Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 10² CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 10² CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 10³ and 3.6 × 10³ CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.     

Phylogenetic and toxinogenic characteristics of Bacillus cereus group members isolated from spices and herbs

The foodborne pathogen Bacillus (B.) cereus is a common contaminant in spices and herbs. To further characterise B. cereus and its closely related group members present in spices and herbs, we analysed presumptive B. cereus strains isolated from six different condiments with view to B. cereus group species, phylogenetic affiliation and toxinogenic potential.Of a total of 59 isolates 44 were identified as B. cereus sensu stricto (s.s.), four as B. toyonensis-like, five as B. thuringiensis, one as B. weihenstephanensis, two as B. pseudomycoides/B. mycoides and three as undefined B. cereus group species. A maximum of three different species occurred simultaneously in the same spice sample. The isolates comprised 33 multilocus (ML) sequence types (STs), which can be assigned to three different phylogenetic groups. Except two B. pseudomycoides/B. mycoides strains, all isolates were able to produce enterotoxins and one strain the emetic toxin cereulide as detected by an immunoassay and LC-MS, respectively. The prevalence of toxin genes was 96.6% for nheA, 94.9% for hblD, 50.8% for cytK-2 and 1.7% for ces. The emetic strain was characterised by ST 869, which for the first time was assigned to an emetic B. cereus (s.s.) strain and is not part of the previously known two emetic MLST clusters.Our results demonstrate that not only B. cereus (s.s.) but also toxin producing B. thuringiensis, B. weihenstephanensis and B. toyonensis-like strains could be detected in condiments. For some isolates MLST revealed disagreements between phylogenetic relationship and the classification as B. weihenstephanensis and B. mycoides based on previously described species markers.     

Distribution and expression of the enterotoxin genes of Bacillus cereus in food products from Jiangxi Province,China

Bacillus cereus is a major foodborne pathogen that can cause a type of diarrhea. Diarrheal syndrome results from the ingestion of food products contaminated with enterotoxin-producing B. cereus. This study investigated the presence of four enterotoxins genes in 130 B. cereus isolated from various food products from Jiangxi, China. The expression levels of the enterotoxin genes in three B. cereus strains of different origins were subsequently analyzed. All of the B. cereus strains harbor at least 1 enterotoxin gene, whereas 34 strains harbor 2 enterotoxin genes, 44 strains harbor 3 enterotoxin genes, and 47 strains carry all of the four enterotoxin genes. The detection rates of the cytK, hblD, nheA, and entFM enterotoxin genes in all B. cereus isolates were 71%, 43%, 92%, and 99%, respectively. Moreover, the food matrix significantly affected the expression of these enterotoxin genes. The majority of the enterotoxin genes were also downregulated in four food matrices, indicating that the relative expression of certain enterotoxin genes, especially the entFM gene, was lower in a real food matrix than in laboratory broths. Hence, these data revealed that B. cereus is a serious health hazard and that the food matrix may influence the virulence expression of B. cereus. Our results provide better insights into the physiology of the microorganism grown in food products.     

Quantification and differentiation of Bacillus cereus group species in spices and herbs by real-time PCR

Spores of Bacillus (B.) cereus group species are frequent contaminants in foodstuffs including spices and herbs. However, the distribution of individual B. cereus group species is unknown as standard cultural methods are insufficient for differentiation. Real-time PCR is an alternative method to detect, differentiate and quantify B. cereus group species in food.In our study we applied a combination of previously described real-time PCR assays to detect and quantify the B. cereus group (excluding B. cytotoxicus) with simultaneous discrimination of B. pseudomycoides and cry1-positive B. thuringiensis as well as differentiation of B. weihenstephanensis from B. cereus group species with non-rhizoid colony morphology. For testing food matrices, which can also include PCR inhibiting substances, an internal amplification control was included. In total, five DNA extraction kits were tested on pure spore suspensions to select the one with the best recovery rate when coupled to real-time PCR. The Qiagen DNeasy Blood & Tissue Kit performed best with a limit of detection (LOD) of approximately 100 cfu/ml for spores of each B. cereus, B. weihenstephanensis, B. thuringiensis and B. pseudomycoides strain. However, applied to allspice, paprika, pepper and oregano artificially contaminated with B. cereus spores the LOD was ≥10⁵ cfu/g. In contrast, using the open-formula cetyltrimethylammonium bromide (CTAB) method LODs of 10² to 10³ cfu/g were achieved for paprika, pepper and oregano. For allspice, the LOD was 10⁶ cfu/g.Our quantitative multiplex real-time PCR coupled to DNA extraction by the CTAB method provides a sensitive culture independent technique with the potential to quantitatively detect and differentiate B. cereus group species in several spices and herbs.     

Growth and inhibition by spices of growth from spores of enterotoxigenic Bacillus cereus in cooked rice

Bacillus cereus is a pathogenic spore-forming bacterium implicated in cases of diarrheal and emetic type of foodborne illness. We previously found that enterotoxigenic B. cereus is widely present in retail spices. Here we analyzed the spore heat resistance of nine diarrheal strains isolated from spices. Seven had D_95°C values ranging from 0.64 to 3.53 min while two emetic strains had D_95°C values of 7.04 min and 6.64 min. The ability of selected strains to grow in cooked rice at temperatures 20 °C, 17 °C and 12 °C was determined as well as their toxin expression capability. After 48 h, B. cereus levels of 1.26 × 10⁷ and 3.8 × 10⁷ CFU/g were obtained in cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 10⁴ CFU/g even after 48 h of incubation. The increase in the aerobic, mesophilic bacterial population (APC) and B. cereus population naturally present in 0.1% crushed pepper added to cooked rice was determined over a period of 48 h at 20 °C and 17 °C. Levels of B. cereus in pepper/rice samples reached a maximum of 1600 MPN/g at 20 °C while the APC was 2.4 × 10⁸/g at this temperature. When thyme, containing the same initial natural level of B. cereus, was added to rice in place of pepper, more than a five-fold greater level of B. cereus was detected at 20 °C. Since thyme contained initial APC of 2.5 log₁₀ less than pepper we conclude that the high APC functions in a competitive-exclusion manner, minimizing the growth of B. cereus and potentially other agents of foodborne illness. This is particularly relevant in instances of temperature abuse of foods and may explain the low incidence of foodborne illness due to B. cereus despite its widespread presence shown in previous surveys of market spices.     

Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 10² CFU/mL, as low as 7.5 × 10⁰ CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 10¹ CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 10³ CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples.     

Extracellular enzyme production and enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains isolated from cheese in Turkey

The aim of the present study was to investigate the biochemical characteristics, extracellular enzyme production and enterotoxigenic genes contents of 6 Bacillus cereus and 22 Bacillus thuringiensis strains, isolated from 100 cheese samples in Turkey. Crystal morphologies of B. thuringiensis strains were found either spherical (n = 12) or spherical and irregular-shaped (n = 10) by phase contrast microscopy. B. cereus and B. thuringiensis strains were found to produce extracellular enzymes, respectively: gelatinase (83% and 91%), DNase (83% and 77%), lecithinase (83% and 95%), protease on skim milk agar (100% and 100%), protease on milk agar (100% and 91%), protease on casein agar (83% and 77%), xylanase (100% and 45%), and cellulase (0% and 41%), and amylase (83% and 27%). All of the strains, except for Bt-D1, hydrolyzed Tween 20 (96%), but not Tween 80 or tributyrin. Pectinolytic activity was obtained to be the least frequent (4%). PCR analysis showed that all strains contained nheA, nheB, nheC and hblD genes. The hblA and hblC genes were present in 2 and 4 of B. thuringiensis strains, respectively. The bceT gene was detected in 1 B. cereus and 9 B. thuringiensis strains. The entFM gene was detected more frequently in B. thuringiensis (82%) than in B. cereus strains (50%). To our knowledge, this is the first report about the isolation and identification of enterotoxigenic B. cereus and B. thuringiensis strains from cheese samples in Turkey.     

Investigation of the diversity and safety of the predominant Bacillus pumilus sensu lato and other Bacillus species involved in the alkaline fermentation of cassava leaves for the production of Ntoba Mbodi

The objective of the study was to investigate the identity, diversity, and safety of the Bacillus population involved in the fermentation of cassava (Manihot esculenta Crantz) leaves for the production of Ntoba Mbodi, a Congolese food. Ninety bacteria were identified by phenotyping and genotyping using ITS-PCR, rep-PCR, and sequencing of the 16S rRNA, gyrA, gyrB and rpoB genes. Moreover, the isolates were screened for the presence of genes coding for haemolytic (HblC, HblD) and non-haemolytic enterotoxins (NheA, NheB and NheC), cytotoxin K (CytK) and emetic toxin (EM1) as well as their ability to produce haemolysin.The investigations revealed the predominance (72.2%) of species of the Bacillus pumilus group i.e. B. safensis (48), B. pumilus (7), and B. pumilus sensu lato (10). Other species of Bacillus including B. cereus sensu lato (11), B. megaterium (4), B. subtilis (4), B. amyloliquefaciens (2), B. siamensis (2), B. licheniformis (1) and Lysinibacillus louembei (1) were also identified. Haemolytic, non-haemolytic and cytokin toxin genes were detected in the B. cereus strains which were also able to produce haemolysin. The emetic toxin gene was not detected in any isolates. The toxin genes screened were not detected in any of the non B. cereus species.     

Antimicrobial herb and spice compounds in food

Herbs and spices containing essential oils (EOs) in the range of 0.05–0.1% have demonstrated activity against pathogens, such as Salmonella typhimurium, Escherichia coli O157:H7, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus, in food systems. Application of herbs, spices and EOs with antimicrobial effects comparable to synthetic additives is still remote for three major reasons: limited data about their effects in food, strong odor, and high cost. Combinations of techniques have been successfully applied in several in-food and in vitro experiments. This paper aims to review recent in-food applications of EOs and plant-origin natural antimicrobials and recent techniques for screening such compounds.     

Culture-independent quantification of pathogenic bacteria in spices and herbs using real-time polymerase chain reaction

A culture-independent method was developed for quantification of pathogenic bacteria in spices and herbs. The method is based on DNA extraction using cetyltrimethylammonium bromide (CTAB) and on real-time polymerase chain reaction (PCR). When evaluated with spices (black pepper, paprika) and herbs (oregano, parsley) artificially contaminated with Staphylococcus aureus, Salmonella enterica or Escherichia coli, the method demonstrated quantitative response with linear calibration lines and quantification limits of 10²–10⁴ CFU/g. The developed method is suitable for rapid microbiological analysis of spices and herbs, taking 8–9 h.     

In vitro antimicrobial activity of less-utilized spice and herb extracts against selected food-borne bacteria

In this study we compared the antimicrobial activities of extracts from four under-utilized spices and herbs including goraka (Garcinia quaesita), galangal (Alpinia galanga), lemon iron bark (Eucalyptus staigerana) and mountain pepper (Tasmannia lanceolata) to the three common spices and herbs pepper (Piper nigrum), rosemary (Rosmarinus officinalis), and oregano (Oreganum vulgare). Different extraction solvents were used (water, ethanol and hexane) and extracts were tested against four food-borne bacteria (Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes and Staphylococcus aureus) using agar disc diffusion and broth dilution assays. Solvent type greatly influenced the antimicrobial activity of the spice and herb extracts except for those of P. nigrum, which had little or no activity. In general the spice and herb extracts with antimicrobial activity were more effective against Gram-positive than Gram-negative bacteria. Extracts from the under-utilized herbs and spices had significant activity. In particular, A. galanga hexane and ethanol extracts and E. staigerana ethanol and water extracts had strong antimicrobial activity against S. aureus and/or L. monocytogenes. Interestingly the minimal inhibitory concentrations determined using the broth dilution method and the diameter of inhibition zones using the disc diffusion assay were not strongly correlated (r² ranged from 0.10 to 0.70) in most extracts, suggesting that choosing just one method for antimicrobial testing may lead to indefinite conclusions. The total phenolic content of two extracts from each spice and herb was assayed to establish any relationship between antimicrobial activity and phenolic compound levels, however this was found to poorly correlated (r² < 0.30). This study has demonstrated that simple extracts of novel under-utilized herbs and spices have potential antimicrobial activity against food-borne bacterial species. Further it is indicated that the antimicrobial activity in some herbs and spices may be due to the presence of substances other than phenolic compounds.     

Short communication: Fate of major foodborne pathogens and Bacillus cereus spores in sterilized and non-sterilized Korean turbid rice wine (Makgeolli)

The objective of this study was to examine the fate of foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus) and B. cereus spores in Korean turbid rice wine (Makgeolli). Samples of sterilized and non-sterilized turbid rice wine were inoculated with each of the vegetative bacteria or B. cereus spores at 3–4 log CFU/ml. The samples were stored at 5 °C or 22 °C, and bacterial survival was monitored over 28 days. Despite the harsh environment (alcohol content: 6–7% and pH: 3.43–3.98), long-term survival of pathogens was observed. Survival time was different depending on the type of beverage (pathogens survived longer in sterilized wine than in non-sterilized wine), cellular state (spores survived longer than vegetative cells), species (B. cereus survived longer than other species), and storage temperature (pathogens survived longer at 5 °C then at 22 °C). The number of B. cereus spores remained constant at both temperatures. The vegetative B. cereus population declined rapidly within 1 day, but then remained steady for up to 28 days (1.20–1.55 log CFU/ml in sterile wine). These results indicate that B. cereus formed spores that survived for a long time; therefore, it is possible that B. cereus may exist as spores in turbid rice wine. E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus survived for up to 28, 14, 14, and 14 days, respectively, in sterilized wine at 5 °C. Thus, the health implications of the long-term survival of pathogens in alcoholic beverages should be carefully considered. The results provide new information that may be useful in predicting the potential microbiological hazards associated with turbid rice wine.     

In vitro assessment of synthetic phenolic antioxidants for inhibition of foodborne Staphylococcus aureus,Bacillus cereus and Pseudomonas fluorescens

Minimal inhibitory concentrations (MICs) of seven synthetic phenolic compounds, five commonly used as antioxidants (TBHQ, BHA, BHT, propyl gallate and octyl gallate) and two as antimicrobials (propyl 4-hydroxybenzoate and n-heptyl 4-hydroxybenzoate) were assessed against several strains of two Gram-positive (Staphylococcus aureus and Bacillus cereus) and one Gram-negative (Pseudomonas fluorescens) bacteria, by using a standardized microdilution assay (ISO 20776-1, 2006). Octyl gallate was the most effective compound against the three genera/species of bacteria considered simultaneously (with the exception of four strains of B. cereus, which were resistant for this compound) with MIC values (≤100 μg/ml) lower than the concentrations usually used as antioxidants. TBHQ and n-heptyl 4-hydroxybenzoate were also effective in the control of S. aureus at very low concentrations (MIC of 3.1 μg/ml and 12.5 μg/ml, respectively). Propyl 4-hydroxybenzoate was the most inhibitory phenolic compound against all strains of B. cereus and both tested parabens (propyl- and heptyl-) were not effective for P. fluorescens (MIC > 1600 μg/ml). B. cereus was the bacterial genera that showed more intra-species variation, distinguishing two clearly groups of sensitivity among the strains to octyl gallate and n-heptyl 4-hydroxybenzoate (“sensitive” with mean MICs of 42.8 and 4.2 μg/ml, respectively; and “resistant” with MICs >1600 and >800 μg/ml, respectively). According to all that, octyl gallate would be an interesting phenolic compound for the food industry, not only because of its recognized antioxidant properties but also because of their effectivity as antimicrobial against S. aureus, B. cereus and P. fluorescens.     

Assessment of microbiological quality and safety of marinated pork products from German retail during shelf life

Foodborne diseases are of major concern for public health. Here we assess the microbiological quality and safety of marinated pork steaks (n = 300) and marinades (n = 30) which were used for the production of marinated steaks by analyzing quantitative microbiological parameters and foodborne pathogens. Salmonella spp. and Listeria monocytogenes were isolated from about 2%, Staphylococcus aureus from 8% and Bacillus cereus from 21% of the steaks. One steak was MRSA-positive and one contained EHEC/STEC. B. cereus was the only pathogen detected in the marinades. Similar toxin patterns of B. cereus strains from meat and marinades suggested that a contamination of meat with B. cereus occurred via marinades.     

The microbiological quality of commercial herb and spice preparations used in the formulation of a chicken supreme ready meal and microbial survival following a simulated industrial heating process

The microbiological status of thirty herb and spice preparations used in the manufacture of ready meals was determined. The effect of a simulated manufacturing process with subsequent cold storage was evaluated on spices having highest microbial loads either suspended in water, or added to a ready meal. Total aerobic mesophilic bacteria count indicated that 20% of the spices had >6 log CFU/g. Spore-forming bacteria and thermophiles (2–6 log CFU/g) were detected in 80% of samples. Pseudomonas spp. and Enterobacteriaceae (2–6 log CFU/g) were detected in 33% or 23% of spices, respectively. Molds were detected in 50% of samples (1–3 log CFU/g), while yeasts were detected in two samples only. Bacillus cereus was detected only in samples of marjoram. The simulated manufacturing treatment with subsequent cold storage indicated a degree of bacterial survival with a possible protective effect of the food matrix. Overall, the heat processing steps applied during manufacture of chilled ready meals may not always be sufficient to eliminate the indigenous microflora especially in spices of poor microbiological quality.     

Isolation, identification and monitoring of contaminant bacteria in Iranian Kefir type drink by 16S rDNA sequencing

Iranian Kefir type drink (IKTD) is a highly consumed, traditional Iranian, fermented milk product. To improve monitoring procedures for food safety 32 industrial Kefir type drinks from 4 brands and 8 different production dates as well as 32 samples from pasteurized milk of the same Kefir manufacturers and air of the production sites were analyzed for contaminations. 16S rDNA extraction from Kefir samples as well as 16S rDNA obtained from samples incubated on Columbia agar were analyzed using PCR/DGGE, cloning, sequencing and phylogenetic classification. Already DGGE analysis indicated contaminations including Bacillus strains. Subsequently analysis of cultured clones indicated contaminations with Bacillus sp. including Bacillus cereus, Bacillus thuringiensis and Paenibacillus sp. in 9 (28%) from all analyzed samples. Also 38% of pasteurized milk samples were contaminated with B. cereus. The average count of B. cereus was 74 ± 19 cfu/ml. B. cereus and B. thuringiensis were found as contaminant bacteria in the air of the all manufacturing sites. These results suggest that milk is one of the most important sources of contamination with Bacillus sp., especially B. cereus for Kefir products in Iran. But bacterial contamination in Kefir samples might also originate from the air of the production sites. 16S rDNA analysis accelerates monitoring strategies.     

Mycological quality and mycotoxin contamination of Sri Lankan peppers (Piper nigrum L.) and subsequent exposure assessment

The aim of the study was to characterize the toxigenic moulds and to screen different mycotoxins in peppers (Piper nigrum L.) of Sri Lankan origin. Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Penicillium spp. were found to be the most dominant fungi. Characterization of the moulds was carried out in A. flavus and parasiticus agar (AFPA) and malt extract agar (MEA) in 77 black pepper (BP) and 11 white pepper (WP) samples. In total, 73% of the BP and 64% of the WP samples were contaminated with A. flavus and/or A. parasiticus (AfAp). A BP sample with water activity (a_w) 0.70 recorded the highest count of AfAp (4.3*10⁴ CFU/g). Moreover, 75% of the BP samples exceeded the safe a_w limit (0.65) set by the European Spice Association (ESA). The frequency of occurrence of A. niger in BP was 62% with counts up to 1.3*10³ CFU/g. Penicillium spp. were found in 61% and 55% of the BP and WP samples, respectively. In BP 94% of the samples had a Penicillium contamination below 10³ CFU/g. Other Aspergillus spp, found in peppers included, Aspergillus terrus, Aspergillus tamarii, Aspergillus candidus, Aspergillus penicilloides, Aspergillus sydowii and Aspergillus fumigatus. Mould counts in BP (10²–10⁴ CFU/g) were significantly higher than that of WP (<10² CFU/g). Apart from the occurrence of “classical mycotoxins” of spices, aflatoxins (<LOQ-18 μg/kg) and ochratoxin A (<LOQ-79 μg/kg), other toxins including fumonisin B1 (<LOQ-135 μg/kg), sterigmatocystin (<LOQ-49 μg/kg) and citrinin (<LOQ-112 μg/kg) were detected in peppers. In total, 63% of the BP samples were contaminated with at least one mycotoxin. Mycotoxin contamination in WP was significantly less compared to BP. The exposure to aflatoxins and ochratoxin A by consuming pepper remains harmless considering the existing pepper dietary intake data of the Sri Lankan population.     

Potential of high-throughput sequencing for broad-range detection of pathogenic bacteria in spices and herbs

A broad-range culture-independent method was developed and evaluated regarding its sensitivity of detection of pathogenic bacteria in spices and herbs, with focus on paprika powder. The method involved DNA extraction using cetyltrimethylammonium bromide (CTAB), 16S rDNA amplification using universal bacterial polymerase chain reaction, and high-throughput sequencing on Illumina MiSeq platform. The sensitivity of the method was evaluated with series of model samples contaminated at different levels with Salmonella enterica and Escherichia coli (as representatives of Gram-negative bacteria) and Staphylococcus aureus (as a representative of Gram-positive bacteria). For spices (paprika, black pepper), the method had a screening-level sensitivity with limits of detection in the range of 10⁴–10⁵ CFU/g, and a semi-quantitative response. Low sensitivity (LOD ≥10⁷ CFU/g) was observed with herbs (oregano, parsley). The developed method demonstrated a good potential for microbiological screening of spices, with a prospect of further improvement of sensitivity based on progress in high-throughput sequencing technology.     

Antimicrobial peptides derived from hen egg lysozyme with inhibitory effect against Bacillus species

In food industry, Bacillus species are encountered in deteriorating many food products thus shortening their shelf-life. Moreover, Bacillus cereus and the subtilis group (Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus) have been recognized as food poisoning agents. Lysozyme peptides preparation (LzP) is a commercially available as a natural food preservative. Although, LzP derived from lysozyme yet it showed only 11% of the lysozyme lytic activity. LzP at a concentration of 100 μg ml⁻¹ completely inhibited B. subtilis, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. coagulans, B. amyloliquefaciens, B. polymexa and B. macerans. However, B. cereus and B. stearothermophilus showed a slightly higher resistance. Interestingly, LzP at concentration ⩾10 μg ml⁻¹ showed inhibitory effect on both vegetative and spore forms of B. subtilis. Moreover, LzP was stable at 95 °C for 30 min and at different pH values (4.5–7). In conclusion, LzP may be useful to control growth of Bacillus spoilage organisms.     

Antimicrobial activities of spices and herbs against Salmonella Oranienburg

The detection of Salmonella spp. within dried spices and herbs is challenging due to their potent antimicrobial activity. In the present study nine condiments were investigated whereof oregano and cinnamon led to a complete inhibition of Salmonella Oranienburg at a 1:10 dilution in buffered peptone water, while towards allspice and thyme an adaptation was apparent. At a next step, a tenacity study was set up where the survival of S. Oranienburg in dry condiment samples was followed qualitatively and quantitatively during storage at 25 °C for 365 days. Generally, a higher susceptibility of S. Oranienburg to spices than herbs was determined. Furthermore, to the best of the author’s knowledge, this is the first study presenting a significant antimicrobial effect of paprika/chilli against Salmonella. Nevertheless, S. Oranienburg was able to persist during storage in several condiment samples with a low reduction of log₁₀ <1.5 colony-forming units g⁻¹. Thus, pointing to the outstanding ability of S. Oranienburg to survive dry storage conditions even spiked to condiments, known for their high antimicrobial activity.