Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 °C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5 μg/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254 nm.     

Lactobacillus salivarius: Bacteriocin and probiotic activity

Lactic acid bacteria (LAB) antimicrobial peptides typically exhibit antibacterial activity against food-borne pathogens, as well as spoilage bacteria. Therefore, they have attracted the greatest attention as tools for food biopreservation. In some countries LAB are already extensively used as probiotics in food processing and preservation. LAB derived bacteriocins have been utilized as oral, topical antibiotics or disinfectants. Lactobacillus salivarius is a promising probiotic candidate commonly isolated from human, porcine, and avian gastrointestinal tracts (GIT), many of which are producers of unmodified bacteriocins of sub-classes IIa, IIb and IId. It is a well-characterized bacteriocin producer and probiotic organism. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. This review gives an up-to-date overview of all L. salivarius strains, isolated from different origins, known as bacteriocin producing and/or potential probiotic.     

Characterisation and profiling of Bacillus subtilis,Bacillus cereus and Bacillus licheniformis by MALDI-TOF mass fingerprinting

The Bacillus genus includes species such as Bacillus cereus, Bacillus licheniformis and Bacillus subtilis, some of which may be pathogenic or causative agents in the spoilage of food products. The main goal of this work was to apply matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass fingerprinting to the classification of these Bacillus species. Genetic analyses were also compared to phyloproteomic analyses. A collection of 57 Bacillus strains isolated from fresh and processed food and from culture collections were studied and their mass spectra compiled. The resulting mass fingerprints were compared and characteristic peaks at the strain and species levels were assigned. The results showed that MALDI-TOF was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate classification of Bacillus species, especially for differentiating B. subtilis and B. cereus from Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. MALDI-TOF was also found to provide valuable information at both intra- and interspecies levels in the Bacillus species studied.     

Studies on the low-salt Chinese potherb mustard (Brassica juncea, Coss.) pickle. I—The effect of a homofermentative l(+)-lactic acid producer Bacillus coagulans on starter culture in the low-salt Chinese potherb mustard pickle fermentation

A homofermentative l(+)-lactic acid producer Bacillus coagulans B179 has been evaluated as starter cultures for low-salt potherb mustard pickle process. This study examined the impact of inoculation by B. coagulans B179 as starter culture on the spontaneous lab-scale fermentation process of potherb mustard pickle. Three different salt concentrations trials, i.e., 5 g/100 g, 8 g/100 g and 10 g/100 g, respectively, were carried out. As a result, the addition of starter culture of B. coagulans B179 to 5 g/100 g salinity fermentation showed an inhibiting effect upon the population of non-lactic acid bacteria (LAB) and marked increase in population of LAB and production of titratable acidity but did not significantly influence the pickle end products quantity of acid produced by fermentation as compared with a treatment without inoculation. However, in the higher salt concentrations, i.e., 8 g/100 g and 10 g/100 g NaCl trials, inoculation had no impact on the fermentation of the pickle. From the standpoint of fermentation, as a functional starter culture, inoculated B. coagulans B179 can effectively improve desired native LAB growth and inhibit the growth of undesired fungi, which will thereby allow it to shorten fermentation period and enhance the pickle quality in the low-salt potherb mustard pickle production.     

Inhibition of Bacillus cereus and Bacillus weihenstephanensis in raw vegetables by application of washing solutions containing enterocin AS-48 alone and in combination with other antimicrobials

Enterocin AS-48 is a broad-spectrum cyclic antimicrobial peptide produced by Enterococcus faecalis. In the present study, the bacteriocin was tested alone and in combination with other antimicrobials for decontamination of Bacillus inoculated on alfalfa, soybean sprouts and green asparagus. Washing with enterocin AS-48 solutions reduced viable cell counts of Bacillus cereus and Bacillus weihenstephanensis by 1.0–1.5 and by 1.5–2.38 log units right after application of treatment, respectively. In both cases, the bacteriocin was effective in reducing the remaining viable population below detection levels during further storage of the samples at 6 °C, but failed to prevent regrowth in samples stored at 15 or 22 °C. Application of washing treatments containing enterocin AS-48 in combination with several other antimicrobials and sanitizers (cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric acid, peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite) greatly enhanced the bactericidal effects. The combinations of AS-48 and sodium hypochlorite, peracetic acid or hexadecylpyridinium chloride provided the best results. After application of the combined treatments on alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis, viable bacilli were not detected or remained at very low concentrations in the treated samples during a 1-week storage period at 15 °C. Inhibition of B. cereus by in situ produced bacteriocin was tested by cocultivation with the AS-48 producer strain E. faecalis A-48-32 inoculated on soybean sprouts. Strain A-48-32 was able to grow and produce bacteriocin on sprouts both at 15 and 22 °C. At 15 °C, growth of B. cereus was completely inhibited in the cocultures, while a much more limited effect was observed at 22 °C. The results obtained for washing treatments are very encouraging for the application of enterocin AS-48 in the decontamination of sprouts. Application of washing treatments containing AS-48 alone can serve to reduce viable cell counts of bacilli in samples stored under refrigeration, while application of combined treatments should be recommended to avoid proliferation of the surviving bacilli under temperature-abuse conditions.     

Short communication: Presence of neutral metallopeptidase (npr) gene and proteolytic activity of Bacillus cereus isolated from dairy products

The control of proteolytic microorganisms is one of the main challenges of the dairy industry, due to their spoilage activity that jeopardizes the quality of their products. Seventy-four Bacillus cereus strains isolated from powdered, UHT, and pasteurized milks were tested for the presence of the neutral metallopeptidase (npr) gene and proteolytic activity at 7, 10, 25, 30, and 37°C. All strains had the npr gene, and proteolytic activity increased with the incubation temperature. The obtained results highlight the relevance of B. cereus as a spoiling agent in the dairy industry in terms of its genetic predisposition for proteolytic capacity, especially at room temperature.     

Effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against the spoilage bacterium Lactobacillus diolivorans in apple juice

Enterocin AS-48 was tested in apple juice against the cider-spoilage, exopolysaccharide-producing strain Lactobacillus diolivorans 29 in combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 μs and bipolar mode). A response surface methodology was applied to study the bactericidal effects of the combined treatment, with AS-48 concentration and HIPEF treatment time as process variables. At subinhibitory bacteriocin concentrations, microbial inactivation by the combined treatment increased as the bacteriocin concentration and the HIPEF treatment time increased (from 0.5 to 2.0 μg/ml and from 100 to 1000 μs, respectively). Highest inactivation (4.87 logs) was achieved by 1000 μs HIPEF treatment in combination with 2.0 μg/ml AS-48. While application of treatments separately did not protect juice from survivors during storage, survivors to the combined treatment were inactivated within the following 24 h of storage, and the treated samples remained free from detectable lactobacilli for at least 15 days at temperatures of 4 °C as well as 22 °C. The combined treatment could be useful for inactivation of exopolysaccharide-producing L. diolivorans in apple juice.     

Emetic toxin producing Bacillus cereus Korean isolates contain genes encoding diarrheal-related enterotoxins

Bacillus cereus can cause the diarrheal and emetic type of food poisoning but the symptoms of emetic food poisoning caused by B. cereus occasionally include emesis and diarrhea. The enterotoxin characteristics of emetic toxin (cereulide) producing B. cereus were needed to be determined. Therefore, forty B. cereus strains isolated from various sources in Korea were investigated for the presence of enterotoxin genes. All strains were confirmed to produce the emetic toxin using HPLC-MS methods. The rates of the nheABC, hblCDA, entFM and cytK genes amongst emetic toxin producing B. cereus strains were 82.5, 7.5, 50.0 and 27.5%, respectively. Pattern III harbored nheABC and entFM genes and pattern V processed entFM gene and were shown to be the major patterns, being present in 55.0% (21 of 40) of the emetic toxin producing B. cereus strains. Our findings revealed that 34 (85.0%) of 40 emetic toxin producing B. cereus strains isolated in Korea have the potential to cause diarrheal and emetic type of food poisoning, simultaneously. Thus, emetic toxin and enterotoxin genes should be constantly screened to provide insight into B. cereus food poisoning.     

Inactivation of Bacillus subtilis spores in soybean milk by radio-frequency flash heating

An apparatus to pasteurize soybean milk using radio-frequency flash heating (RF-FH) was developed. An electrode surface was covered with a 50-μm thick Teflon film, and 28 MHz RF-FH was applied to soybean milk flowing through the electrode.     

Enzymatic properties of transglutaminase produced by a new strain of Bacillus circulans BL32 and its action over food proteins

The aim of this study was to physicochemically characterize transglutaminase (TGase) from Bacillus circulans BL32, a strain recently isolated from the Amazon basin region, for its application in food systems. The effects of pH and temperature on the enzyme activity were determined by Central Composite Rotatable Design (CCRD), with maximal TGase activities obtained for pH between 5.7 and 8.7 and temperatures of 25-45 °C. This microbial TGase showed to be remarkably stable: over 90% of its activity was retained after 120 min of incubation at 50 °C. The Ca²⁺ and Mg²⁺ cations enhanced enzyme activity and its thermal stability when in concentrations of up to 2 and 1 mol L⁻¹, respectively. Casein, isolated soy protein, and hydrolysed animal protein were treated with this TGase. The decrease in the amount of free amino groups, especially for casein, showed the cross-linking of protein catalysed by this enzyme, while the emulsifying properties of these proteins were improved with treatment. These results suggest that this microbial TGase has a good potential to be used in food and other industrial applications.     

Effect of enterocin AS-48 in combination with biocides on planktonic and sessile Listeria monocytogenes

Enterocin AS-48 was tested on a cocktail of Listeria monocytogenes strains in planktonic and sessile states, singly or in combination with biocides benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide, triclosan, poly-(hexamethylen guanidinium) hydrochloride, chlorhexidine, hexachlorophene, and the commercial sanitizers P3 oxonia and P3 topax 66. Combinations of sub-inhibitory bacteriocin concentrations and biocide concentrations 4 to 10-fold lower than their minimum inhibitory concentrations (MIC) completely inhibited growth of the planktonic listeriae. Inactivation of Listeria in biofilms formed on polystyrene microtiter plates required concentrations of enterocin AS-48 greater than 50 μg/ml, and biocide concentrations ten to 100-fold higher. In combination with enterocin AS-48 (25 or 50 μg/ml), microbial inactivation increased remarkably for all biocides except P3 oxonia and P3 topax 66 solutions. Polystyrene microtiter plates conditioned with enterocin solutions (0.5-25 μg/ml) decreased the adherence and biofilm formation of the L. monocytogenes cell cocktail, avoiding biofilm formation for at least 24 h at a bacteriocin concentration of 25 μg/ml.     

Inactivation of Bacillus stearothermophilus spores in egg patties by pressure-assisted thermal processing

The use of pressure-assisted thermal processing (PATP) to inactivate bacterial spores in shelf-stable low-acid foods, without diminishing product quality, has received widespread industry interest. Egg patties were inoculated with Bacillus stearothermophilus spores (10⁶ spores/g) and the product was packaged in sterile pouches by heat sealing. Test samples were preheated and then PATP-treated at 105 °C at various pressures and pressure-holding times. Thermal inactivation of spores was studied at 121 °C using custom-fabricated aluminum tubes; this treatment served as a control. Application of PATP at 700 MPa and 105 °C inactivated B. stearothermophilus spores, suspended in egg matrix rapidly, (4 log reductions in 5 min) when compared to thermal treatment at 121 °C (1.5 log reduction in 15 min). Spore inactivation by PATP progressed rapidly (3 log reductions at 700 MPa and 105 °C) during pressure-hold for up to 100 s, but greater holding times (up to 5 min) had comparatively limited effect. When PATP was applied to spores in water suspension or egg patties, D values were not significantly different. While thermal inactivation of spores followed first-order kinetics, PATP inactivation exhibited nonlinear inactivation kinetics. Among the nonlinear models tested, the Weibull model best described PATP inactivation of B. stearothermophilus spores in the egg product.     

Formation of pyrazines and 2-acetyl-1-pyrroline by Bacillus cereus

The production of alkylpyrazines and 2-acetyl-1-pyrroline by different Bacillus cereus strains, which has been previously reported, was studied in detail. B. cereus ATCC 27522 produced the highest amounts of flavour compounds when grown as surface cultures on plate count agar. Pyrazine, methylpyrazine, 2,5-dimethylpyrazine, trimethylpyrazine and 3-ethyl-2,5-dimethylpyrazine were produced in low amounts. Since they were also detected in control flasks, an enzymatic formation was concluded to be unlikely. Only the production of 3-ethyl-2,5-dimethylpyrazine was in all cases significantly different from the control. Detailed precursor studies revealed that the production of 2-acetyl-1-pyrroline by B. cereus ATCC 27522 proceeds via acetylation of 1-pyrroline, a metabolic degradation product of proline and ornithine. Comparison of results obtained from dynamic headspace and simultaneous steam distillation – solvent extraction showed that the use of a non-thermal extraction method is essential to obtain reliable results on the biological formation of these Maillard flavour compounds.     

Toxin producing Bacillus cereus persist in ready-to-reheat spaghetti Bolognese mainly in vegetative state

The potential of Bacillus cereus to cause a diarrheal toxico-infection is related to its ability to perform de novo enterotoxin production in the small intestine. A prerequisite for this is presence of sufficient numbers of B. cereus that have survived gastro-intestinal passage. It is known that the percentage of survival is much smaller for vegetative cells in comparison to spores and it is therefore important to know the state in which B. cereus is ingested. The results of the current study performed on twelve B. cereus strains, comprising both diarrheal and emetic type, indicate that exposure via contaminated foods mainly concerns vegetative cells. Inoculated vegetative cells grew to high counts, with the growth dynamic depending on the storage temperature. At 28 °C growth to high counts resulted in spore formation, in general, after 1 day of storage. One strain was an exception, producing spores only after 16 days. At 12 °C obtained high counts did not result in spore formation for 11 of 12 tested strains after two weeks of storage. The highest counts and time to sporulation were different between strains, but no difference was observed on the group level of diarrheal and emetic strains. The spore counts were always lower than vegetative cell counts and occurred only when food was obviously sensory spoiled (visual and odor evaluation). Similar observations were made with food inoculated with B. cereus spores instead of vegetative cells. Although the prospect of consuming spores was found very weak, the numbers of vegetative B. cereus cells were high enough, without obvious sensory deviation, to survive in sufficient level to cause diarrheal toxico-infection.     

The survivability of Bacillus anthracis (Sterne strain) in processed liquid eggs

In this study, we investigated the survival and inactivation kinetics of a surrogate strain of Bacillus anthracis (Sterne strain) in whole egg (WE), egg white (EW), sugared egg yolk (YSU), and salted egg yolk (YSA) at low (−20, 0, and 5 °C), moderate (15, 20, 25, 30, 35, and 40 °C), and high storage temperatures (45, 50, 55, and 60 °C). Outgrowth of the spores was measured as lag phase duration (LPD). Replication of vegetative cells was measured in terms of growth rate (GR) and maximum population density (MPD). Spore inactivation was recorded as inactivation rate and percent reduction in viable count. In general, spore viability decreased at low and high temperatures and increased at moderate temperatures. At 0 and 5 °C, a 60–100% reduction in spore viability was seen within 2–3 weeks in WE and YSU, 0–30% in YSA, and 50–100% in EW. At −20 °C, however, no drop in spore titer was observed in YSU and EW but a 20% drop in titer was seen in YSA and 50% in WE within 2–3 weeks. At high temperatures, WE, EW, and YSA produced a 20–50% drop in the spore titer within 1–4 h whereas YSU showed 100% inactivation within 0.75 h. At moderate storage temperatures, as the temperature increased from 15 to 40 °C, LPD decreased from 13.5 to 0.75 h and MPD reached 0.27–2.2 × 10⁹ CFU/ml in YSU and WE, respectively. Markedly lower growth was observed in YSA (LPD = 24–270 h, MPD = 9 × 10⁵ CFU/ml) and spores were inactivated completely within 1–6 h in EW. The survivability and inactivation data of B. anthracis in liquid egg products reported in this investigation will be helpful in developing risk assessment models on food biosecurity.     

Preparation of eicosapentaenoic acid concentrates from sardine oil by Bacillus circulans lipase

An extracellular lipase derived from Bacillus circulans, isolated from marine macroalga, Turbinaraia conoides, was used to prepare n-3 polyunsaturated fatty acid (PUFA) concentrates from sardine oil triglycerides. The enzyme was purified 132-fold with specific activity of 386 LU/mg. The purified lipase was able to enrich sardine oil with 37.7 ± 1.98% 20:5n-3 and 5.11 ± 0.14% 18:3n-3 in the triglyceride fraction after 3 h of hydrolysis. Lower hydrophobic constants of n-3 fatty acids (18:3n-3_logP = 5.65; 20:5n-3_logP = 5.85, respectively) than n-6 (20:4n-6_logP = 6.16) resulted in higher hydrolytic resistance of the former toward lipase, leading to their enrichment in the triglyceride fraction. Lipase-catalysed hydrolysis of sardine oil for 3 h, followed by urea complexation, provided free fatty acids containing 51.3 ± 4.65% 20:5n-3. The purified methyl ester of 20:5n-3 (68.29 ± 2.15%) from the urea concentrate was attained by chromatography on argentated neutral alumina.     

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10² CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.