Factors affecting the removal of aflatoxin M1 from food model by Lactobacillus and Bifidobacterium strains

This paper describes the ability of six dairy strains of Lactobacillus and Bifidobacterium to remove aflatoxin M₁ (AFM₁) from phosphate-buffered saline (PBS) and reconstituted milk. Bacteria were incubated in both PBS and reconstituted milk containing 5, 10 and 20 ng mL^?1 for 0, 4 and 24 h at 37°C. After centrifugation the concentration of AFM₁ was determined in the supernatant fraction using high-performance liquid chromatography. The binding abilities of AFM₁ by viable (10⁸ CFU mL^?1) and heat-killed Lactobacillus and Bifidobacterium strains in PBS ranged from 10.22 to 26.65% and 14.04 to 28.97%, respectively. Similarly, AFM₁-binding capacity in reconstituted milk was found to range from 7.85 to 25.94% and from 12.85 to 27.31% for viable and heat-killed bacteria, respectively within 4 h. While B. bifidum Bb 13 was the best binder, the poorest removal was achieved by L. acidophilus NCC 68. Binding was reversible, and a small proportion of AFM₁ was released back into the solution. The toxin concentration and incubation period had no effect on the removal of AFM₁ by bacteria both in PBS and reconstituted milk.     

Determination of wine microbiota using classical method, polymerase chain method and Step One Real-Time PCR during fermentation process

The aim of our study was the identification of grape, must and wine microbiota during the fermentation process using a classical microbiological method and Real-Time PCR. The changes in different groups of microorganisms were monitored in total counts of bacteria, lactobacilli and yeasts. Microbiological parameters were observed during the current collection and processing of grapes in 2009. Samples were taken during the fermentation process in wine enterprises and a private vineyard. During this period 30 samples of wine among Müller Thurgau, Cabernet Sauvignon, Chardonnay, Tramin and Red Bio-wine were examined. Samples were collected from stages of grape-must unfiltered, grape-must filtered, the beginning of fermentation, fermentation, late fermentation and young wine. The highest total counts of bacteria ranged from 0.00 to 176 ± 15 CFU.mL(-1) in the wine of Müller Thurgau, the highest number of yeast ranged from 0.00 to 150 ± 9 CFU.mL(-1) in the wine of Müller Thurgau and the number of Lactobacillus spp. ranged from 0.00 to 92 ± 5 CFU.mL(-1) in the sample of Cabernet Sauvignon wine. The presence and sensitivity of Gram-positive and Gram-negative bacterial species Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus crispatus and Lactobacillus salivarius were detected using Real-Time PCR (RTQ PCR). Susceptibility of Enterococcus faecium varied in different isolates from 1 to 10(6) CFU.mL(-1), the sensitivity of the species Lactobacillus acidophilus in different isolates of the wine samples ranged from 1 to 10(5) CFU.mL(-1). We also monitored representation of species Lactobacillus crispatus, which were captured by RTQ PCR sensitivity and ranged from 1 to 10(5) CFU.mL(-1). Identification of the species Lactobacillus salivarius in each of isolates by RTQ PCR method showed the presence of these bacteria in the range of 1 to 10(4) CFU.mL(-1).     

Occurrence of aflatoxin M1 in human milk samples in Vojvodina,Serbia: Estimation of average daily intake by babies

The objectives of the study were to determine the aflatoxin M1 content in human milk samples in Vojvodina, Serbia, and to assess the risk of infants' exposure to aflatoxins food contamination. The growth of Aspergillus flavus and production of aflatoxin B1 in corn samples resulted in higher concentrations of AFM1 in milk and dairy products in 2013, indicating higher concentrations of AFM1 in human milk samples in 2013 and 2014 in Serbia. A total number of 60 samples of human milk (colostrum and breast milk collected 4–8 months after delivery) were analyzed for the presence of AFM1 using the Enzyme Linked Immunosorbent Assay method. The estimated daily intake of AFM1 through breastfeeding was calculated for the colostrum samples using an average intake of 60 mL/kg body weight (b.w.)/day on the third day of lactation. All breast milk collected 4–8 months after delivery and 36.4% of colostrum samples were contaminated with AFM1. The greatest percentage of contaminated colostrum (85%) and all samples of breast milk collected 4–8 months after delivery had AFM1 concentration above maximum allowable concentration according to the Regulation on health safety of dietetic products. The mean daily intake of AFM1 in colostrum was 2.65 ng/kg bw/day. Results of our study indicate the high risk of infants' exposure, who are at the early stage of development and vulnerable to toxic contaminants.     

Alternative antimicrobial compounds to control potential Lactobacillus contamination in bioethanol fermentations

Antibiotics are commonly used to control microbial contaminants in yeast-based bioethanol fermentation. Given the increase in antibiotic-resistant bacteria, alternative natural antimicrobials were evaluated against the potential contaminant, Lactobacillus. The effects of nisin, ?-polylysine, chitosan (CS) and lysozyme were screened against 5 Lactobacillus strains. A standard broth- microdilution method was used in 96-well plates to assess the minimal inhibitory concentration (MIC). L. delbrueckii subsp lactis ATCC479 exhibited maximal MICs with CS, ?-polylysine and nisin (1.87, 0.3125 and 0.05 mg/mL, respectively). Nisin reduced most Lactobacillus strains by 6 log CFU/mL after 48 hours with the exception of L. casei. Synergism occurred when ethylenediaminetetraacetic acid (EDTA) was added with nisin. An MIC of 0.4 mg/mL of nisin combined with the EDTA at an MIC of 1 mg/ml markedly suppressed L .casei by 6 log CFU/mL. In conclusion, alternative antimicrobials proved to be a potential candidate for controlling bacterial contamination in the fermentation process. Synergistic effect of nisin with EDTA successfully inhibited the nisin-resistant contaminant, L. casei.     

Antimicrobial potential of commercial silver nanoparticles and the characterization of their physical properties toward probiotic bacteria isolated from fermented milk products

The application of nanotechnology in the agriculture and food sector is relatively recent compared to its usage in drug delivery or pharmaceuticals. Therefore, this paper presents a study of the effect of silver nanoparticles on probiotic bacteria based on the example of Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12 and Streptococcus thermophilus ST-Y31 isolated from fermented milk products. Probiotic bacteria are one of the most crucial groups of bacteria for the food industry, because of their claimed health-promoting properties. Studies have shown that the type and concentration of silver nanoparticle solutions have a significant impact on the tested probiotic bacteria which are profitable for the digestive system. In the presence of all tested silver nanoparticles, St. thermophilus ST-Y31 growth was inhibited significantly by the dilution method as opposed to the disk-diffusion method. Both the disk-diffusion and the dilution methods showed no significant differences between L. acidophilus LA-5 and B. animalis subsp. lactis BB-12. The concentrations 2 μg mL^?1 and 0.25 μg mL^?1 had the highest antibacterial activity and statistically significant impacts on the tested probiotic strains. To our knowledge, this is the first report on potential antimicrobial effect of nanosilver against the health-promoting probiotic bacteria L. acidophilus LA-5, B. animalis subsp. lactis BB-12 and St. thermophilus ST-Y31 isolated from fermented milk products.     

Denitrification using PBS as carbon source and biofilm support in a packed-bed bioreactor

Biodegradable polymer was used as carbon source and biofilm support for nitrate removal from aqueous solution as an attractive alternative for biological denitrification. The objective of this paper was to investigate the denitrification performance and microbial community of a packed-bed bioreactor using poly (butanediol succinate) (PBS), a biodegradable polymer, as carbon source and biofilm support. NO₃–N concentration was determined by UV spectrophotometer. NO₂–N concentration was assayed by hydrochloric acid naphthyl ethylenediamine spectrophotometry method. Total organic carbon (TOC) was measured using a TOC analyzer. The morphology of the samples was observed using an environmental scanning electron microscope (ESEM). The microbial community was analyzed by pyrosequencing method. The experimental results showed that an average removal efficiency of nitrate was 95 %. ESEM observation and FTIR analysis indicated the changes of PBS granules before and after microbial utilization. Pyrosequencing results showed that Betaproteobacteria predominated, and most of PBS-degrading denitrifying bacteria were assigned to the family Comamonadaceae. Denitrifying bacteria accounted for 13.02 % in total population. The PBS granules were suitable support and carbon source for denitrifying bacteria.     

Methodology for management of endosulfan contaminated eluent.

Management of Endosulfan contaminated eluent (24 mg/l) resulting from a treatment process to remove Endosulfan from water with wood charcoal, was attempted using various methods viz. volatilisation, hydrolysis and sorption by viable cell bacteria with and without acclimatisation. Volatilisation failed in giving better result, as Endosulfan was not considerably volatile. It could achieve a removal efficiency of 1.4-2%. Hydrolysis resulted in 28.4% and 17.9% removal of Endosulfan in acidic and alkaline media, respectively. Viable cell bacteria (aerobic) without prior acclimatization showed efficiency of 89.7% and after prior acclimatisation showed 96% removal efficiency. Sorption by the acclimatized biomass was found a suitable method for the removal of Endosulfan at a concentration of 24 mg/l.     

石油降解菌的筛选及其降解能力的研究

从广州石化污水处理厂废水中自行分离出 30株除油菌 ,用市售的 90 # 柴油作为油品进行筛选 ,所得菌种用于处理石化厂物理隔油后的废水。通过研究含油量和接种量对除油率、COD的去除率和pH值的影响以及酸、碱、盐对除油率和COD去除率的影响来比较这些菌种对石化废水的处理效果。结果表明 ,6 # 菌株除油和去除有机物的效果都比较好 ,除油率约在 70 %左右 ,最高为 83.6 7% ,COD去除率约为 5 5 %左右 ,最高为 6 0 .0 1% ;5 # 菌株对环境要求较高 ,在碱性环境下表现出较好的除油和去除有机物的能力 ,除油率和COD去除率分别为 5 5 %和 5 0 %左右。实验菌株在消除石化废水的异味方面也有一定的效果。     

Extracellular antimutagenic activities of selected probiotic Bifidobacterium and Lactobacillus spp. as a function of growth phase

The capabilities of selected strains from genera Lactobacillus and Bifidobacterium to produce extracellular bioactive compounds with antimutagenic properties against benzo[a]pyrene (BaP) and sodium azide (SA) were tested as a function of growth phase. The bacterial supernatants from exponential and stationary phases were characterized with different patterns of antimutagenic activity against the two mutagens. All lactobacilli exhibited either no effect or low antimutagenicity against BaP during exponential growth. Higher antimutagenic activities of lactobacilli supernatants were observed in the stationary phase against SA as well. An exception was Lactobacillus sakei 23K which expressed a relatively low percent of inhibition of mutagenesis (PI = 28.14 +/- 7.41) in the exponential phase and no antimutagenic activity in the stationary phase. Of the bifidobacteria, only Bifidobacterium adoleascentis ATCC 15703 exhibited higher antimutagenecity against BaP in the exponential phase. The same bacterial supernatants however, did not possess any antimutagenicity against SA in either the exponential or stationary phases. B. bifidum ATCC 11863 did not express any significant differences in its activity against either BaP or SA in the exponential or stationary phases. Only B. breve ATCC 15700 expressed a high antimutagenic effect against SA in the stationary phase but exhibited no effect during exponential growth. Overall, bacterial antimutagenic responses were associated with growth phase and type of mutagen.     

水质净化高效复合微生态制剂的研制

在室内模拟条件下,采用正交实验的方法对光合细菌、枯草芽孢杆菌和反硝化细菌的复配比例进行了研究,筛选一种用于水产养殖水质净化的高效复合微生态制剂。结果表明,当光合细菌(菌细胞浓度约为2×109CFU/mL)、枯草芽孢杆菌(菌细胞浓度约为8×108CFU/mL)和反硝化细菌(菌细胞浓度约为8×108CFU/mL)按菌液体积比为1∶2∶1进行复配利于水中溶解氧的提高和COD、氨氮、亚硝态氮、硝态氮的降解。验证实验表明,筛选组合各指标均优于商品微生态制剂和空白对照,其中溶解氧含量显著高于商品微生态制剂EM和复合芽孢菌处理,在实验第5天对COD的降解率为95%,显著优于EM和复合芽孢菌处理的66.3%和47.9%,实验第7天对氨氮、亚硝态氮和硝态氮的降解率分别达到70%、89%和56%。     

Application of QuEChERS method and gas chromatography-mass spectrometry for the analysis of cypermethrin residue in milk

Analytical methods for the isolation and determination of cypermethrin in milk, based on the solid-phase microextraction (SPME) and QuEChERS methods (Quick, Easy, Cheap, Effective, Rugged, and Safe) are presented. The SPME technique was not appropriate to analyse cypermethrin in milk, even establishing the best extraction conditions, polydimethylsiloxane fiber, 60 min time extraction, 60 °C temperature extraction, addition of salt (NaCl) and stirring rate. The extraction efficiency was low probably because of the matrix constituents. The QuEChERS method involves the extraction of the analyte with acetonitrile and simultaneous liquid-liquid partitioning formed by adding anhydrous MgSO(4) plus NaCl, followed by the removal of residual water and cleanup using a procedure called dispersive solid-phase extraction, in which anhydrous MgSO(4) plus PSA and C18 are mixed with 1 mL of acetonitrile extract. The detection and quantification limits were 0.01 and 0.04 mg kg(-1), respectively, and the percentage recovery obtained ranged from 92 to 105% with relative standard deviations below 7%.     

Binding and detoxification of chlorpyrifos by lactic acid bacteria on rice straw silage fermentation

This investigation examined the reduction of pesticide residues on straw inoculated with lactic acid bacteria (LAB) during ensiling. Lactobacillus casei WYS3 was isolated from rice straw that contained pesticide residues. Non-sterilized rice straw, which was inoculated with L. casei WYS3, showed increased removal of chlorpyrifos after ensiling, compared with rice straw that was not inoculated with L. casei WYS3 or sterilized rice straw. In pure culture, these strains can bind chlorpyrifos as indicated by high-performance liquid chromatography analysis. Viable L. casei WYS3 was shown to bind 33.3–42% of exogenously added chlorpyrifos. These results are similar to those of acid-treated cells but less than those of heat-treated cells, which were found to bind 32.0% and 77.2% of the added chlorpyrifos respectively. Furthermore, gas chromatography–mass spectrometry analysis determined that L. casei WYS3 detoxified chlorpyrifos via P-O-C cleavage. Real-time polymerized chain reaction analysis determined that organophosphorus hydrolase gene expression tripled after the addition of chlorpyrifos to LAB cultures, compared with the control group (without chlorpyrifos). This paper highlights the potential use of LAB starter cultures for the detoxification and removal of chlorpyrifos residues in the environment.     

Identification of lactic acid bacteria isolated from wine using real-time PCR

Different lactic acid bacteria strains have been shown to cause wine spoilage, including the generation of substances undesirable for the health of wine consumers. The aim of this study was to investigate the occurrence of selected species of heterofermentative lactobacilli, specifically Lactobacillus brevis, Lactobacillus hilgardii, and Lactobacillus plantarum in six different Slovak red wines following the fermentation process. In order to identify the dominant Lactobacillus strain using quantitative (real time) polymerized chain reaction (qPCR) method, pure lyophilized bacterial cultures from the Czech Collection of Microorganisms were used. Six different red wine samples following malolactic fermentation were obtained from selected wineries. After collection, the samples were subjected to a classic plate dilution method for enumeration of lactobacilli cells. Real-time PCR was performed after DNA extraction from pure bacterial strains and wine samples. We used SYBR® Green master mix reagents for measuring the fluorescence in qPCR. The number of lactobacilli ranged from 3.60 to 5.02 log CFU mL^?1. Specific lactobacilli strains were confirmed by qPCR in all wine samples. The number of lactobacilli ranged from 10³ to 10⁶ CFU mL^?1. A melting curve with different melting temperatures (T_m) of DNA amplicons was obtained after PCR for the comparison of T_m of control and experimental portions, revealing that the most common species in wine samples was Lactobacillus plantarum with a T_m of 84.64°C.     

Biological denitrification in drinking water treatment using the seaweed Gracilaria verrucosa as carbon source and biofilm carrier.

Chemical and microbiological aspects were investigated with regard to biological denitrification of drinking water using the seaweed Gracilaria verrucosa as the carbon and energy substrate and as physical support for the microbial flora in semibatch, fixed-bed reactors. Complete removal of nitrate (100 mg/L) was readily achieved without accumulation of nitrite. Microbiological analysis indicated that the effluent of the reactor contained high numbers of bacteria (>10(6)/mL total count). Among the 44 bacterial strains isolated directly from the samples or isolated after enrichment at 37 degrees C, 25 different fatty acid profiles were found, indicating a complex microflora, including potential pathogens.     

Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B(1) adsorption ability for use in poultry feedstuffs

In this study the aflatoxin B(1) (AFB(1)) removal capacity, the tolerance to salivary and gastrointestinal conditions, autoaggregation and coaggregation with pathogenic bacteria of Saccharomyces cerevisiae strains isolated from broiler feces, were evaluated. Only four of twelve isolated strains were identified as Saccharomyces cerevisiae using molecular techniques. The results obtained in AFB(1) binding studies indicated that the amount of AFB(1) removed was both strain and mycotoxin-concentration dependent. Therefore, a theoretical model was applied in order to select the most efficient strain to remove AFB(1) in a wide range of mycotoxin concentration. The results indicated that S. cerevisiae 08 and S. cerevisiae 01 strains were the most efficient microorganisms in the mycotoxin removal. Viability on simulated salivary and gastrointestinal conditions was investigated and S. cerevisiae 08 strain showed the best results, achieving 98% of total survival whereas S. cerevisiae 01 reached only 75%. Autoaggregation and coaggregation assays showed S. cerevisiae 08 as the most appropriate strain, mainly because it was the unique strain able to coaggregate with the four bacterial pathogens assayed. Consequently, S. cerevisiae 08 is the best candidate for future in vivo studies useful to prevent aflatoxicosis. Further quantitative in vitro and in vivo studies are required to evaluate the real impact of yeast-binding activity on the bioavailability of AFB(1) in poultry. However, this study could be useful in selecting efficient strains in terms of AFB(1) binding and provide an important contribution to research into microorganisms with potential probiotic effects on the host.     

Application of QuEChERS method and gas chromatography-mass spectrometry for the analysis of cypermethrin residue in milk

Analytical methods for the isolation and determination of cypermethrin in milk, based on the solid-phase microextraction (SPME) and QuEChERS methods (Quick, Easy, Cheap, Effective, Rugged, and Safe) are presented. The SPME technique was not appropriate to analyse cypermethrin in milk, even establishing the best extraction conditions, polydimethylsiloxane fiber, 60 min time extraction, 60 °C temperature extraction, addition of salt (NaCl) and stirring rate. The extraction efficiency was low probably because of the matrix constituents. The QuEChERS method involves the extraction of the analyte with acetonitrile and simultaneous liquid-liquid partitioning formed by adding anhydrous MgSO₄ plus NaCl, followed by the removal of residual water and cleanup using a procedure called dispersive solid-phase extraction, in which anhydrous MgSO₄ plus PSA and C18 are mixed with 1 mL of acetonitrile extract. The detection and quantification limits were 0.01 and 0.04 mg kg^?1, respectively, and the percentage recovery obtained ranged from 92 to 105% with relative standard deviations below 7%.     

Isolation and characterization of 3-nitrophenol-degrading bacteria associated with rhizosphere of Spirodela polyrrhiza

Introduction

The accelerated biodegradation of 3-nitrophenol (3-NP) in the rhizosphere of giant duckweed (Spirodela polyrrhiza) was investigated.

Materials and methods

Biodegradation of 3-nitrophenol in the rhizosphere of a floating aquatic plant, S. polyrrhiza, was investigated by using three river water samples supplemented with 10?mg?l^?1 of 3-NP. Isolation and enrichment culture of 3-NP-degrading bacteria were performed in basal salts medium containing 3-NP (50?mg?l^?1). The isolated strains were physiologically and phylogenetically characterized by using an API20NE kit and 16S rRNA gene sequencing.

Results and discussion

Accelerated removal of 3-NP (100%) was observed in river water samples with S. polyrrhiza compared with their removal in plant-free river water. Also, 3-NP persisted in an autoclaved solution with aseptic plants, suggesting that the accelerated 3-NP removal resulted largely from degradation by bacteria inhabiting the plant rather than from adsorption and uptake by the plant. We successfully isolated six and four strains of 3-NP-degrading bacteria from the roots of S. polyrrhiza and plant-free river water, respectively. Phylogenetic analysis based on 16S rRNA gene divided the 3-NP-degrading bacteria into two taxonomic groups: the genera Pseudomonas and Cupriavidus. The strains belonging to the genus Cupriavidus were only isolated from the roots of duckweed. All strains isolated from the roots utilized 3-NP (0.5?mM) as a sole carbon and energy source, indicating that they could have contributed to the accelerated degradation of 3-NP in the rhizosphere of S. polyrrhiza.

Conclusions

The rhizoremediation using S. polyrrhiza and its rhizosphere bacteria can be an effective strategy for cleaning up the 3-NP-contaminated surface waters.     

Sanitation assessment of wastewater treated by stabilization ponds for potential reuse in agricultural irrigation sanitation assessment.

Wastewater reuse has become an important alternative to agricultural irrigation; on the other hand, it poses concern with regard to public health. Total coliform and Escherichia coli concentration, presence of helminth eggs and Salmonella, and physical-chemical parameters were evaluated in raw and treated wastewater. Chemical and biochemical oxygen demand removal efficiency was 74.6 and 77.9%, respectively. As for organic nitrogen, total phosphorus, and total suspended solids, total efficiency removal was 17.4, 12.5, and 32.9%, respectively. The average density of total coliforms and E. coli was 3.5 x 10(9) and 1.8 x 10(8) MPN/100 mL and 1.1 x 10(7) MPN/100 mL and 3.9 x 10(5) MPN/100 mL for raw and treated wastewater, respectively. Ascaris eggs were observed in 80.8% of the samples collected, and viable eggs in 42.3% of the samples. Salmonella was detected in 36.4% of the samples. The values observed in treated wastewater did not show the adequate bacteriological quality, as recommended by World Health Organization (Geneva, Switzerland). Therefore, additional measures should be taken to achieve an improved microbiological and parasitological quality.     

Assessment of vancomycin resistance transfer among enterococci of clinical importance in milk matrix

Abstract

Dissemination of vancomycin resistance in enterococci has been associated with horizontal transfer of mobile genetic elements. Aim of the study was to evaluate if milk matrix is a suitable environment to support transferability of vancomycin resistance (vanA) gene from clinical vancomycin-resistant Enterococcus faecium to vancomycin-sensitive Enterococcus faecalis. Enterococci strains were firstly screened for the presence of cpd (inducible sex pheromone determinant) gene, vanA and tetL genes (vancomycin and tetracycline resistance markers, respectively) and the gelE (extracellular metalloendopeptidase) gene to define the mating pairs. Based on these selection markers, we investigated the transferability of eight plasmid-borne vanA harbored by E. faecium (vanA+, cpd-, tetL- and gelE-) into two E. faecalis (vanA-, cpd+, tetL?+?and gelE+) recipient strains in milk matrix. The strains were mated in a 1:1 ratio in 7% reconstituted milk and incubated at 37?°C. Transconjugants emerged from all 16 matings within 2?h of incubation and were evidenced by dual antibiotic resistance (vancomycin and tetracycline). The vancomycin-resistance of trasconjugants was maintained even after ten subsequent passages on nonselective medium. Transconjugants were positive for vanA, tetL and gelE genes. This study indicates milk matrix as suitable environment to support gene exchange between Enterococcus species.     

连续式生物吸收工艺脱除二氧化硫

运用连续式生物吸收处理工艺,以废糖蜜发酵液作为碳源进行了微生物法去除SO2气体的研究,在简单粗放的实验条件下,研究了脱硫脱硫弧菌对较大气量SO2气体的去除效果,并对产物H2S在第二级生物反应器中的去除率进行了测定。实验结果表明,随着进气量由0.18 m3/h增大至5 m3/h,脱硫率会降低,但是随之提高搅拌速度和补料速度后,脱硫率又恢复到较高水平,当搅拌速度为590 r/min时,5 L生物反应液可以处理5 m3/h的SO2气体,1#反应器SO2去除率和2#反应器H2S去除率分别达到92%和98%以上。在气量增至5 m3/h时,1#和2#反应器补料流速分别为175 mL/h和200 mL/h时,没有亚硫酸盐和硫化物的积累,pH值和菌体浓度稳定,系统运行良好。