Heat-inactivated Cells Research Articles

A Rapid Total Bacterial Count Method for Food Samples using Syringe Filters and Lectin-Conjugated Semiconductor Nanorods.

Total bacterial count in food is one of important food safety criteria. The current plate count method (Heterotrophic Plate Count) for food analysis requires microbiology lab facilities and at least 2 days turnover time. We developed a rapid fluorescence-based total bacterial count method that utilises semiconductor nanorods (SNRs) conjugated with a lectin Griffonia simplicifolia II (GSII-SNRs) to stain bacterial cells captured on syringe filters, via the common N-acetylglucosamine molecules on bacterial cell wall. This "Filter-and-Stain" detection method has a rapid turnover time of 20 min. The fluorescence emission can be seen under UV light with minimum interference from food sample background. The fluorescence intensity quantified through image analysis is proportional to the bacterial concentration with a limit of detection of 1000 CFU/mL, for total bacterial count assessment in food safety. Moreover, the GSII-SNRs do not bind to heat inactivated bacterial cells, and thus can differentiate live and dead bacteria. Our method has been validated with representative food (coffee powder, raw spinach leaves, and ready-to-eat tomato salsa) to demonstrate its high potential for on-site food safety assessment, especially in places with no immediate access to microbiology labs.

Dendritic Cells Loaded With Heat Shock Inactivated Glioma Stem Cells Enhance Antitumor Response of Mouse Glioma When Combining With CD47 Blockade.

For glioma patients, the long-term advantages of dendritic cells (DCs) immunization remain unknown. It is extremely important to develop new treatment strategies that enhance the immunotherapy effect of DC-based vaccines. DCs exposed to glioma stem cells (GSCs) are considered promising vaccines against glioma. Glioma stem cells were isolated from mouse glioma GL261 cells (GCs). Both were subjected to severe (47°C) and mild (42°C) heat shock to induce immunogenic cell death (ICD). Membrane mobilization of calreticulin (CRT) and release of heat shock proteins (HSPs) were detected by flow cytometry. Dendritic cells were then exposed to heat-inactivated cells and co-culturing of T cells tested for immunotherapeutic efficacy in vitro. In vivo, we investigated the GSC targeting effect of the GSC-DC vaccine combined with CD47 blockade. Heat shock induced ICD in GCs and GSCs, as indicated by significant release of calreticulin, HSP70, and HSP90. Heat shock condition ICD lysates induce maturation and activation-associated marker expression on monocyte-derived DCs. Accordingly, DCs pulsed with GCs and GSCs inactivated reduced colony formation, sphere formation, migration, and invasion of glioma and GSCs in vitro. Glioma stem cell-DC vaccine in combination with anti-CD47 antibody significantly enhanced survival in mice with glioma, induced production of interferon (IFN)-γ, and enhanced T-cell expansion in vivo. Of note, DCs pulsed with inactivated GSCs were more effective to control tumor growth than DCs pulsed with inactive GCs. Severe heat shock induces ICD in vitro. These data showed that administration of anti-CD47 antibody combined with GSC-DC vaccine may represent an effective immunotherapeutic strategy for cancer patients in clinical.

Limosilactobacillus vaginalis Exerts Bifidogenic Effects: A Novel Postbiotic Strategy for Infant Prebiotic Supplementation.

Infant microbiota shaping strictly influences newborns' well-being and long-term health, and babies born by cesarean-section and formula-fed generally show low microbial gut diversity and are more prone to develop various disorders. The supplementation with beneficial microbes of vaginal origin or derivatives (postbiotics, including heat-inactivated cells) represents a valid strategy to drive the correct gut microbiota shaping. Here, we explored for the first time the bifidogenic activity of a heat-killed vaginal strain (Limosilactobacillus vaginalis BC17), in addition to the assessment of its safety. L. vaginalis BC17 whole genome was sequenced by Nanopore technology and highlighted the absence of antibiotic resistance genes and virulence factors, indicating the strain safety profile for human health. MIC values confirmed that L. vaginalis BC17 is susceptible to widely employed antibiotics. Heat-killed BC17 cells significantly enhanced the planktonic growth of Bifidobacterium spp. For the first time, stimulating effects were observed also toward biofilm formation of bifidobacteria and their pre-formed biofilms. Conversely, heat-killed BC17 cells exerted antibacterial and anti-biofilms activities against Gram-positive and Gram-negative pathogens. Lyophilized heat-killed BC17 cells were formulated in a sunflower oil suspension (1010 heat-killed cell/g) intended for infant oral intake. This possessed optimal technological (i.e., re-dispersibility and stability) and functional properties (i.e., bifidogenic activity) that were maintained even after pre-digestion in acidic conditions.

Brevibacterium sp. strain CS2: A potential candidate for arsenic bioremediation from industrial wastewater

A multiple metal-resistant Brevibacterium sp. strain CS2, isolated from an industrial wastewater, resisted arsenate and arsenate upto 280 and 40 mM. The order of resistance against multiple metals was Arsenate > Arsenite > Selenium = Cobalt > Lead = Nickel > Cadmium = Chromium = Mercury. The bacterium was characterized as per morphological and biochemical characteristics at optimum conditions (37 ℃ and 7 pH). The appearance of brownish color precipitation was due to the interaction of silver nitrate confirming its oxidizing ability against arsenic. The strain showed arsenic processing ability at different temperatures, pH, and initial arsenic concentration which was 37% after 72 h and 48% after 96 h of incubation at optimum conditions with arsenite 250 mM/L (initial arsenic concentration). The maximum arsenic removal ability of strain CS2 was determined for 8 days, which was 32 and 46% in wastewater and distilled water, respectively. The heat-inactivated cells of the isolated strain showed a bioremediation efficiency (E) of 96% after 10 h. Genes cluster (9.6 kb) related to arsenite oxidation was found in Brevibacterium sp. strain CS2 after the genome analysis of isolated bacteria through illumine and nanopore sequencing technology. The arsenite oxidizing gene smaller subunit (aioB) on chromosomal DNA locus (Prokka_01508) was identified which plays a role in arsenite oxidation for energy metabolism. The presence of arsenic oxidizing genes and an efficient arsenic oxidizing potential of Brevibacterium sp. strain CS2 make it a potential candidate for green chemistry to eradicate arsenic from arsenic-contaminated wastewater.

Vaccination with a ZNF2oe Strain of Cryptococcus Provides Long-Lasting Protection against Cryptococcosis and Is Effective in Immunocompromised Hosts.

Systemic cryptococcosis is fatal without treatment. Even with the current antifungal therapies, this disease kills 180,000 of 225,000 infected people annually. Exposure to the causative environmental fungus Cryptococcus neoformans is universal. Either reactivation of a latent infection or an acute infection after high exposure to cryptococcal cells can result in cryptococcosis. Currently, there is no vaccine to prevent cryptococcosis. Previously, we discovered that Znf2, a transcription factor that directs Cryptococcus yeast-to-hypha transition, profoundly affects cryptococcal interaction with the host. Overexpression of ZNF2 drives filamentous growth, attenuates cryptococcal virulence, and elicits protective host immune responses. Importantly, immunization with cryptococcal cells overexpressing ZNF2, in either live or heat-inactivated form, offers significant protection to the host from a subsequent challenge by the otherwise lethal clinical isolate H99. In this study, we found that the heat-inactivated ZNF2oe vaccine offered long-lasting protection with no relapse upon challenge with the wild-type H99. Vaccination with heat-inactivated ZNF2oe cells provides partial protection in hosts with preexisting asymptomatic cryptococcal infection. Importantly, once animals have been vaccinated with heat-inactivated or live short-lived ZNF2oe cells, they are protected against cryptococcosis even when their CD4+ T cells are depleted at the time of fungal challenge. Remarkably, vaccination with live, short-lived ZNF2oe cells in CD4-depleted hosts still provides strong protection to these hosts with preexisting immunodeficiency at the time of vaccination. This work raises hope for developing effective vaccines with long-lasting protection for individuals who are immunocompromised or could become immunocompromised later in life.

In vitro detoxification of aflatoxin B1 and ochratoxin A by lactic acid bacteria isolated from Algerian fermented foods

Wheat occupies the first place in Algerian’s diet. However, because of the risk of mycotoxin contamination, its consumption can cause serious health concern to Algerian population. This study aimed to determine the in vitro detoxification properties of lactic acid bacteria (LAB) strains toward aflatoxin B1 (AFB1) and ochratoxin A (OTA), the most prevalent mycotoxins in wheat and derived products in Algeria. Eleven LAB strains isolated from Algerian fermented foods were characterized and identified using API 50 CHL and 16S rDNA sequencing methods. In order to study the ability of LAB strains to remove AFB1 and OTA, viable and heat inactivated cells of two LAB strains (Lab-L4/al and Lab-L1) selected among the eleven isolated and identified ones, as well as of the reference strain Lactobacillus plantarum (LP) R1096 were incubated individually in Citrate Phosphate Buffer (CPB 0.1 M, pH 6 or 5) containing AFB1 and/or OTA at a concentration each of 40 ng/mL for 24 h at 25 °C. Free mycotoxin concentrations were analyzed by HPLC-FLD. The isolated LAB strains were identified as Lactococcus lactis ssp lactis 1, Lactococcus lactis ssp lactis 2 and Lactobacillus paracasei ssp paracasei using API 50 CHL kit, whereas with the molecular method, the strains were identified as Enterococcus faecium and Enterococcus durans. Both viable and nonviable cells of Lab-L4/al and Lab-L1, as well as LP R1096, were able to remove AFB1 and OTA, with efficiency varying between the strains and higher for AFB1 with nonviable cells (>50 %). Removal ability of viable Lab-L4/al and LP R1096 cells increased with a decrease in pH from 6 to 5, while pH had no effect on the amounts of mycotoxins removed by viable Lab-L1 cells. In conclusion, the tested LAB strains were able to reduce the amounts of AFB1 and OTA in vitro conditions. This result suggests that these LAB strains could be used as additives or formative agents to reduce mycotoxin levels in fermented wheat foods such as sourdough bread.

Bioremoval of Cylindrospermopsis raciborskii cells and cylindrospermopsin toxin in batch culture by the yeast Aureobasidium pullulans.

This study describes the ability of a yeast strain, Aureobasidium pullulans KKUY0701 isolated from eutrophic lake to eliminate Cylindrospermopsis raciborskii and cylindrospermopsin (CYN) toxin. The anti-cyanobacterial activity of this yeast strain was evaluated by growing with living cells and filtrate of C. raciborskii. CYN bioremoval was assayed using living and heat-inactivated yeast cells. Both living cells and filtrate of this yeast strain were able to suppress the growth of C. raciborskii, with total cell death occurring at day 2 and day 3, respectively. Living and inactivated yeast cells, but not yeast filtrate, reduced CYN concentrations released into cyanobacterial cultures, indicating that this toxin might be removed from the culture medium via absorption onto yeast surface rather than enzymatic biodegradation. The adsorption experiments also confirmed the elimination of CYN by living and heat-inactivated yeast. Nevertheless, inactivated yeast exhibited higher capacity (K = 3.3) and intensity (n = 1.4) than living yeast (K = 1.9, n = 1) for CYN adsorption. The study suggests that this yeast strain could be employed for bioremediation of Cylindrospermopsis blooms in freshwaters. Additionally, heat-inactivated yeast biomass could be used in slow sand filters for elimination of CYN in drinking water treatment plants.

Simultaneous biodegradation of harmful Cylindrospermopsis raciborskii and cylindrospermopsin toxin in batch culture by single Bacillus strain.

This study investigates the capability of a Bacillus flexus strain isolated from decayed cyanobacterial blooms for the bioremediation of Cylindrospermopsis raciborskii and cylindrospermopsin (CYN) toxin. The algicidal activity of this strain was tested by co-cultivation with C. raciborskii cultures. CYN biodegradation was investigated in the presence of living and heat-inactivated bacterial cells or bacterial filtrate. Living bacterial cells inhibited C. raciborskii growth after 2 days of incubation with complete cell death at day 5. Bacterial filtrate caused a rapid reduction in C. raciborskii growth at the first day, with complete cell lysis at day 3. Only living cells of SSZ01 caused reduction in CYN released into the medium during the bacterial decay of C. raciborskii cells. The biodegradation rate of CYN by SSZ01 relied on initial toxin concentrations. The highest rate (42 μg CYN L-1 day-1) was obtained at the higher initial concentration (300 μg L-1), and the lowest (4μg CYN L-1 day-1) was at lower concentration (50 μg L-1). These results suggest that this bacterial strain could be employed to bioremediate cyanobacterial blooms in freshwaters. Also, the application of this bacterium in slow sand filters would give possibilities for degradation and bioremediation of cyanotoxins in drinking water treatment plants.

A Tentative Study of the Effects of Heat-Inactivation of the Probiotic Strain Shewanella putrefaciens Ppd11 on Senegalese Sole (Solea senegalensis) Intestinal Microbiota and Immune Response.

Concerns about safety, applicability and functionality associated with live probiotic cells have led to consideration of the use of non-viable microorganisms, known as paraprobiotics. The present study evaluated the effects of dietary administration of heat-inactivated cells of the probiotic strain Shewanella putrefaciens Ppd11 on the intestinal microbiota and immune gene transcription in Solea senegalensis. Results obtained were evaluated and compared to those described after feeding with viable Pdp11 cells. S. senegalensis specimens were fed with basal (control) diet or supplemented with live or heat inactivated (60 °C, 1 h) probiotics diets for 45 days. Growth improvement was observed in the group receiving live probiotics compared to the control group, but not after feeding with a probiotic heat-inactivated diet. Regarding immune gene transcription, no changes were observed for tnfα, il-6, lys-c1, c7, hsp70, and hsp90aa in the intestinal samples based on the diet. On the contrary, hsp90ab, gp96, cd4, cd8, il-1β, and c3 transcription were modulated after probiotic supplementation, though no differences between viable and heat-inactivated probiotic supplemented diets were observed. Modulation of intestinal microbiota showed remarkable differences based on the viability of the probiotics. Thus, higher diversity in fish fed with live probiotic cells, jointly with increased Mycoplasmataceae and Spirochaetaceae to the detriment of Brevinemataceae, was detected. However, microbiota of fish receiving heat-inactivated probiotic cells showed decreased Mycoplasmataceae and increased Brevinemataceae and Vibrio genus abundance. In short, the results obtained indicate that the viable state of Pdp11 probiotic cells affects growth performance and modulation of S. senegalensis intestinal microbiota. On the contrary, minor changes were detected in the intestinal immune response, being similar for fish receiving both, viable and inactivated probiotic cell supplemented diets, when compared to the control diet.

Mycotoxin Removal by Lactobacillus spp. and Their Application in Animal Liquid Feed.

The removal of mycotoxins from contaminated feed using lactic acid bacteria (LAB) has been proposed as an inexpensive, safe, and promising mycotoxin decontamination strategy. In this study, viable and heat-inactivated L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells were investigated for their ability to remove aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEA), and deoxynivalenol (DON) from MRS medium and PBS buffer over a 24 h period at 37 °C. LAB decontamination activity was also assessed in a ZEA-contaminated liquid feed (LF). Residual mycotoxin concentrations were determined by UHPLC-FLD/DAD analysis. In PBS, viable L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells removed up to 57% and 30% of ZEA and DON, respectively, while AFB1 and OTA reductions were lower than 15%. In MRS, 28% and 33% of ZEA and AFB1 were removed, respectively; OTA and DON reductions were small (≤15%). Regardless of the medium, heat-inactivated cells produced significantly lower mycotoxin reductions than those obtained with viable cells. An adsorption mechanism was suggested to explain the reductions in AFB1 and OTA, while biodegradation could be responsible for the removal of ZEA and DON. Both viable LAB strains reduced ZEA by 23% in contaminated LF after 48 h of incubation. These findings suggest that LAB strains of L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T may be applied in the feed industry to reduce mycotoxin contamination.

Isolation of a Novel Kluyveromyces marxianus Strain QKM-4 and Evidence of Its Volatilome Production and Binding Potentialities in the Biocontrol of Toxigenic Fungi and Their Mycotoxins

To overcome the economic losses associated with fungi and their toxic metabolites, environmentally safe and efficient approaches are needed. To this end, biological control using yeasts and safe bacterial strains and their products are being explored to replace synthetic fungicides. In the present study, the biocontrol effect of a yeast strain of Kluyveromyces marxianus, QKM-4, against the growth and mycotoxin synthesis potential of key toxigenic fungi was evaluated. In vitro assays were performed to find the application of yeast volatile organic compounds (VOCs) against fungal contamination on important agricultural commodities. The removal of ochratoxin A (OTA) and deoxynivalenol (DON) by living and heat-inactivated yeast cells was also explored. VOCs produced by strain QKM-4 were able to significantly limit the fungal growth of 17 fungal species belonging to genera Aspergillus, Penicillium, and Fusarium. Yeast VOCs were able to reduce OTA biosynthesis potential of Penicillium verrucosum and Aspergillus carbonarius by 99.6 and 98.7%, respectively. In vivo application of QKM-4 VOCs against Fusarium oxysporum and A. carbonarius infection on tomatoes and grapes, respectively, determined a complete inhibition of fungal spore germination. GC/MS-based analysis of yeast VOCs identified long-chain alkanes, including nonadecane, eicosane, docosane, heptacosane, hexatriacontane, and tetracosane. In vitro testing of the mycotoxin-binding potential of the living and heat-inactivated QKM-4 cells showed a reduction of OTA and DON up to 58 and 49%, respectively, from artificially contaminated buffers. Our findings clearly demonstrate the strong antifungal potential of K. marxianus QKM-4 and propose this strain as a strong candidate for application in agriculture to safeguard food and feed products.

Growth inhibition of Microcystis aeruginosa and adsorption of microcystin toxin by the yeast Aureobasidium pullulans, with no effect on microalgae.

This study evaluates the inhibitory effect of a yeast strain, Aureobasidium pullulans KKUY0701, isolated from decayed cyanobacterial bloom against harmful cyanobacterium Microcystis aeruginosa and determines the ability of this strain to remove microcystin (MC) toxin from the water. The antialgal activity of this yeast strain was assayed by co-cultivation with M. aeruginosa, diatom, and green algal species. The MC adsorption experiment was conducted in the presence of living and heat-inactivated yeast cells. Both yeast cells and filtrates caused a rapid reduction in the growth of M. aeruginosa, with complete death and cell lysis occurring after 3days. The yeast strain did not exhibit any inhibitory effect on either green algae or diatoms. Both living and heat-inactivated yeast cells were capable of adsorption of MC on their surfaces. Inactivated yeast exhibited higher adsorption capacity and lower intensity than living yeast for the adsorption of MC toxin. The results of this study suggest that this yeast strain could be employed to selectively reduce cyanobacterial blooms in freshwaters. Moreover, the application of heat-inactivated yeast's biomass for toxin adsorption gives new possibilities in drinking water treatment plants.

Comparative Study on Multiway Enhanced Bio- and Phytoremediation of Aged Petroleum-Contaminated Soil

Bioremediation and phytoremediation of soil polluted with petroleum hydrocarbons (PHs) are an effective and eco-friendly alternative to physicochemical methods of soil decontamination. These techniques can be supported by the addition of effective strains and/or surface-active compounds. However, to obtain maximum efficacy of bioremediation, the interactions that occur between the microorganisms, enhancement factors and plants need to be studied. Our study aimed to investigate the removal of petroleum hydrocarbons from an aged and highly polluted soil (hydrocarbon content about 2.5%) using multiway enhanced bio- and phytoremediation. For this purpose, 10 enhanced experimental groups were compared to two untreated controls. Among the enhanced experimental groups, the bio- and phytoremediation processes were supported by the endophytic strain Rhodococcus erythropolis CDEL254. This bacterial strain has several plant growth-promoting traits and can degrade petroleum hydrocarbons and produce biosurfactants. Additionally, a rhamnolipid solution produced by Pseudomonas aeruginosa was used to support the total petroleum hydrocarbon loss from soil. After 112 days of incubation, the highest PH removal (31.1%) was observed in soil planted with ryegrass (Lolium perenne L. cv. Pearlgreen) treated with living cells of the CDEL254 strain and rhamnolipid solution. For non-planted experimental groups, the highest PH loss (26.1%) was detected for soil treated with heat-inactivated CDEL254 cells and a rhamnolipid solution. In general, the differences in the efficacy of the 10 experimental groups supported by plants, live/dead cells of the strain tested and rhamnolipid were not statistically significant. However, each of these groups was significantly more effective than the appropriate control groups. The PH loss in untreated (natural attenuation) and soils that underwent phytoremediation reached a value of 14.2% and 17.4%, respectively. Even though the CDEL254 strain colonized plant tissues and showed high survival in soil, its introduction did not significantly increase PH loss compared to systems treated with dead biomass. These results indicate that the development of effective biological techniques requires a customized approach to the polluted site and effective optimization of the methods used.

Diversity of cyclic antimicrobial lipopeptides from Bacillus P34 revealed by functional annotation and comparative genome analysis

Cyclic lipopeptides (CLPs) from Bacillus strains have demonstrated a wide range of bioactivities making them interesting candidates for different applications in the pharmaceutical, food and biotechnological industries. Genome sequencing, together with phylogenetic analysis of the Bacillus sp. P34, isolated from a freshwater fish gut, showed that the bacterial strain belongs to the Bacillus velezensis group. In silico investigation of metabolic gene clusters of nonribosomal peptide synthetases (NRPS) revealed the genetic elements associated with the synthesis of surfactin, fengycin and iturin family component bacillomycin. Further, an assay was conducted to investigate the production of CLPs in the presence of heat inactivated bacterial cultures or fungal spores. Maximum fengycin concentration was observed at 24 h (2300−2700 mg/mL), while maximum iturin amounts were detected at 48 h (250 mg/mL) in the presence of heat-inactivated spores of Aspergillus niger. Heat-inactivated cells of Listeria monocytogenes caused a reduction of both fengycin and iturin amounts. The production of fengycins A and B and the iturin family component bacillomycin L was confirmed by mass spectrometry analyses. This study reinforces the potential of B. velezensis P34 as a valuable strain for biotechnological production of CLPs recognized as important antimicrobial substances.

Improved Production of Two Anti-Candida Lipopeptide Homologues Co- Produced by the Wild-Type Bacillus subtilis RLID 12.1 under Optimized Conditions.

Antifungal cyclic lipopeptides, bioactive metabolites produced by many species of the genus Bacillus, are promising alternatives to synthetic fungicides and antibiotics for the biocontrol of human pathogenic fungi. In a previous study, the co- production of five antifungal lipopeptides homologues (designated as AF1, AF2, AF3, AF4 and AF5) by the producer strain Bacillus subtilis RLID 12.1 using unoptimized medium was reported; though the two homologues AF3 and AF5 differed by 14 Da and in fatty acid chain length were found effective in antifungal action, the production/ yield rate of these two lipopeptides determined by High-Performance Liquid Chromatography was less in the unoptimized media. In this study, the production/yield enhancement of the two compounds AF3 and AF5 was specifically targeted. Following the statistical optimization (Plackett-Burman and Box-Behnken designs) of media formulation, temperature and growth conditions, the production of AF3 and AF5 was improved by about 25.8- and 7.4-folds, respectively under static conditions. To boost the production of these two homologous lipopeptides in the optimized media, heat-inactivated Candida albicans cells were used as a supplement resulting in 34- and 14-fold increase of AF3 and AF5, respectively. Four clinical Candida auris isolates had AF3 and AF5 MICs (100 % inhibition) ranging between 4 and 16 μg/ml indicating the lipopeptide's clinical potential. To determine the in vitro pharmacodynamic potential of AF3 and AF5, time-kill assays were conducted which showed that AF3 (at 4X and 8X concentrations) at 48h exhibited mean log reductions of 2.31 and 3.14 CFU/ml of C. albicans SC 5314, respectively whereas AF5 at 8X concentration showed a mean log reduction of 2.14 CFU/ml. With the increasing threat of multidrug-resistant yeasts and fungi, these antifungal lipopeptides produced by optimized method promise to aid in the development of novel antifungal that targets disease-causing fungi with improved efficacy.

Evaluation of biofilm of Vibrio anguillarum for oral vaccination of Asian seabass, Lates calcarifer (BLOCH, 1790)

The present study evaluated the biofilm (BF) of Vibrio anguillarum for oral vaccination of Asian seabass, Lates calcarifer. An 80-day experiment was carried out in circular fiber-reinforced plastic (FRP) tanks using free cell (FC) and BF of Vibrio anguillarum with triplicate in each. Heat-inactivated FC and BF cells at 107, 1010 and 1013 CFU/g fish/d were fed to fish for 20 days, agglutination antibody titer estimated at each 10 days interval up to 60-day post vaccination. As compared to FC and control there was a significant increase in agglutinating antibody titer in the biofilm vaccinated fishes. Among the 3 doses, BF at 1010 cfu/g fish/d was considered the ideal dose for vaccination. Relative percentage survival (RPS) was higher in biofilm vaccinated fish (85.4%) compared to that with free cells (27.0%). The study demonstrated the better performance of V. anguillarum biofilm oral vaccine compared that with free cell vaccine in L. calcarifer. The study further supports better performance of biofilm vaccine model with one more bacterial pathogen in a high carnivore fish.

Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry.

The determination of cell viability is essential to many areas of life sciences and biotechnology. Typically, cell viability measurements are based on the optical analysis of stained cells, which requires additional labeling steps and is hard to implement online. Frequency-dependent impedance flow cytometry (IFC) provides a label-free, fast, and reliable alternative to determine cell viability at the single cell level based on the Coulter principle. Here, we describe the application of IFC to eukaryotic cell cultures and compare the results to commonly used staining methods. Yeast cell parameters were assessed in normal and heat-inactivated cells as well as in alcoholic fermentation and long-term batch cultures providing a precise and fast determination of the cell viability and further quantitative measures of the cell culture status. As an important new application, we have investigated recombinant protein production in the widely used baculovirus insect cell expression system. The IFC analysis revealed the presence of a subpopulation of cells, which correlates with the protein expression yield, but it is not detectable with conventional optical cell counters. We tentatively identify this subpopulation as cells in the late phase of infection. Their detection can serve as a predictor for the optimal time point of harvest. The IFC technique should be generally applicable to many eukaryotic cell cultures in suspension, possibly also implemented online.

Characterization of Deoxynivalenol Detoxification by Lactobacillus paracasei LHZ-1 Isolated from Yogurt

Deoxynivalenol (DON) is a potent mycotoxin produced by many Fusarium spp. that invade grains during the growth and storage seasons. Lactic acid bacteria have been reported to be capable of removing several toxins, thereby providing an effective detoxification method for possible contaminated substrates. The present study mainly focused on investigating the detoxification characteristics of DON by a Lactobacillus paracasei LHZ-1 strain, which was recently isolated from yogurt with a strong promise of removing DON from liquid culture. The results obtained showed that the cell wall of L. paracasei LHZ-1 can remove up to 40.7% of 50 μg/mL DON, whereas only 10.5 and 8.9% are removed by the culture supernatant or cellular lysate, respectively. Laser scanning confocal microscopy helped to identify the mechanism of DON detoxification by L. paracasei LHZ-1 through cellular adsorption, where DON was found to bind to the surface of bacterial cells to form complexes. In stability tests, about 39 or 99% of bound DON, either to viable bacterial cells or heat-inactivated cells, respectively, was released by methanol extractions, which indicated that the binding force between viable cells and DON could be stronger than it is in heat-inactivated cells. Adsorption kinetics demonstrated that approximately 33% of DON was removed within 20 h, with a maximum adsorption capacity of approximately 50.5 μg/mL in phosphate-buffered solution.

Biocontrol features of Clavispora lusitaniae against Penicillium digitatum on lemons

Penicillium digitatum is the main pathogen which causes postharvest decays in lemons, and the use of synthetic fungicides is the principal way of controlling these postharvest infections. In our previous work, identifying biological alternatives to the use of fungicides, a killer strain of Clavispora lusitaniae 146, which exhibited highly protective activity against green mold on lemons, was isolated from citrus plants. The aim of the present work was to study the ability of C. lusitaniae 146 to colonize wounds in lemon fruit and its effect on P. digitatum spore germination. Additionally, the protection efficacy of yeast strain 146 against an imazalil-resistant P. digitatum as well as yeast survival on commercial waxes were also evaluated. Results indicated that C. lusitaniae 146 exhibited an evident ability to colonize wounds in lemons and continue to grow at both low and room temperatures. In addition, the use of heat-inactivated cells showed a certain degree of efficacy against fruit decays. The ability of C. lusitaniae 146 to inhibit spore germination and its high efficiency against both sensitive and resistant fungi to fungicides were also demonstrated. Lastly, it was found that C. lusitaniae 146 could survive in natural waxes for 7 d at 25 °C and 40 d at 8 °C. Thus, our studies confirmed the potential industrial use of C. lusitaniae 146 as an effective biocontrol agent against postharvest infections of lemons caused by P. digitatum.

Development of rapid dipstick assay for food pathogens, Salmonella, by optimized parameters.

Salmonella is among the very important pathogens threating the human and animal health. Rapid and easy detection of these pathogens is crucial. In this context, antibody (Ab) based lateral flow assays (LFAs) which are simple immunochromatographic point of care test kits were developed by gold nanoparticles (GNPs) as labelling agent for Salmonella detection. For that purpose some critical parameters such as reagent concentrations on the capture zones, conjugate concentrations and ideal membrane type needed for LFAs for whole cell detection were tested for naked eye analysis. Therefore, prepared LFAs were applied to the live and heat inactivated cells when they were used alone or included in different bacterial mixtures. Among the test platforms, membrane 180 (M180) was found as an ideal membrane and 36nm GNPs showed highly good labelling in the developed LFAs. Diluted conjugates and low concentrations of reagents affected the test signal negatively. Salmonella was detected in different bacterial mixtures, selectively in 4-5min. The best recognized species by used Ab were S. enteritidis and S. infantis. 5 × 105 S. typhimurium cells were also determined as a limit of detection of this study with mentioned parameters.