Pojava i određivanje Thermoanaerobacterium i Thermoanaerobacter u konzerviranoj hrani u limenkama

In order to determine the reason for loss of vacuum in canned food, obligately anaerobic, spore forming thermophilic organisms were isolated from shelf-stable canned food containing vegetables, noodles and potatoes as main ingredients. Thermophilic bacteria from 44 canned food samples that had been stored under anaerobic conditions at 37 °C for at least 7 days were isolated. In addition, organic fertilizer used for the cultivation of some of the foods’ ingredients was examined and anaerobic, thermophilic bacteria could also be isolated from this source. Identification of bacterial strains was carried out by partial and complete 16S-rRNA-gene sequencing. Some of the obtained gene sequences showed a high level of similarity to existing 16S-rRNA gene sequences towards strains of the genera Thermoanaerobacter, Thermoanaerobium and Thermoanaerobacterium respectively, which have not yet been reported to be of importance as food spoilers. In the course of identification of these thermophilic bacteria we developed genera specific PCR-based approaches for detecting isolates belonging to the genera Thermoanaeroacterium and Thermoanaerobacter. Direct capturing of free DNA from contaminated samples using oligonucleotides coupled with paramagentic beads allowed the reduction of the detection time to six hours with a lower limit of 104 cells/mL.Da bi se odredio uzrok nestanka vakuuma u limenkama konzervirane hrane, obligatni anaerobi, termofilni organizmi koji stvaraju spore, izolirani su iz hrane u limenkama s glavnim sastojcima: povrće, rezanci i krumpir. Izolirane su termofilne bakterije iz 44 uzorka limenki uskladištenih pod anaerobnim uvjetima pri 37 °C barem 7 dana. Osim toga, ispitana su organska gnojiva upotrijebljena za uzgoj navedenog povrća pa su i iz tog izvora izolirane anaerobne termofilne bakterije. Identifikacija bakterijskih sojeva provedena je djelomičnim i potpunim sekvencioniranjem 16S-rRNA gena. Neke od dobivenih genskih sekvencija pokazale su visoki stupanj sličnosti s postojećim sekvencijama 16S-rRNA gena sojeva rodova Thermoanaerobacter, Thermoanaerobium i Thermoanaerobacterium. Do sada još nije bila ustanovljena važnost tih sojeva kao onečišćavača hrane. Tijekom identifikacije navedenih termofilnih bakterija autori su razvili genetički specifičan pristup utemeljen na PCR za određivanje izolata koji pripadaju rodovima Thermoanaerobacterium i Thermoanaerobacter. Izravno vezanje slobodne DNA iz onečišćenih uzoraka, koristeći oligonukleotide povezane s paramagnetskim zrncima omogućilo je smanjenje vremena detekcije na 6 sati s donjom granicom od 104 stanica/mL

Size-Dependent Variability in Flow and Viscoelastic Behavior of Levan Produced by Gluconobacter albidus TMW 2.1191

Levan is a fructan-type exopolysaccharide which is produced by many microbes from sucrose via extracellular levansucrases. The hydrocolloid properties of levan depend on its molecular weight, while it is unknown why and to what extent levan is functionally diverse depending on ist size. The aim of our study was to gain deeper insight into the size-dependent functional variability of levan. For this purpose, levans of different sizes were produced using the water kefir isolate Gluconobacter albidus TMW 2.1191 and subsequently rheologically characterized. Three levan types could be identified, which are similarly branched, but differ significantly in their molecular size and rheological properties. The smallest levan (10$^{8}$ Da) produced at pH ≥ 4.5 were shear-thinning, and the levan produced at pH 5.0 showed a gel-like behavior at 5% (w/v). A third (intermediate) levan variant was obtained through production in buffers at pH 4.0 and exhibited the properties of a viscoelastic fluid up to concentrations of 15% (w/v). Our study reveals that the rheological properties of levan are determined by its size and polydispersity, rather than by the amount of levan used or the structural composition

SxsA, a novel surface protein mediating cell aggregation and adhesive biofilm formation of Staphylococcus xylosus

Biofilm formation of staphylococci has been an emerging field of research for many years. However, the underlying molecular mechanisms are still not fully understood and vary widely between species and strains. The aim of this study was to identify new effectors impacting biofilm formation of two Staphylococcus xylosus strains. We identified a novel surface protein conferring cell aggregation, adherence to abiotic surfaces, and biofilm formation. The S. xylosus surface protein A (SxsA) is a large protein occurring in variable sizes. It lacks sequence similarity to other staphylococcal surface proteins but shows similar structural domain organization and functional features. Upon deletion of sxsA, adherence of S. xylosus strain TMW 2.1523 to abiotic surfaces was completely abolished and significantly reduced in TMW 2.1023. Macro- and microscopic aggregation assays further showed that TMW 2.1523 sxsA mutants exhibit reduced cell aggregation compared with the wildtype. Comparative genomic analysis revealed that sxsA is part of the core genome of S. xylosus, Staphylococcus paraxylosus, and Staphylococcus nepalensis and additionally encoded in a small group of Staphylococcus cohnii and Staphylococcus saprophyticus strains. This study provides insights into protein-mediated biofilm formation of S. xylosus and identifies a new cell wall-associated protein influencing cell aggregation and biofilm formation.Peer Reviewe

High pressure thermal inactivation of Clostridium botulinum type E endospores – kinetic modeling and mechanistic insights

Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT) processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores. We investigated the inactivation of C. botulinum type E spores by (near) isothermal HPT treatments at 300–1200 MPa at 30–75°C for 1 s to 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone), large heat susceptible (HPT-induced germinated) or lysozyme-dependently germinable (damaged coat layer) spore fractions were not detected. Inactivation followed first order kinetics. Dipicolinic acid release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective) physiologic-like (similar to nutrient-induced) germination at ≤450 MPa/≤45°C and non-physiological germination at >500 MPa/>60–70°C. Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores compared with the resistance of spores from other C. botulinum types could allow for the implementation of milder processes without endangering food safety

Effect of High Pressure and Heat on Bacterial Toxins

Even though the inactivation of microorganisms by high pressure treatment is a subject of intense investigations, the effect of high pressure on bacterial toxins has not been studied so far. In this study, the influence of combined pressure/temperature treatment (0.1 to 800 MPa and 5 to 121 °C) on bacterial enterotoxins was determined. Therefore, heat-stable enterotoxin (STa) of cholera toxin (CT) from Vibrio cholerae, staphylococcal enterotoxins A-E, haemolysin BL (HBL) from Bacillus cereus, and Escherichia coli (STa) were subjected to different treatment schemes. Structural alterations were monitored in enzyme immunoassays (EIAs). Cytotoxicity of the pressure treated supernatant of toxigenic B. cereus DSM 4384 was investigated with Vero cells. High pressure of 200 to 800 MPa at 5 °C leads to a slight increase of the reactivity of the STa of E. coli. However, reactivity decreased at 800 MPa and 80 °C to (66±21) % after 30 min and to (44±0.3) % after 128 min. At ambient pressure no decrease in EIA reactivity could be observed after 128 min. Pressurization (0.1 to 800 MPa) of heat stable monomeric staphylococcal toxins at 5 and 20 °C showed no effect. A combined heat (80 °C) and pressure (0.1 to 800 MPa) treatment lead to a decrease in the immuno-reactivity to 20 % of its maximum. For cholera toxin a significant loss in latex agglutination was observable only at 80 °C and 800 MPa for holding times higher than 20 min. Interestingly, the immuno-reactivity of B. cereus HBL toxin increased with the increase of pressure (182 % at 800 MPa, 30 °C), and high pressure showed only minor effects on cytotoxicity to Vero cells. Our results indicate that pressurization can increase inactivation observed by heat treatment, and combined treatments may be effective at lower temperatures and/or shorter incubation time

Effect of High Pressure and Heat on Bacterial Toxins

Even though the inactivation of microorganisms by high pressure treatment is a subject of intense investigations, the effect of high pressure on bacterial toxins has not been studied so far. In this study, the influence of combined pressure/temperature treatment (0.1 to 800 MPa and 5 to 121 °C) on bacterial enterotoxins was determined. Therefore, heat-stable enterotoxin (STa) of cholera toxin (CT) from Vibrio cholerae, staphylococcal enterotoxins A-E, haemolysin BL (HBL) from Bacillus cereus, and Escherichia coli (STa) were subjected to different treatment schemes. Structural alterations were monitored in enzyme immunoassays (EIAs). Cytotoxicity of the pressure treated supernatant of toxigenic B. cereus DSM 4384 was investigated with Vero cells. High pressure of 200 to 800 MPa at 5 °C leads to a slight increase of the reactivity of the STa of E. coli. However, reactivity decreased at 800 MPa and 80 °C to (66±21) % after 30 min and to (44±0.3) % after 128 min. At ambient pressure no decrease in EIA reactivity could be observed after 128 min. Pressurization (0.1 to 800 MPa) of heat stable monomeric staphylococcal toxins at 5 and 20 °C showed no effect. A combined heat (80 °C) and pressure (0.1 to 800 MPa) treatment lead to a decrease in the immuno-reactivity to 20 % of its maximum. For cholera toxin a significant loss in latex agglutination was observable only at 80 °C and 800 MPa for holding times higher than 20 min. Interestingly, the immuno-reactivity of B. cereus HBL toxin increased with the increase of pressure (182 % at 800 MPa, 30 °C), and high pressure showed only minor effects on cytotoxicity to Vero cells. Our results indicate that pressurization can increase inactivation observed by heat treatment, and combined treatments may be effective at lower temperatures and/or shorter incubation time

Crescimento inicial de Pinus taeda L. relacionado a doses de N, P e K.

This study aimed to evaluate the effect N, P and K on the growth of young (19 month-old) Pinus taeda. The experiment took place near Cambará do Sul county, with UTM coordinates of 562125 E and 6777386 N. The statistical methodology was the confounding technique, for three factors, with four levels, totalizing 64 treatments, allocated in four blocks. The following variables were measured: total height, crown height, steam diameter and crown diameter. The results pointed out a significant linear quadratic effect for P, indicating that P fertilization is essential on the initial development of Pinus taeda plants, with a maximum cylindric volume achieved at 64 and 87 kg ha-1 doses of P2O5 and K2O respectively.O presente trabalho teve como objetivo estudar os efeitos de diferentes doses de N, P e K no crescimento inicial de plantas de Pinus taeda, aos 19 meses de idade. O experimento localiza-se próximo do Município de Cambará do Sul, tendo como coordenadas UTM "centrais" 565125 E e 6777386 N. O solo da região é classificado como Cambissolo Humico alumínico típico, textura argilosa. A metodologia estatística utilizada foi a técnica do confundimento, para três fatores, com quatro níveis, totalizando 64 tratamentos, distribuídos em quatro blocos. Foram medidas as seguintes variáveis no campo: altura total, altura de copa, diâmetro à altura do colo e diâmetro de copa. Os resultados indicam que foi obtida resposta linear e quadrática significativa para o P, evidenciando a importância da aplicação desse nutriente no crescimento inicial das plantas de Pinus taeda, com máximo ganho em volume cilíndrico, nas doses de 64 e 87 kg ha-1 de P2O5 e K2O respectivamente

Genomic analysis reveals Lactobacillus sanfranciscensis as stable element in traditional sourdoughs

Sourdough has played a significant role in human nutrition and culture for thousands of years and is still of eminent importance for human diet and the bakery industry. Lactobacillus sanfranciscensis is the predominant key bacterium in traditionally fermented sourdoughs

Proteomic Analysis of Lactobacillus nagelii in the Presence of Saccharomyces cerevisiae Isolated From Water Kefir and Comparison With Lactobacillus hordei

Water kefir is a slightly alcoholic and traditionally fermented beverage, which is prepared from sucrose, water, kefir grains, and dried or fresh fruits (e.g., figs). Lactobacillus (L.) nagelii, L. hordei, and Saccharomyces (S.) cerevisiae are predominant and stable lactic acid bacteria and yeasts, respectively, isolated from water kefir consortia. The growth of L. nagelii and L. hordei are improved in the presence of S. cerevisiae. In this work we demonstrate that quantitative comparative proteomics enables the investigation of interactions between LAB and yeast to predict real-time metabolic exchange in water kefir. It revealed 73 differentially expressed (DE) in L. nagelii TMW 1.1827 in the presence of S. cerevisiae. The presence of the yeast induced changes in the changes in the carbohydrate metabolism of L. nagelii and affected reactions involved in NAD+/NADH homeostasis. Furthermore, the DE enzymes involved in amino acid biosynthesis or catabolism predict that S. cerevisiae releases glutamine, histidine, methionine, and arginine, which are subsequently used by L. nagelii to ensure its survival in the water kefir consortium. In co-culture with S. cerevisiae, L. nagelii profits from riboflavin, most likely secreted by the yeast. The reaction of L. nagelii to the presence of S. cerevisiae differs from that one of the previously studied L. hordei, which displays 233 differentially expressed proteins, changes in citrate metabolism and an antidromic strategy for NAD+/NADH homeostasis. So far, aggregation promotion factors, i.e., formation of a specific glucan and bifunctional enzymes were only detected in L. hordei