicity risks toward cells demonstrated that 0.0125–0.02% of CNMs did not cause toxicity, but 0.4% of CNMs caused cytotoxicity. Additionally, CNMs did not induced genotoxicity either. CNMs protected against E. coli O157-induced barrier dysfunction by increasing transepithelial electrical resistance, decreasing lactate dehydrogenase and promoting the protein expression of occludin. CNMs were further found to ameliorate inflammation via modulation of tumor factor α, toll-like receptor 4 and nuclear factor κB (NF-κB) expression via inhibition of mitogen-activated protein kinase and NF-κB activation and improved antioxidant activity. Taken together, CNMs could protect the host against E. coli O157-induced intestinal barrier damage and inflammation, showing that CNMs have great advantages and potential application as novel antimicrobial polymers in the food industry as food biopreservatives, bringing new hope for the treatment of bacterial infections.