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.     

Effect of the medium characteristics and the heating and cooling rates on the nonisothermal heat resistance of Bacillus sporothermodurans IC4 spores

In recent years, highly thermo-resistant mesophilic spore-forming bacteria belonging to the species Bacillus sporothermodurans have caused non-sterility problems in industrial sterilization processes. The aim of this research was to evaluate the effect of the heating medium characteristics (pH and buffer/food) on the thermal inactivation of B. sporothermodurans spores when exposed to isothermal and non-isothermal heating and cooling treatments and the suitability of non-linear Weibull and Geeraaerd models to predict the survivors of these thermal treatments. Thermal treatments were carried out in pH 3, 5 and 7 McIlvaine buffer and in a courgette soup. Isothermal survival curves showed shoulders that were accurately characterized by means of both models. A clear effect of the pH of the heating medium was observed, decreasing the D₁₂₀ value from pH 7 to pH 3 buffer down to one third. Differences in heat resistance were similar, regardless of the model used and were kept at all temperatures tested. The heat resistance in courgette soup was similar to that shown in pH 7 buffer. When the heat resistance values obtained under isothermal conditions were used to predict the survival in the non-isothermical experiments, the predictions estimated the experimental data quite accurately, both with Weibull and Geeraerd models.     

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.     

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.     

Extraction optimization of a hydrocolloid extract from cress seed (Lepidium sativum) using response surface methodology

Extraction conditions for maximum values of yield, viscosity and minimum protein content of hydrocolloid extract from Lepidium sativum seed were investigated using response surface methodology. A Central Composite Face Design (CCFD) with four independent variables: temperature (25–85 °C); pH (3–10); extraction time (10–25 min) and water to seed ratio (10:1–80:1) was used to study the response variables (yield, viscosity and protein content). Data analysis showed that all the variables significantly (p < 0.05) affected the extraction yield and viscosity, whereas the effect of water to seed ratio on protein content was not significant (p > 0.05). Applying a desirability function method the optimum parameters were: extraction temperature 35 °C, pH 10, water to seed ratio of 30:1 and an extraction time of 15 min. At this optimum point, apparent viscosity, yield and protein content were 0.2 Pa s, 6.46% and 0.57%, respectively. The experimental values were very close to the predicted values and were not statistically different (p > 0.05).     

Optimisation of the solvent extraction of bioactive compounds from Parkia speciosa pod using response surface methodology

Central composite design was employed to optimise the buffer-to-solids ratio (X₁: 20–50 ml/g), incubation temperature (X₂: 35–55 °C) and time (X₃: 100–200 min), obtaining extracts from Parkia speciosa pod with high total phenolic and flavonoid contents and high antioxidant activities. Analysis of variance showed that the contribution of a quadratic model was significant for the responses. An optimisation study using response surface methodology was performed and 3D response surfaces were plotted from the mathematical models. The optimal conditions based on combination responses were: X₁ = 20 ml/g, X₂ = 35–36 °C and X₃ = 100–102 min. These optimum conditions yielded total phenolic contents of 664–668 mg gallic acid equivalents/100 g, total flavonoid contents of 47.4–49.6 mg pyrocatechol equivalents/100 g, %DPPH_sc of 81.2–82.1%, %ABTS_sc of 78.2–79.8% and FRAP values of 3.2–3.3 mM. Close agreement between experimental and predicted values was found. This methodology could be applied in the extraction of bioactive compounds in the natural product industry.     

Optimization of subcritical fluid extraction of seed oil from Nitraria tangutorum using response surface methodology

Subcritical fluid extraction (SFE) technology was used to extract oil from Nitraria tangutorum seed. The best possible combination of extraction parameters was found using response surface methodology (RSM) in a three-variable, three-level Box-Behnken experimental design (BBD). The optimum extraction parameters were an extraction time of 40 min, an extraction pressure of 0.60 MPa, an extraction temperature of 44 °C and a raw material particle size of 0.45 mm. Conventional solvent extraction and supercritical CO₂ fluid extraction were comparatively used. The yield of seed oil obtained using SFE was 12.92%, which was similar to or higher than the other methods. The chemical compositions of the seed oil, determined by GC–MS, indicate that its unsaturated fatty acids content was 97%. SFE proved to be an effective technique for extracting oil from N. tangutorum.     

Optimization of the extraction of flavanols and anthocyanins from the fruit pulp of Euterpe edulis using the response surface methodology

The response surface methodology (RSM) was employed to optimize the ultrasound extraction of flavanols and anthocyanins from the pulp of jussara (Euterpe edulis), using a second-order polynomial equation to describe the experimental data for total flavanol (TF), total phenolic (TP), and total monomeric anthocyanin (TMA) contents, as well as the total antioxidant activity (TAA). A central composite design with two-variables (extraction time and solid to liquid ratio) was then applied. The optimized conditions that maximized the yields of flavanol-enriched extract were a solvent methanol/0.1 M HCl, solid to liquid ratio of between 1:50 and 1:100 and extraction time of 15 min. For anthocyanin-enriched extracts the respective optimal parameters were a solvent methanol/1.5 M HCl, solid to liquid ratio of between 1:30 and 1:50 and extraction time of 24 h. The results showed good fits with the proposed model for both the flavanol-enriched extract (R² = 0.94) and for the anthocyanin-enriched extract (R² = 0.99).     

Clostridial spore germination versus bacilli: genome mining and current insights

Bacilli and clostridia share the characteristic of forming metabolically inactive endospores. Spores are highly resistant to adverse environmental conditions including heat, and their ubiquitous presence in nature makes them inevitable contaminants of foods and food ingredients. Spores can germinate under favourable conditions, and the following outgrowth can lead to food spoilage and foodborne illness. Germination of spores has been best studied in Bacillus species, but the process of spore germination is less well understood in anaerobic clostridia. This paper describes a genome mining approach focusing on the genes related to spore germination of clostridia. To this end, 12 representative sequenced Bacillus genomes and 24 Clostridium genomes were analyzed for the distribution of known and putative germination-related genes and their homologues. Overall, the number of ger operons encoding germinant receptors is lower in clostridia than in bacilli, and some Clostridium species are predicted to produce cortex-lytic enzymes that are different from the ones encountered in bacilli. The in silico germination model constructed for clostridia was linked to recently obtained experimental data for selected germination determinants, mainly in Clostridium perfringens. Similarities and differences between germination mechanisms of bacilli and clostridia will be discussed.     

Optimization of ionic liquid based ultrasonic assisted extraction of puerarin from Radix Puerariae Lobatae by response surface methodology

Ionic liquid (IL) based ultrasonic assisted extraction (ILUAE) was developed for the effective extraction of puerarin from Radix Puerariae Lobatae (RPL). The ILUAE parameters including the type of ILs, IL concentration, RPL amount, ultrasonic power and time were optimized by single-factor experiment and response surface methodology. Under the optimized experimental conditions, the best results were obtained using RPL amount 0.43 g in 10 mL 1.06 mol L⁻¹ 1-butyl-3-methylimidazolium bromide aqueous solution, ultrasonic time 27.43 min and ultrasonic power 480 W. Scanning electron microscope images of RPL samples were obtained to provide visual evidence of the sonication effect. Compared with the conventional ultrasonic assisted extraction and refluent extraction, the proposed ILUAE offered shorter extraction time and remarkable higher efficiencies due to the higher penetration ability and solubility of IL and the cavitation phenomenon produced in the solvent by the passage of an ultrasonic wave, which further supported the suitability of the proposed approach.     

Response of Bacillus cereus spores to high hydrostatic pressure and moderate heat

The effects of high pressure (400-600 MPa) and moderate heat (60-80 °C) treatments at various process times (10-20 min) on the reduction of Bacillus cereus As 1.1846 spores, suspended in milk buffer were investigated. In the present work, response surface methodology (RSM) was employed, and a quadratic equation of high hydrostatic pressure inactivation was built with RSM. By analyzing response surface plots and corresponding contour plots and by solving the quadratic equation, experimental values were shown to be significantly in agreement with predicted values, since the adjusted determination coefficient was 0.9752 and the level of significance was P < 0.0001. Optimum process parameters for a six-log cycle reduction of B. cereus spores were obtained: pressure, 540.0 MPa; temperature, 71 °C; and pressure-holding time, 16.8 min. The adequacy of the model equation in predicting optimum response values was verified effectively using experimental test data that was not used in the development of the model.     

Production of a diacylglycerol-enriched palm olein using lipase-catalyzed partial hydrolysis: Optimization using response surface methodology

Partial hydrolysis using Lipozyme RMIM lipase in a solvent-free system was used to produce a diacylglycerol (DAG)-enriched palm olein. Response surface methodology (RSM) was applied to model and optimize the reaction conditions namely water content (30–70 wt% of enzyme mass), enzyme load (5–15 wt% of oil mass), reaction temperature (45–85 °C) and reaction time (6–16 h). Well fitting models were successfully established for both DAG yield (R² = 0.8788) and unhydrolysed triacylglycerol (TAG) (R² = 0.8653) through multiple linear regressions with backward elimination. Chi-square test indicated that there were no significant (P > 0.05) differences between the observed and predicted values for both models. All reaction conditions had positive effects on DAG yield and negative effects on unhydrolysed TAG. Optimal reaction conditions were: 50 wt% water content, 10 wt% enzyme load, 65°C of reaction temperature and 12 h of reaction time. The process was further up-scaled to a 9 kg production in a continuous packed bed bioreactor. Results indicated that upscaling was possible with a similar DAG yield (32 wt%) as in lab scale. Purification of the DAG oil using short path distillation yielded a DAG-enriched palm olein with 60 wt% DAG and 40 wt% TAG which is suitable for margarine, spread or shortening applications.     

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.     

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.     

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.     

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.     

Optimization of the process variables for the preparation of processed paneer using response surface methodology

Models capable of predicting the quality and yield of processed paneer have been developed using response surface methodology to determine the optimum processing conditions. Three and four-dimensional response surfaces were drawn and the individual contour plots of the different responses were superimposed, and regions meeting the maximum sensory score (29.50–30.75) and yield (9.50–10.50 g/100 g) were identified at 70.1±2.0 °C at 4.85±0.23 pH and 2.908±0.113 g/kg stabilizer concentration. These predicted values for optimum process conditions were in good agreement with experimental data.     

Response surface methodology for autolysis parameters optimization of shrimp head and amino acids released during autolysis

Protein hydrolysates were prepared from the head waste of Penaens vannamei, a China seawater major shrimp by autolysis method. Autolysis conditions (viz., temperature, pH and substrate concentration) for preparing protein hydrolysates from the head waste proteins were optimized by response surface methodology (RSM) using a central composite design. Model equation was proposed with regard to the effect of temperature, pH and substrate concentration. Substrate concentration at 23% (w/v), pH at 7.85 and temperature at 50 °C were found to be the optimal conditions to obtain a higher degree of hydrolysis close to 45%. The autolysis reaction was nearly finished in the initial 3 h. The amino acid compositions of the autolysis hydrolysates prepared using the optimized conditions in different time revealed that the hydrolysates can be used as a functional food ingredient or flavor enhancer. Endogenous enzymes in the shrimp heads had a strong autolysis capacity (AC) for releasing threonine, serine, valine, isoleucine, tyrosine, histidine and tryptophan. Endogenous enzymes had a relatively lower AC for releasing cystine and glycine.     

Optimization of medium composition for enhanced chitin extraction from Parapenaeus longirostris by Lactobacillus helveticus using response surface methodology

Chitin extraction by biological way, using the lactobacilli Lactobacillus helveticus, is a non-polluting method and offers the opportunity to preserve the exceptional qualities of chitin and its derivatives. However, the major disadvantage of the fermentative way is the low efficiency of demineralization and deproteinization. The aim of our study is to improve the yield of extraction.     

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.