Oxygen and air cold plasma for the inactivation of Bacillus cereus in low-water activity soy powder.

Cold plasma (CP) technology is a promising alternative to thermal treatments for the microbial decontamination of foods with low-water activity. The aim of this work is study the application of low-pressure CP (0.35 mbar) for the inactivation of Bacillus cereus in a soybean powder matrix using O2 and synthetic air as ionizing gases. The parameters tested were an input power of 100, 200 and 300 W and an exposure time of 10 to 30 min. The excited reactive species formed were monitored by optical emission spectroscopy, and survival data were analyzed using the Weibull mathematical model. Treatments with both gases were effective in inactivating B. cereus. Air plasma resulted in a maximum 3.71-log reduction in bacterial counts at 300 W and 30 min, while O2 plasma showed the strongest inactivation ability, achieving levels higher than 5 log cycles at 300 W and > 25 min. This is likely due to the strong antimicrobial activity of oxygen-derived radicals together with carbon monoxide as an oxidation by-product. In addition, the Weibull distribution function accurately modeled the inactivation of B. cereus. Cold plasma technology is a promising approach for the decontamination of bacteria in low-water activity foods.

Effects of a co-bacterial agent on the growth, disease control, and quality of ginseng based on rhizosphere microbial diversity.

BACKGROUND: The ginseng endophyte Paenibacillus polymyxa Pp-7250 (Pp-7250) has multifaceted roles such as preventing ginseng diseases, promoting growth, increasing ginsenoside accumulation, and degrading pesticide residues, however, these effects still have room for improvements. Composite fungicides are an effective means to improve the biocontrol effect of fungicides, but the effect of Pp-7250 in combination with its symbiotic bacteria on ginseng needs to be further investigated, and its mechanism of action has not been elucidated. In this study, a series of experiments was conducted to elucidate the effect of Paenibacillus polymyxa and Bacillus cereus co-bacterial agent on the yield and quality of understory ginseng, and to investigate their mechanism of action. RESULTS: The results indicated that P. polymyxa and B. cereus co-bacterial agent (PB) treatment improved ginseng yield, ginsenoside accumulation, disease prevention, and pesticide degradation. The mechanism is that PB treatment increased the abundance of beneficial microorganisms, including Rhodanobacter, Pseudolabrys, Gemmatimonas, Bacillus, Paenibacillus, Cortinarius, Russula, Paecilomyces, and Trechispora, and decreased the abundance of pathogenic microorganisms, including Ellin6067, Acidibacter, Fusarium, Tetracladium, Alternaria, and Ilyonectria in ginseng rhizosphere soil. PB co-bacterial agents enhanced the function of microbial metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, biosynthesis of amino acids, carbon fixation pathways in prokaryotes, DNA replication, and terpenoid backbone biosynthesis, and decreased the function of microbial plant pathogens and animal pathogens. CONCLUSION: The combination of P. polymyxa and B. cereus may be a potential biocontrol agent to promote the resistance of ginseng to disease and improve the yield, quality, and pesticide degradation.

Isolation and Identification of the Antibacterial Compounds Produced by Maillard Reaction of Xylose with Phenylalanine or Proline.

Maillard reaction products (MRPs) of xylose with phenylalanine and xylose with proline exhibit high antibacterial activity. However, the active antibacterial compounds in MRPs have not yet been identified or isolated. This study aimed to isolate the active compounds in the two antibacterial MRPs. The organic layer of the MRP solution was separated and purified using silica gel chromatography and high-performance liquid chromatography. The chemical structures of the isolated compounds were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds inhibited the growth of Bacillus cereus and Salmonella Typhimurium at 25 °C for 7 days at a concentration of 0.25 mM. Furthermore, the isolated compounds inhibited the growth of naturally occurring microflora of lettuce and chicken thighs at 25 °C for 2 days at a concentration of 0.5-1.0 mM. The antibacterial compounds found in MRPs demonstrated a wide range of effectiveness and indicated their potential as alternative preservatives.

Inhibition and Mechanism of Citral on Bacillus cereus Vegetative Cells, Spores, and Biofilms.

Bacillus cereus causes food poisoning by producing toxins that cause diarrhea and vomiting and, in severe cases, endocarditis, meningitis, and other diseases. It also tends to form biofilms and spores that lead to contamination of the food production environment. Citral is a potent natural antibacterial agent, but its antibacterial activity against B. cereus has not been extensively studied. In this study, we first determined the minimum inhibitory concentrations and minimum bactericidal concentrations, growth curves, killing effect in different media, membrane potential, intracellular adenosine triphosphate (ATP), reactive oxygen species levels, and morphology of vegetative cells, followed by germination rate, morphology, germination state of spores, and finally biofilm clearance effect. The results showed that the minimum inhibitory concentrations and minimum bactericidal concentrations of citral against bacteria ranged from 100 to 800 µg/mL. The lag phase of bacteria was effectively prolonged by citral, and the growth rate of bacteria was slowed down. Bacteria in Luria-Bertani broth were reduced to below the detection limit by citral at 800 µg/mL within 0.5 h. Bacteria in rice were reduced to 3 log CFU/g by citral at 4000 µg/mL within 0.5 h. After treatment with citral, intracellular ATP concentration was reduced, membrane potential was altered, intracellular reactive oxygen species concentration was increased, and normal cell morphology was altered. After treatment with citral at 400 µg/mL, spore germination rate was reduced to 16.71%, spore morphology was affected, and spore germination state was altered. It also had a good effect on biofilm removal. The present study showed that citral had good bacteriostatic activity against B. cereus vegetative cells and its spores and also had a good clearance effect on its biofilm. Citral has the potential to be used as a bacteriostatic substance for the control of B. cereus in food industry production.

Comparison of plate counting with flow cytometry, using four different fluorescent dye techniques, for the enumeration of Bacillus cereus in milk.

This study aimed to compare the performance of flow cytometry methods with plate counting for the enumeration of bacteria, using Bacillus cereus as a model organism. It was found that the cFDA-propidium iodide, CellROX™ Green-propidium iodide, and DiOC2 dye techniques had similar accuracy to plate counting, while the SYTO 24-propidium iodide dye technique was not as accurate. The four dye techniques had comparable precision to plate counting, with the CellROX™ Green-propidium iodide dye having the greatest precision. The consistency of the position and shape of the cell clusters on the flow cytometry plots, and the extent of separation of the cell from background clusters, was greatest with the DiOC2 and CellROX™ Green-propidium iodide dyes. Furthermore, the DiOC2 and CellROX™ Green-propidium iodide dyes performed well, even when a sample was measured containing reconstituted whole milk powder at a 10-1 dilution, without the use of sample preparation to specifically remove the milk constituents prior to measurement. Given gating of only one cell cluster was required to be managed with the DiOC2 dye, to determine the viable number of cells, it was found that the DiOC2 dye had the greatest ease-of-use. Overall, results indicated that the DiOC2 dye is an ideal candidate for the enumeration of viable bacteria in dairy samples on a high-throughput, routine basis.

Novel Bacillus and Prestia isolates from Dwarf century plant enhance crop yield and salinity tolerance.

Soil salinity is a major environmental stressor impacting global food production. Staple crops like wheat experience significant yield losses in saline environments. Bioprospecting for beneficial microbes associated with stress-resistant plants offers a promising strategy for sustainable agriculture. We isolated two novel endophytic bacteria, Bacillus cereus (ADJ1) and Priestia aryabhattai (ADJ6), from Agave desmettiana Jacobi. Both strains displayed potent plant growth-promoting (PGP) traits, such as producing high amounts of indole-3-acetic acid (9.46, 10.00 µgml-1), ammonia (64.67, 108.97 µmol ml-1), zinc solubilization (Index of 3.33, 4.22, respectively), ACC deaminase production and biofilm formation. ADJ6 additionally showed inorganic phosphate solubilization (PSI of 2.77), atmospheric nitrogen fixation, and hydrogen cyanide production. Wheat seeds primed with these endophytes exhibited enhanced germination, improved growth profiles, and significantly increased yields in field trials. Notably, both ADJ1 and ADJ6 tolerated high salinity (up to 1.03 M) and significantly improved wheat germination and seedling growth under saline stress, acting both independently and synergistically. This study reveals promising stress-tolerance traits within endophytic bacteria from A. desmettiana. Exploiting such under-explored plant microbiomes offers a sustainable approach to developing salt-tolerant crops, mitigating the impact of climate change-induced salinization on global food security.

Meta-analysis of antimicrobial activity of Allium, Ocimum, and Thymus spp. confirms their promising application for increasing food safety.

Biopreservation strategies such as the use of Mediterranean plant extracts to ensure food safety are promising to deal with the emergence of antimicrobial resistances and the overreliance on food chemical additives. In the last few decades, antimicrobial susceptibility testing (AST) for evaluating the in vitro antibacterial potential of plant extracts against the most relevant foodborne pathogens has been widely reported in the literature. The current meta-analysis aimed to summarise and analyse the extensive evidence available in the literature regarding the in vitro antimicrobial capability of Allium, Ocimum and Thymus spp. extracts against foodborne pathogens. A systematic review was carried out to gather data on AST results of these extracts against Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli and Bacillus cereus, including inhibition diameters (ID) and minimum inhibitory concentrations (MIC). A total of 742 records were gathered from a raw collection of 2,065 articles. Weighted mixed-effect linear models were adjusted to data to obtain pooled ID, pooled MIC and the relationship between both model estimations and observations. The pooled results revealed B. cereus as the most susceptible bacteria to Allium sativum (pooled ID = 20.64 ± 0.61 mm) by diffusion methods and S. aureus (pooled MIC = 0.146 mg/mL) by dilution methods. Diffusion methods did not yield conclusive results for Ocimum spp. extracts; however, the lowest pooled MIC was obtained for S. aureus (0.263 mg/mL). Among the foodborne pathogens evaluated, B. cereus showed the highest sensitivity to Thymus spp. extracts by both diffusion and dilution methods (pooled ID = 28.90 ± 2.34 mm and MIC = 0.075 mg/mL). The methodology used for plant extraction was found to not significantly affect MIC values (p > 0.05). Overall, the antimicrobial effectiveness of the studied extracts against Gram-positive and Gram-negative bacteria was demonstrated. Finally, the robustness of the meta-regression model was confirmed, also revealing an inversely proportional correlation between the ID and MIC measurements (p < 0.0001). These results provide a robust scientific basis on the factors affecting the in vitro antimicrobial efficacy of extracts from Mediterranean plants. They also provide valuable information for stakeholders involved in their industrial application in food, including producers, regulatory agencies and consumers which demand green-labelled foods.

Inactivation of Bacillus cereus spores by ohmic heating: Efficiency and changes of spore biological properties.

Bacillus cereus spores pose a significant concern during food processing due to their high resistance to environmental stress. Ohmic heating (OH) is an emerging and alternative heating technology with potential for inactivating such spores. This study evaluated the inactivation effects and the biological property changes of Bacillus cereus spores during OH treatments. OH effectively inactivated spores in milk, orange juice, broth, rice soup, and buffer solution in less time than oil bath heating (OB). A decrease in NaCl content improved spore inactivation at the same temperature. Spores were more sensitive to acid at 80-85 °C with OH treatment. Furthermore, OH at 10 V/cm and 50 Hz could reduce the spore resistance and inhibit an increase in spore hydrophobicity and spore aggregation. Both heating methods resulted in significant dipicolinic acid (DPA) leakage and damage to the cortex and inner membranes of the spores. However, OH at 10 V/cm and 50 Hz had the lowest DPA leakage and inflicted the least damage to the inner membrane. The damage to the spore's inner membrane was considered the primary reason for inactivation by OB and OH treatments. Still, OH at 10 V/cm and 50 Hz might also block the germination or outgrowth of treated spores or cause damage to the spore core.

Inactivation mechanism of phenyllactic acid against Bacillus cereus spores and its application in milk beverage.

Phenyllactic acid (PLA) as a natural phenolic acid exhibits antibacterial activity against non-spore-forming bacteria, while the inhibitory effect against bacterial spore remained unknown. Herein, this study investigated the inactivation effect of PLA against Bacillus cereus spores. The results revealed that the minimum inhibitory concentration of PLA was 1.25 mg/mL. PLA inhibited the outgrowth of germinated spores into vegetative cells rather than germination of spores. PLA disrupted the spore coat, and damaged the permeability and integrity of inner membrane. Moreover, PLA disturbed the establishment of membrane potential due to the inhibition of oxidative metabolism. SEM observations further visualized the morphological changes and structural disruption caused by PLA. Besides, PLA caused the degradation of DNA of germinated spores. Finally, PLA was applied in milk beverage, and showed promising inhibitory effect against B. cereus spores. This finding could provide scientific basis for the application of PLA against spore-forming bacteria in food industry.

Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

Impact of Bacillus cereus SPB-10 on Growth Promotion of Wheat (Triticum aestivum L.) Under Arsenic-Contaminated Soil.

This study investigates the impact of bacteria on arsenic reduction in wheat plants, highlighting the potential of microbe-based eco-friendly strategies for plant growth. In the present study, bacterial isolate SPB-10 was survived at high concentration against both form of arsenic (As3+ and As5+). SPB-10 produced 5.2 g/L and 11.3 g/L of exo-polysaccharide at 20 ppm of As3+ and As5+, respectively, whereas qualitative examination revealed the highest siderophores ability. Other PGP attributes such as IAA production were recorded 52.12 mg/L and 95.82 mg/L, phosphate solubilization was 90.23 mg/L and 129 mg/L at 20 ppm of As3+ and As5+, respectively. Significant amount of CAT, APX, and Proline was also observed at 20 ppm of As3+ and As5+ in SPB-10. Isolate SPB-10 was molecularly identified as Bacillus cereus through 16S rRNA sequencing. After 42 days, wheat plants inoculated with SPB-10 had a 25% increase in shoot length and dry weight, and 26% rise in chlorophyll-a pigment under As5+ supplemented T4 treatment than control. Reducing sugar content was increased by 24% in T6-treated plants compared to control. Additionally, SPB-10 enhanced the content of essential nutrients (NPK), CAT, and APX in plant's-leaf under both As3+ and As5+ stressed conditions after 42 days. The study found that arsenic uptake in plant roots and shoots decreased in SPB-10-inoculated plants, with the maximum reduction observed in As5+ treated plants. Bio-concentration factor-BCF was reduced by 90.89% in SPB-10-inoculated treatment T4 after 42 days. This suggests that Bacillus cereus-SPB-10 may be beneficial for plant growth in arsenic-contaminated soil.

Antibacterial activity of phytopathogenic Streptomyces strains against bacteria associated to clinical diseases / Atividade antimicrobiana de Streptomyces fitopatogênicas contra bactérias associadas a doenças de importância clínica

The genus Streptomyces is associated with the ability to produce and excrete a variety of bioactive compounds, such as antibiotic, antifungal and antiviral. Biological active polyketide and peptide compounds with applications in medicine, agriculture and biochemical research are synthesized by PKS-I and NRPS genes. The evaluation of the presence of these genes associated with the biosynthesis of secondary metabolites in different phytopathogenic Streptomyces strains were performed using degenerated primers. The positive signal was observed in 58/63 Streptomyces strains for NRPS gene, 43/63 for PKS-I, and for PKS-II all the 63 strains showed positive signal of amplification. These strains also were tested with double layer agar-well technique against bacterial with clinical importance, and it was possible to observe the Streptomyces spp. strains were able to inhibit the growth of 14, 20, 13 and 3 isolates Gram-positive and Gram-negative bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 11775) respectively. The Streptomyces sp. strains IBSBF 2019 and IBSBF 2397 showed antibacterial activity against all four bacteria-target tested.(AU)

O gênero Streptomyces apresenta alta capacidade de produzir e excretar uma grande variedade de compostos biologicamente ativos, como antibióticos, antifúngicos e antivirais. Compostos biologicamente ativos de policetídeos e peptídeos com aplicações na medicina, agricultura e pesquisas bioquímicas são sintetizados pelos genes PKS-I e NRPS. A avaliação da presença desses genes associados à biossíntese de metabólitos secundários em diferentes linhagens de Streptomyces fitopatogênicas foi realizada através do uso de primers degenerados. O sinal positivo foi observado em 58/63 linhagens de Streptomyces para o gene NRPS, 43/63 para o gene PKS-I e, para o gene PKS-II, todas as 63 linhagens apesentaram o sinal positivo de amplificação. Essas linhagens também foram testadas através da técnica de dupla camada contra bactérias de importância clínica e foi possível observar que as linhagens de Streptomyces spp. foram capazes de inibir o crescimento de 14, 20, 13 e 3 isolados de bactérias Gram-positivas e Gram-negativas, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) e Escherichia coli (ATCC 11775), respectivamente. As linhagens de Streptomyces sp. ISBSF 2019 e 2397 apresentaram atividade antibacteriana contra todas as bactérias-alvo testadas.(AU)

Growth of enterotoxin producing Bacillus cereus in meat substrate at 10 ºC and 30 ºC

The behaviour of enterotoxin-producing Bacillus cereus in meat was investigated by inoculating spore suspensions of five cultures into meat substrate (pH 5.8) and incubating at 10ºC and 30ºC. The bacterial populations were evaluated after different times by plate counts in nutrient agar. All the cultures presented growth at 30ºC with the generation time varying from 28.8 to 36.0 minutes. Three cultures also presented growth at 10ºC with generation times between 10.16 and 28.38 h. Considering the results, it was concluded that meat kept at abusive temperatures would be subject to development of this microorganism.

Microrganismos heterotróficos mesófilos e bactérias do grupo do Bacillus cereus em leite integral submetido a ultra alta temperatura / Mesophilic heterotrophic microorganisms and spore forming bacteria from Bacillus cereus group in ultra high temperature milk

Cento e dez amostras de 11 diferentes marcas de leite ultra alta temperatura (UAT), comercializadas em São José do Rio Preto - SP, foram submetidas à contagem de microrganismos heterotróficos mesófilos viáveis e à pesquisa de bactérias do grupo do Bacillus cereus. A população de microrganismos mesófilos variou de <1,0 10² UFC/ml a >1,0 10(6) UFC/ml. Bactérias do grupo do Bacillus cereus foram verificadas em 13 (11,8 por cento) amostras. Os resultados evidenciaram elevada população de microrganismos indicadores mesófilos.

Efecto de las microondas sobre la sobrevivencia de bacterias esporuladas inoculadas en carne molida / Microwave effect on the survival of sporulated bacteria inoculated in minced meat

Debido a la creciente tendencia de cocinar y recalentar en horno de microondas platillos a base de carne y a la posibilidad de que estos transmitan enfermedades de origen bacteriano, se evaluó la capacidad de sobrevivencia de esporas de bacterias inoculadas en carne molida y tratadas en horno de microonda. Las tortas de carne fueron inoculadas con un número conocido de esporas de Bacillus cereus o Clostridium perfringens y posteriormente cocinadas en horno de microondas Amana de 2450 Hz. Se utilizó la técnica descrita por Vanderzant y Splittstoesser para determinar la tase de sobrevivencia y la actividad de la enxima fosfatasa ácida como parámetro de conocimiento. Las esporas de B. cereus mostraron una tendencia a disminuir en número, conforme aumenta el tiempo de exposición a la radiación, pero si llegar a desaparecer totalmente. Las esporas de C. perfringens disminuyen en número al aumentar el tiempo de exposición, pero presentan un segundo aumento asociado a la germinación de esporas sobrevivientes.

Inhibition of fooborne pathogens by bacteriocin-producing leuconostocs and lactobacillus sake isolated from "lingüiça frescal"

Two strains of lactic acid bacteria, isolated from "lingüiça" (a typical Brazilian meat product) stored under refrigeration, produced antagonistic substances active against selected foodborne pathogens. The proteinaceous nature of the inhibitors was demonstrated and so they were classified as bacteriocins. The inhibition due to acid production and phages was ruled out. The bacteriocin produced by Leuconostoc sp was active against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. The bacteriocin produced by Lactobacillus sake was active against Listeria monocytogenes and Staphylococcus aureus. Gram negative were bacteria not inhibited by the bacteriocins produced.

Multiplicacion del Bacillus Cereus inoculado en Leche en Polvo reconstituida / Multiplication of Bacillus cereus inoculated into reconstituted powder milk

Se estudio el crecimiento del Bacillus cereus en muestras de leche en polvo reconstituida, inoculadas experimentalmente. Las muestras se incubaron a diversas temperaturas y los conteos se hicieron a diferentes intervalos de tiempo. La leche refrigerada a 7 C durante 24 h mantiene el mismo nivel de concentracion de B. cereus que presentaba al inicio; la multiplicacion del germen se retarda mas a 25 C que a 31 C. La ingestion de la leche despues de 6 h a 31 C o despues de 12 h a 25 C puede ser nociva para la salud