Incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia

The purpose of this study was to evaluate the incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia. The distribution of Bacillus species in raw milk, pasteurised milk and UHT milk were 47.5%, 27.5% and 25%, respectively. Seven Bacillus species, including Bacillus pumilus (10%), Bacillus subtilis (12.5%), Brevibacillus brevis (10%), Bacillus cereus (22.5%), Bacillus sphaericus (7.5%), Bacillus licheniformis (12.5%) and Bacillus sporothermodurans (25%) were identified in different milk samples. Bacillus cereus was predominant in raw and pasteurised milk. Although B. sporothermodurans was the predominant sporogenous micro‐organism in UHT milk, B. cereus, B. sphaericus and B. licheniformis were also present. This study showed that there is a high degree of diversity, both phenotypic and genotypic, among Bacillus isolates from Tunisian milk and the persistence of spoilage risk in UHT milk.     

Occurrence,heat and antibiotic resistance profile of Bacillus cereus isolated from raw cow and processed milk in Mezam Division,Cameroon

Bacillus cereus is the aetiologic agent of two distinct forms of food poisoning: the diarrhoeal and emetic syndromes. Little data exist on the prevalence of B. cereus in raw milk and milk products sold in Cameroonian towns. This study was aimed at investigating the occurrence, heat and antibiotic resistance of B. cereus isolated from raw milk and selected milk products in Mezam division, Cameroon. Bacillus cereus was isolated by inoculating samples onto mannitol‐egg yolk‐polymyxin B agar. Isolates were characterised morphologically and biochemically. The occurrence of B. cereus in raw milk (8.22%) was less than that in milk powder (13.33%). Bacillus cereus was not isolated from fermented milk. There was no significant difference (P < 0.05) between the B. cereus load in raw milk (2.6 × 10³ cfu/mL) and milk powder (3.0 × 10² cfu/mL). All the isolates showed haemolysin activity and were sensitive to tetracycline, gentamicin, chloramphenicol and nalidixic acid, but resistant to penicillin, ampicillin and trimethoprim/sulphamethoxazole. The detection of drug‐resistant, haemolysin‐positive isolates should serve as a warning for an impending health hazard following consumption of untreated milk. Heat resistance of isolates was assessed by determining the decimal reduction time; D‐value (time to inactivate 90% of the B. cereus spores); and the heat sensitivity, z (temperature increase leads to a tenfold reduction in the D‐value). The values for D₁₀₀ ranged from 0.5 to 3.5 min, and z‐values ranged from 10.0 to 32.6 °C. These results could be used in the dairy industry to evaluate the importance of heat treatment on B. cereus inactivation and calculation of process efficiency.     

Bacillus subtilis HJ18‐4 from Traditional Fermented Soybean Food Inhibits Bacillus cereus Growth and Toxin‐Related Genes

Bacillus subtilis HJ18‐4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad‐spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18‐4. Expression of B. cereus toxin–related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18‐4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18‐4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18‐4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus.     

The ability of Schisandra chinensis fruit to inhibit the growth of foodborne pathogenic bacteria and the viability and heat resistance of Bacillus cereus spores

The objective of this study was to investigate the influence of Schisandra chinensis fruit on the growth of spoilage and pathogenic bacteria and on the viability and heat resistance of Bacillus cereus spores. Schisandra chinensis fruit was extracted with one of three different solvents (50% ethanol, 100% ethanol and distilled water), and the extracts exhibited antimicrobial activity against all the bacteria tested. Particularly, the ethanol extracts of S. chinensis fruit had the strongest activity, in a concentration‐dependent manner. Fractionation of extracts by ion chromatography revealed that the antimicrobial activity of S. chinensis fruit is mainly due to organic acids such as citric acid and malic acid. Meanwhile, S. chinensis fruit extract (10%) significantly reduced the viability and heat resistance of B. cereus spores. Therefore, this study suggests that S. chinensis fruit extract has potential as a natural food preservative and/or sanitising agent for the reduction of spoilage and pathogenic contamination.     

Angiotensin converting enzyme‐inhibitory activity and antimicrobial effect of fermented camel milk (Camelus dromedarius)

This study aimed to determine the angiotensin converting enzyme‐inhibitory activity and antimicrobial effect of fermented camel milk. Samples were prepared either using Lactobacillus acidophilus and Streptococcus thermophilus or Lactobacillus helveticus and Str. thermophilus and labelled as S1 and S2, respectively. The IC₅₀ values of S1 and S2 samples ranged between 113–200 and 70–133 μg/mL, respectively. The antimicrobial effects of S1 and S2 samples against Bacillus cereus, Salmonella Typhimurium and Staphylococcus aureus were apparent after 12 h of incubation and continued until 15 days of storage, whereas unfermented camel milk exhibited no antimicrobial effects against any of the tested pathogens.     

Evaluation of in vitro probiotic potential of phytase‐producing bacterial strain as a new probiotic candidate

Sixty‐three phytase‐producing bacterial strains were isolated from natural sources, and their probiotic potential was evaluated. Of these, only fifteen strains were selected on the basis of confirmatory plate assay. Among these, five phytase‐producing strains exhibiting potent probiotic properties were identified as Bacillus cereus P1, Bacillus subtilis P6, B. subtilis P7, Pseudomonas aeruginosa P12 and P. aeruginosa P15 on the basis of morphological, biochemical and molecular characteristics. Maximum phytase activity (2.74 EU mL^?1) with potential probiotic properties, that is more than 70% survivability at pH 2.0, up to 2.0% bile salt tolerance, sporulation efficiency of more than 80% and survival in anaerobic condition (94.31%), was revealed by B. subtilis P6 as compared to the well‐established commercial probiotic strains Lactobacillus sporogenes and Lactobacillus casei. Thus, phytase‐producing B. subtilis P6 with promising probiotic features can be used in food and animal feed applications for betterment of mankind after further validation.     

Occurrence and antibiotic resistance of Escherichia coli,Staphylococcus aureus and Bacillus cereus in raw milk and dairy products in Turkey

In this survey, 150 samples of raw milk, white cheese and ice cream from three different dairy‐processing plants in Ankara were analysed to find out if they were contaminated with Escherichia coli, Staphylococcus aureus or Bacillus cereus. The highest contamination percentages were found in raw milk samples as follows: B. cereus (90%), E. coli (74%) and S. aureus (56%) followed by cheese (70% B. cereus, 60% E. coli, and 48% S. aureus) and ice cream (56% E. coli, 36% S. aureus and 20% B. cereus). The survey showed that 2% of cheese samples were contaminated with E. coli O157. It was also found that the numbers of S. aureus and E. coli in raw milk, cheese and ice cream samples exceeded the numbers permitted under the Turkish Food Codex (TFC). The number of B. cereus in raw milk, cheese and ice cream samples was lower than the limit given in the TFC standards. The study also showed that E. coli and S. aureus exhibit resistance to ampicillin, penicillin, tetracycline, erythromycin, gentamicin and trimethoprim/sulfamethoxazole. Escherichia coli isolates also showed resistance to chloramphenicol and ciprofloxacin but none of them exhibited resistance to cefotaxime. All S. aureus isolates were found to be susceptible to cefotaxime, chloramphenicol, and ciprofloxacin. Bacillus cereus isolates were found to be resistant to ampicillin, penicillin and trimethoprim/sulfamethoxazole and sensitive to cefotaxime, chloramphenicol, ciprofloxacin erythromycin, gentamicin and tetracycline.     

Prevalence and risk factors for Bacillus cereus in raw milk in Inner Mongolia,Northern China

The prevalence of Bacillus cereus in raw milk from dairy herds in Inner Mongolia and associated risk factors were determined. In total, 160 raw milk samples collected from 2014 to 2015 and examined by culture methods to isolate B. cereus. Fifty‐five samples (34.38%) were positive. The prevalence of B. cereus in raw milk was significantly (P < 0.01) lower in winter (15%) than in summer (50%). The highest prevalence of B. cereus in raw milk was seen in samples collected from small‐ to medium‐sized dairy herds in summer. The results of this study indicated that B. cereus could be a common pathogen in Inner Mongolia because of its high prevalence. It is essential for dairy farmers and producers to strengthen hygienic practices in summer to control B. cereus contamination in raw milk. These results will be useful in establishing proper management procedures to reduce the prevalence of B. cereus in raw milk.     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.     

Purification of two bacteriocins produced by Enterococcus faecalis DBFIQ E24 strain isolated from raw bovine milk

Enterococcus faecalis DBFIQ E24 was formerly characterised as a producer of antibacterial and antifungal substances. This strain is vancomycin sensitive, nonhaemolytic and gelatinase negative. SDS‐PAGE revealed the presence of an active band located between 1060 and 3500 Da. After the application of a three‐step purification procedure, two antimicrobial peptides were isolated. One of them is mostly active against Bacillus cereus with a molecular mass of 1364 Da, and the other one with a molecular mass of 1686 Da is mainly active against Escherichia coli. These results confirm the technological potential of E. faecalis DBFIQ E24 strain as a GRAS food biopreservative.     

Bacillus cereus in personal care products: risk to consumers

Bacillus cereus is ubiquitous in nature and thus occurs naturally in a wide range of raw materials and foodstuffs. B. cereus spores are resistant to desiccation and heat and able to survive dry storage and cooking. Vegetative cells produce several toxins which on ingestion in sufficient numbers can cause vomiting and/or diarrhoea depending on the toxins produced. Gastrointestinal disease is commonly associated with reheated or inadequately cooked foods. In addition to being a rare cause of several acute infections (e.g. pneumonia and septicaemia), B. cereus can also cause localized infection of post‐surgical or trauma wounds and is a rare but significant pathogen of the eye where it may result in severe endophthalmitis often leading to loss of vision. Key risk factors in such cases are trauma to the eye and retained contaminated intraocular foreign bodies. In addition, rare cases of B. cereus‐associated keratitis (inflammation of the cornea) have been linked to contact lens use. Bacillus cereus is therefore a microbial contaminant that could adversely affect product safety of cosmetic and facial toiletries and pose a threat to the user if other key risk factors are also present. The infective dose in the human eye is unknown, but as few as 100 cfu has been reported to initiate infection in a susceptible animal model. However, we are not aware of any reports in the literature of B. cereus infections in any body site linked with use of personal care products. Low levels of B. cereus spores may on occasion be present in near‐eye cosmetics, and these products have been used by consumers for many years. In addition, exposure to B. cereus is more likely to occur through other routes (e.g. dustborne contamination) due to its ubiquity and resistance properties of spores. The organism has been recovered from the eyes of healthy individuals. Therefore, although there may be a perceived hazard, the risk of severe eye infections as a consequence of exposure through contaminated near‐eye cosmetics is judged to be vanishingly small. It is unlikely that more stringent microbiological standards for near‐eye cosmetics will have any impact on the risk of severe eye infections caused by B. cereus, as these are not linked to use of personal care products.     

Membrane‐disruptive property of a novel antimicrobial peptide from anchovy (Engraulis japonicus) hydrolysate

A novel antimicrobial peptide named Pep39 has been isolated from anchovy hydrolysate using ÄKTA protein liquid chromatography and reverse‐phase high‐performance liquid chromatography. Its amino acid sequence was RLFRHAFKAVLRL with a molecular weight of 1626.92. Pep39 demonstrated antimicrobial ability against Escherichia coli ATCC 25922, Salmonella typhi ATCC 50013, Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 9372, with minimal inhibitory concentration (MIC) ranging from 4 to 32 μg mL^?1. No cytotoxicity to mouse red blood cells was observed when its concentration was lower than 30 μg mL^?1. Pep39 induced the influx of fluorescent probe 8‐Anilino‐1‐naphthalenesulfonic acid and the outflow of β‐galactosidase by increasing E. coli outer and inner membrane permeabilities, respectively. Flow cytometric analysis also demonstrated that Pep39 disrupted E. coli cell membrane. These results suggested that the antimicrobial mechanism of Pep39 involved cytoplasmic membrane damage.     

Purification and characterization of a peptide from Bacillus licheniformis showing dual antimicrobial and emulsifying activities

Bacillus licheniformis strain P40 produces a bacteriocin-like substance (BLS) that inhibits important pathogenic and food spoilage bacteria such as Listeria monocytogenes, Streptococcus spp., Bacillus cereus, and Erwinia carotovora. The antimicrobial peptide produced by B. licheniformis P40 was purified by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-100 and reversed phase chromatography on Source-RPC. The purification was about 100-fold with a yield of 0.3%. The molecular mass of the purified BLS about 800 Da, as determined by mass spectroscopy. The BLS was resistant for up to 100 °C and pH ranging 3–10, but lost its activity when treated with proteases and trichloroacetic acid. Reaction with ninhydrin produced the yellowish color instead the characteristic purple. Data from infrared spectroscopy also indicate the peptide is cyclic, resembling the lipopeptides surfactin and lichenisin. The BLS also showed emulsifying properties with several hydrophobic compounds, and dual antimicrobial and emulsifying activity in a meat model system. This antimicrobial peptide presents potential for use in as a food biopreservative or biodetergent.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Antimicrobial activity of Zataria multiflora Boiss. essential oil incorporated with whey protein based films on pathogenic and probiotic bacteria

The antimicrobial potential of whey protein isolate (WPI) edible films containing 1–4% (v/v) Zataria multiflora Boiss. essential oil (EO) on food‐borne pathogenic bacteria (Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Bacillus cereus) and probiotic bacteria (Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus casei subsp. casei) was evaluated. WPI films incorporated with 2% (v/v) of this EO inhibited the growth of all tested pathogenic bacteria and gram‐negative bacteria were more sensitive than gram‐positive bacteria. Incorporation of the EO at higher than 2% (v/v) showed significant antimicrobial effects (P < 0.05) for S. enteritidis and L. acidophilus. The growth of all probiotic lactic acid‐producing bacteria also inhibited when 2% of the EO was added. Comparison of an image processing‐based method with conventional method for measuring of inhibitory effects of edible films exhibited high correlations (R² ≥ 0.876) between the two methods. These results revealed that Z. multiflora Boiss. EO is a good antimicrobial additive for some food applications when included into WPI edible films.     

Antibacterial and antifungal activities of selected microalgae and cyanobacteria

In vitro activity of nine cyanobacterial and ten microalgal newly isolated or culture collection strains against eight significant food‐borne pathogens has been evaluated and compared. Water extracts and culture liquids of Gloeocapsa sp. and Synechocystis sp. demonstrated the widest spectrum of activity with minimal inhibitory concentration (MIC) ranging from 1.56 to 12.5 mg mL^?1. Culture liquid of Anabaena sp. had the highest activity (MIC = 0.39 mg mL^?1) but only to Gram‐positive bacteria. Ethanol extracts and fatty acids from all cyanobacteria and microalgae were active against Streptococcus pyogenes and/or Staphylococcus aureus. The fatty acids of Synechocystis sp. inhibited the growth of Bacillus cereus, Escherichia coli and Candida albicans (MIC values of 2.5–1.25 mg mL^?1, respectively). Exopolysaccharides (EPS) of Gloeocapsa sp. were the sample that exhibited activity against all test pathogens with lowest MIC values (0.125–1 mg mL^?1). High activity with a narrower range of susceptible targets demonstrated the exopolysaccharides of Synechocystis sp. and Rhodella reticulata. Antimicrobial activity was proven for phycobiliproteins isolated from Synechocystis sp., Arthrospira fusiformis, Porphyridium aerugineum and Porphyridium cruentum, respectively. In conclusion Gloeocapsa sp. and Synechocystis sp. and especially their exopolysaccharides showed the most promising potential against the examined food pathogens.     

Original article: Modelling the effects of food ingredients and pH on high‐pressure processing inactivation of Bacillus cereus spores: a laboratorial study

The combination of high pressure and heat on Bacillus cereus spores in food matrix was investigated with the purpose of achieving a predictive model of microbial inactivation. The high‐pressure processing (HPP) conditions were fixed at 540 MPa and 71 °C for 16.8 min, which were determined as the optimum conditions considering six‐log‐cycle reductions of B. cereus spores. The effects of soybean protein, sucrose, soybean oil and pH on the inactivation of B. cereus spores by HPP were evaluated, and a quadratic predictive model for the effects of food ingredients and pH on the reductions of B. cereus spores by HPP was built using response surface methodology. The experimental results showed that soybean protein, sucrose and pH significantly affected the reductions of B. cereus spores. The predictive model is significant, because the level of significance was P < 0.0001 and the calculated F‐value is much greater than the tabulated F‐value. The adequacy of the predictive model equation was verified effectively using the experimental test data that were not used in the development of the model.     

Preconditioning of the stainless steel surface affects the adhesion of Bacillus cereus spores

The adhesion of Bacillus cereus on stainless steel, with and without prior conditioning of the surface (water, skimmed milk, and whole milk) was evaluated. Inocula consisting of a pool of spores of four different B. cereus strains isolated from the dairy industry, and spores of B. cereus ATCC 14579 were used. The pool of B. cereus spores adhered in all conditions evaluated. Higher adhesion of B. cereus spores (4.93 log cfu cm⁻²) was observed when using whole milk as conditioning matrix. However, without prior conditioning, lower adhesion was observed (3.01 log cfu cm⁻²) when the pool of B. cereus spores was inoculated on whole milk, suggesting the interaction between milk fat and microorganism on the stainless steel. The pool of B. cereus spores showed higher adhesion to the surface, possibly due to its greater hydrophobicity (66%) when compared with the B. cereus ATCC 14579 spores (47%).