Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology

Spores of Bacillus sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. sporothermodurans spores, only d-glucose, l-alanine, and inosine were considered as strong independent germinants. Both inosine and l-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (l-alanine, d-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. sporothermodurans spores was obtained after incubation of spore for 60 min at 35 °C in presence of 9 and 60 mM of d-glucose and l-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors.     

Origin of bacterial spores contaminating foods

Bacterial spores (= endospores) are common contaminants in foods. Sources of contamination in the food chain may include soil, faeces, animal feeds and food ingredients and processing chain themselves. Sporulation may occur in very diverse environments. The environment of sporulation has a strong influence on spore properties relevant for food quality and safety.     

A mixed-species microarray for identification of food spoilage bacilli

Failure of food preservation is frequently caused by thermostable spores of members of the Bacillaceae family, which show a wide spectrum of resistance to cleaning and preservation treatments. We constructed and validated a mixed-species genotyping array for 6 Bacillus species, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus sporothermodurans, Bacillus cereus and Bacillus coagulans, and 4 Geobacillus species, including Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus toebii and Geobacillus sp., in order to track food spoilage isolates from ingredient to product. The discriminating power of the array was evaluated with sets of 42 reference and 20 test strains. Bacterial isolates contain a within-species-conserved core genome comprising 68-88% of the entire genome and a non-conserved accessory genome comprising 7-22%. The majority of the core genome markers do not hybridise between species, thus they allow for efficient discrimination at the species level. The accessory genome array markers provide high-resolution discrimination at the level of individual isolates from a single species. In conclusion, the reported mixed-species microarray contains discriminating markers that allow rapid and cost-effective typing of Bacillus food spoilage bacteria in a wide variety of food products.     

Modeling germination of fungal spores at constant and fluctuating temperature conditions

The germination of Penicillium expansum and Aspergillus niger spores was monitored microscopically on malt extract agar at isothermal conditions ranging from 0 to 33 °C and 5 to 41.5 °C, respectively. The obtained germination data, expressed as percentage of germination (% P) versus time, were fitted to the modified Gompertz equation for the estimation of the germination kinetic parameters (lag time, λ_g, and germination rate, μ_g), which were further modeled as a function of temperature via the use of Cardinal Models with Inflection (CMI). The effect of temperature on these parameters was similar with that previously reported for mycelium growth kinetics of the tested isolates. The germination of spores was also studied at various dynamic time-temperature conditions including single or sequential temperature shifts. The germination of spores at fluctuating temperatures was predicted using the modified Gompertz equation in conjunction with the CMI models for λ_g and μ_g and based on the assumptions that i) a temperature shift does not result in any additional λ_g and, thus, the total lag can be calculated by adding relative parts of the lag time, and ii) after a temperature shift the germination rate μ_g adapts instantaneously to the new temperature. The comparison between predicted and observed data showed that the germination of spores is strongly affected by the extent of the temperature shift, the percentage of germinated spores at the time of the shift and the fungal species. Apart from the scientific interest in understanding the dynamics of spores' germination, the models developed in this study can be used as tools in effective quality management systems for fungi control in foods.     

Optimization of pressure-induced germination of Bacillus sporothermodurans spores in water and milk

Bacillus sporothermodurans produces highly resistant endospores that can survive ultra-high-temperature treatment in milk. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk sterility. In this work, the rate of spore germination of B. sporothermodurans LTIS27 was measured in distilled water after high-pressure treatments with varying pressure (50–600 MPa), treatment temperature (20–50 °C), pressure-holding time (5–30 min) and post-pressurization incubation time (30–120 min) at 37 °C or 4 °C. The results showed that pressure-induced germination was maximal (62%) after a treatment at 200 MPa and 20 °C and increased with pressure-holding time and post-pressurization incubation time. Treatment temperature had no significant effect on germination. A central composite experimental design with three factors (pressure, pressure-holding time, and post-pressurization incubation time) using response surface methodology was used to optimize the germination rate in distilled water and in skim milk. No factor interaction was observed. Germination was induced at lower pressure and was faster in milk than in distilled water, but complete germination was not reached. The optimum germination obtained with experimental data was 5.0 log cfu/mL in distilled water and 5.2 log cfu/mL in milk from 5.7 log cfu/mL of spores initially present in the suspension. This study shows the potential of using high hydrostatic pressure to induce the germination of B. sporothermodurans spores in milk before a heat treatment.     

Quantification of spore resistance for assessment and optimization of heating processes: A never-ending story

The assessment and optimization of food heating processes require knowledge of the thermal resistance of target spores. Although the concept of spore resistance may seem simple, the establishment of a reliable quantification system for characterizing the heat resistance of spores has proven far more complex than imagined by early researchers. This paper points out the main difficulties encountered by reviewing the historical works on the subject.     

Strategy to inactivate Clostridium perfringens spores in meat products

The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100–200 MPa, 7 min) and elevated temperature (80 °C, 10 min); spore germination at high temperatures (55, 60 or 65 °C); and inactivation of germinated spores with elevated temperatures (80 and 90 °C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 °C, 10 min). Low pressures (100–200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 °C, 10 min), and germinated at temperatures lethal for vegetative cells (≥55 °C) when incubated for 60 min with a mixture of l-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (∼4 decimal reduction) in meat by elevated temperatures (80–90 °C for 20 min) required a long germination period (55 °C for 60 min). However, similar inactivation level was reached with shorter germination period (55 °C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 °C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 °C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 °C in about 20 min and further incubation at 55 °C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 °C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens.     

Influence of the sporulation temperature on the impact of the nutrients inosine and l-alanine on Bacillus cereus spore germination

The effect of sporulation temperature on Bacillus cereus spore germination triggered by the nutrient germinants inosine and l-alanine was investigated. The germination (expressed as % A(600nm) fall) of heat-activated spores of B. cereus strain ATCC14579 produced at 20 and 37 degrees C, and of psychrotrophic strains LM9 and D15 produced at 15 and 37 degrees C was followed in a germination buffer containing inosine at concentrations between 0.01 and 10mmoll(-1), or l-alanine between 1 and 100mmoll(-1). Spores wet-heat resistance at 90 degrees C was also determined. Spores of the three strains produced at the lowest temperatures generally showed a higher germination capacity in response to both inosine and in l-alanine than those produced at 37 degrees C, and were also more susceptible to heat. Low sporulation temperature is confirmed as a detrimental factor to B. cereus spore wet-heat resistance, and conversely gave spores with a sensitivity to the nutrient germinants inosine and l-alanine higher than that of spores formed close to the maximal sporulation temperature.     

Thermophilic bacilli and their importance in dairy processing

The thermophilic bacilli, such as Anoxybacillus flavithermus and Geobacillus spp., are an important group of contaminants in the dairy industry. Although these bacilli are generally not pathogenic, their presence in dairy products is an indicator of poor hygiene and high numbers are unacceptable to customers. In addition, their growth may result in milk product defects caused by the production of acids or enzymes, potentially leading to off-flavours. Dairy thermophiles are usually selected for by the conditions during dairy manufacture. These bacteria are able to grow in sections of dairy manufacturing plants where temperatures reach 40-65 °C. Furthermore, because they are spore formers, they are difficult to eliminate. In addition, they exhibit a wide temperature growth range, exhibit a fast growth rate (generation time of approximately 15-20 min) and tend to readily form biofilms. Many strategies have been tested to remove, prevent and/or delay the formation of thermophilic bacilli biofilms in dairy manufacture, but with limited success. This is, in part, because little is known about the structure and composition of thermophilic bacilli biofilms in general and, more specifically, in milk processing environments. Therefore, new cleaning regimes often do not target the problem optimally. A greater understanding of the structure of thermophilic biofilms within the context of the milk processing environment and their link with spore formation is needed to develop better control measures. This review discusses the characteristics and food spoilage potential, enumeration and identification methods for the thermophilic bacilli, as well as their importance to dairy manufacture, with an emphasis on biofilm development and spore formation.     

Syngas fermentation in a 100-L pilot scale fermentor: Design and process considerations

Fermentation of syngas offers several advantages compared to chemical catalysts such as higher specificity of biocatalysts, lower energy costs, and higher carbon efficiency. Scale-up of syngas fermentation from a bench scale to a pilot scale fermentor is a critical step leading to commercialization. The primary objective of this research was to install and commission a pilot scale fermentor, and subsequently scale-up the Clostridium strain P11 fermentation from a 7.5-L fermentor to a pilot scale 100-L fermentor. Initial preparation and fermentations were conducted in strictly anaerobic conditions. The fermentation system was maintained in a batch mode with continuous syngas supply. The effect of anaerobic fermentation in a pilot scale fermentor was evaluated. In addition, the impact of improving the syngas mass transfer coefficient on the utilization and product formation was studied. Results indicate a six fold improvement in ethanol concentration compared to serum bottle fermentation, and formation of other compounds such as isopropyl alcohol, acetic acid and butanol, which are of commercial importance.     

Inactivation strategy for Clostridium perfringens spores adhered to food contact surfaces

The contamination of enterotoxigenic Clostridium perfringens spores on food contact surfaces posses a serious concern to food industry due to their high resistance to various preservation methods typically applied to control foodborne pathogens. In this study, we aimed to develop an strategy to inactivate C. perfringens spores on stainless steel (SS) surfaces by inducing spore germination and killing of germinated spores with commonly used disinfectants. The mixture of l-Asparagine and KCl (AK) induced maximum spore germination for all tested C. perfringens food poisoning (FP) and non-foodborne (NFB) isolates. Incubation temperature had a major impact on C. perfringens spore germination, with 40 °C induced higher germination than room temperature (RT) (20 ± 2 °C). In spore suspension, the implementation of AK-induced germination step prior to treatment with disinfectants significantly (p < 0.05) enhanced the inactivation of spores of FP strain SM101. However, under similar conditions, no significant spore inactivation was observed with NFB strain NB16. Interestingly, while the spores of FP isolates were able to germinate with AK upon their adhesion to SS chips, no significant germination was observed with spores of NFB isolates. Consequently, the incorporation of AK-induced germination step prior to decontamination of SS chips with disinfectants significantly (p < 0.05) inactivated the spores of FP isolates. Collectively, our current results showed that triggering spore germination considerably increased sporicidal activity of the commonly used disinfectants against C. perfringens FP spores attached to SS chips. These findings should help in developing an effective strategy to inactivate C. perfringens spores adhered to food contact surfaces.     

Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States

To determine the microbial ecology of pasteurized milk within the United States, 2% fat pasteurized fluid milk samples were obtained from 18 dairy plants from 5 geographical areas representing the Northeast, Southeast, South, Midwest, and West. Of the 589 bacterial isolates identified using DNA sequence-based subtyping methods, 346 belonged to genera characterized as gram-positive endospore-forming bacteria (i.e., Bacillus and Paenibacillus). Of the 346 gram-positive endospore-forming bacteria isolated in the present study, 240 were classified into 45 allelic types identical to those previously identified from samples obtained in New York State, indicating the widespread presence of these microbes in fluid milk production and processing systems in the United States. More than 84% of the gram-positive spore-forming isolates characterized at d 1, 7, and 10 were of the genus Bacillus, whereas more than 92% of isolates characterized at d 17 of shelf life were of the genus Paenibacillus, indicating that the predominant gram-positive spoilage genera shifts from Bacillus spp. to Paenibacillus spp. during refrigerated storage.     

Viability and surface properties of spores subjected to a cleaning-in-place procedure: Consequences on their ability to contaminate surfaces of equipment

This study was designed to evaluate how conditions encountered by spores during cleaning-in-place (CIP) procedures affected their surface properties, their viability and ability to contaminate materials. Spores from five Bacillus cereus strains were treated with NaOH at high temperature. Results revealed that high temperatures (exceeding 60 °C) and NaOH concentrations (over 0.5%) were required to significantly decrease spore viability (3–5 log decrease). In these conditions, modifications were also clearly observed by microscopy to various surface structures of spores (appendages, exosporium, and especially to the hair-like nap) but also to their coat. Therefore, the ability of culturable spores to adhere decreased for the majority of strains tested. We then demonstrated that spores in suspension in NaOH could adhere to surfaces of a CIP rig and that the contamination level was controlled by flow pattern. Consequently, re-adhesion along the processing line might occur during CIP procedures and this phenomenon must be taken into account when defining cleaning strategies.     

Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product

Control of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., is economically important to the dairy industry. These microbes form endospores that can survive high-temperature, short-time pasteurization; hence, their presence in raw milk represents a major potential cause of milk spoilage. A previously developed culture-dependent selection strategy and an rpoB sequence-based subtyping method were applied to bacterial isolates obtained from environmental samples collected on a New York State dairy farm. A total of 54 different rpoB allelic types putatively identified as Bacillus (75% of isolates), Paenibacillus (24%), and Sporosarcina spp. (1%) were identified among 93 isolates. Assembly of a broader data set, including 93 dairy farm isolates, 57 raw milk tank truck isolates, 138 dairy plant storage silo isolates, and 336 pasteurized milk isolates, identified a total of 154 rpoB allelic types, representing an extensive diversity of Bacillus and Paenibacillus spp. Our molecular subtype data clearly showed that certain endospore-forming bacterial subtypes are present in the dairy farm environment as well as in the processing plant. The potential for entry of these ubiquitous heat-resistant spoilage organisms into milk production and processing systems, from the dairy farm to the processing plant, represents a considerable challenge that will require a comprehensive farm-to-table approach to fluid milk quality.     

Tracking spore-forming bacterial contaminants in fluid milk-processing systems

The presence of psychrotolerant Bacillus species and related spore formers (e.g., Paenibacillus spp.) in milk has emerged as a key biological obstacle in extending the shelf life of high-temperature, short-time pasteurized fluid milk beyond 14 d. A recently developed rpoB DNA sequence-based subtyping method was applied to characterize spoilage bacteria present in raw milk supplies for 2 processing plants, and to assess transmission of these organisms into pasteurized products. Thirty-nine raw milk samples and 11 pasteurized product samples were collected to represent the processing continuum from incoming truck loads of raw milk to packaged products. Milk samples were held at 6°C for up to 16 d and plated for bacterial enumeration at various times throughout storage. Among the 88 bacterial isolates characterized, a total of 31 rpoB allelic types representing Bacillus and Paenibacillus spp. were identified, including 5 allelic types found in both raw milk and finished product samples. The presence of the same bacterial subtypes in raw and commercially pasteurized milk samples suggests that the raw milk supply represents an important source of these spoilage bacteria. Extension of the shelf life of high-temperature, short-time pasteurized fluid milk products will require elimination of these organisms from milk-processing systems.     

Use of Fourier transform infrared spectroscopy for rapid comparative analysis of Bacillus and Micrococcus isolates

The identification of foodborne microorganisms and their endospores in food products are important for food safety. The present work compares Bacillus (Bacillus licheniformis, Bacillus circulans and Bacillus subtilis) and Micrococcus (Micrococcus luteus) species with Fourier transform infrared (FTIR) spectroscopy. Our results show that there are several characteristic peaks belonging to both the Micrococcus and Bacillus species which can be used for the identification of these foodborne bacteria and their endospores. For Micrococcus species, a new band was observed at 1338 cm⁻¹ which may be due to acetate oxidation via the carboxylic acid cycle. The bands at 1313 cm⁻¹ and 1256 cm⁻¹ can be explained by an exopolymer formation and the other bands at 1074 cm⁻¹ and 550 cm⁻¹, may be due to the glycogen-like storage material in Micrococcus spp. There are also characteristic peaks at 993 cm⁻¹ and 801 cm⁻¹ for these bacterial species. Different Bacillus species also showed characteristic peaks at 1000–500 cm⁻¹ region. Dipicolinic acid (DPA) bands at ∼728 cm⁻¹ and ∼703 cm⁻¹ seen only in B. circulans were the marker of an endospore formation.     

Identification of surfactin producing strains in Soumbala and Bikalga fermented condiments using Polymerase Chain Reaction and Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry methods

In this study, 67 strains were isolated from two fermented condiments from Burkina Faso: Soumbala and Bikalga. Phenotypical methods, biochemical tests and molecular approaches were used to determinate their genus or species. Twenty-two of them belong to the Bacillus genus. Six strains were selected for their antibacterial or antifungal properties. Their ability to produce lipopeptides synthesized by Non Ribosomal Peptide Synthetases was investigated using two different approaches: PCR with specific degenerated primers and Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS) performed on whole cells cultivated on a solid medium. PCR revealed that the six strains contain genes involved in the biosynthesis of surfactins whereas surfactins C₁₄ and C₁₅ were only detected by MALDI-ToF MS in two of the six strains. For the first time, the presence of surfactins C₁₄ and C₁₅ was also identified by MALDI-ToF MS analyses directly performed on Soumbala methanolic crude extracts. The structure of these compounds was confirmed by + MS2 and + MS3 of sample and reference surfactins.     

High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk

The grade A Pasteurized Milk Ordinance specifies minimum processing conditions of 72°C for at least 15 s for high temperature, short time (HTST) pasteurized milk products. Currently, many US milk-processing plants exceed these minimum requirements for fluid milk products. To test the effect of pasteurization temperatures on bacterial numbers in HTST pasteurized milk, 2% fat raw milk was heated to 60°C, homogenized, and treated for 25 s at 1 of 4 different temperatures (72.9, 77.2, 79.9, or 85.2°C) and then held at 6°C for 21 d. Aerobic plate counts were monitored in pasteurized milk samples at d 1, 7, 14, and 21 postprocessing. Bacterial numbers in milk processed at 72.9°C were lower than in milk processed at 85.2°C on each sampling day, indicating that HTST fluid milk-processing temperatures significantly affected bacterial numbers in fluid milk. To assess the microbial ecology of the different milk samples during refrigerated storage, a total of 490 psychrotolerant endospore-forming bacteria were identified using DNA sequence-based subtyping methods. Regardless of processing temperature, >85% of the isolates characterized at d 0, 1, and 7 postprocessing were of the genus Bacillus, whereas more than 92% of isolates characterized at d 14 and 21 postprocessing were of the genus Paenibacillus, indicating that the predominant genera present in HTST-processed milk shifted from Bacillus spp. to Paenibacillus spp. during refrigerated storage. In summary, 1) HTST processing temperatures affected bacterial numbers in refrigerated milk, with higher bacterial numbers in milk processed at higher temperatures; 2) no significant association was observed between genus isolated and pasteurization temperature, suggesting that the genera were not differentially affected by the different processing temperatures; and 3) although typically present at low numbers in raw milk, Paenibacillus spp. are capable of growing to numbers that can exceed Pasteurized Milk Ordinance limits in pasteurized, refrigerated milk.     

Bacillus probiotics

Bacterial spore formers are being used as probiotic supplements for use in animal feeds, for human dietary supplements as well as in registered medicines. Their heat stability and ability to survive the gastric barrier makes them attractive as food additives and this use is now being taken forward. While often considered soil organisms this conception is misplaced and Bacilli should be considered as gut commensals. This review summarises the current use of Bacillus species as probiotics, their safety, mode of action as well as their commercial applications.     

Effects of nisin and reutericyclin on resistance of endospores of Clostridium spp. to heat and high pressure

The effects of high pressure, temperature, and antimicrobial compounds on endospores of Clostridium spp. were examined. Minimal inhibitory concentrations (MIC) of nisin and reutericyclin were determined for vegetative cells and endospores of Clostridium sporogenes ATCC 7955, Clostridium beijerinckii ATCC 8260, and Clostridium difficile 3195. Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 were exposed to 90 °C and 90 °C/600 MPa in the presence of 16 mg L⁻¹ nisin or 6.4 mg L⁻¹ reutericyclin for 0–60 min in a 0.9% saline solution. Dipicolinic acid (DPA) release was measured using a terbium-DPA fluorescence assay, and endospore permeability was assessed using 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. Vegetative cells of C. sporogenes ATCC 7955 exhibited higher sensitivity to nisin relative to endospores, with MIC values 0.23 ± 0.084 mg L⁻¹ and 1.11 ± 0.48 mg L⁻¹, respectively. Nisin increased DPA release when endospores were treated at 90 °C; however, only C. sporogenes ATCC 7955 exhibited higher inactivation, suggesting strain or species specific effects. Reutericyclin did not enhance spore inactivation or DPA release. Use of nisin in combination with high pressure, thermal treatments enhanced inactivation of endospores of Clostridium spp. and may have application in foods.