Stearothermophilus ATCC Research Articles

A STUDY TO INVESTIGATE THE ANTIBACTERIAL EFFECTS, POLYPHENOL CONTENTS AND ANTIOXIDANT ACTIVITIES OF EXTRACTS FROM ANDROGRAPHIS PANICULATA MEDICINAL PLANTS

Andrographis paniculata is a traditional medicinal plant which has been known for various beneficial properties, such as the inhibition of bacterial growth and health enhancement properties. This study was carried out to evaluate the antibacterial effects, polyphenol contents and antioxidant activities of extracts from Andrographis paniculata plant materials, so as to partly explain its traditional application. Plant materials were extracted with 6 solvents: hot water, ethanol, methanol, ethyl acetate, acetone and hexane. We applied the agar-well-diffusion method to evaluate the inhibitory effects of extracts on several bacteria strains, including: Bacillus subtilis ATCC 6633; Geobacillus stearothermophilus ATCC 7953; Pseudomonas aeruginosa ATCC 9027; Staphylococcus aureus ATCC 25023; Staphylococcus aureus ATCC 25923; Escherichia coli ATCC 85922; Escherichia coli ATCC 35218; Escherichia coli ATCC 25922 and Salmonella ATCC 13311. Reagents, such as Folin Ciocalteu and 1,1-diphenyl-2-picrylhydrazyl (DPPH), were used to measure the polyphenol contents and antioxidant activities of the extracts, which were then respectedly converted to mg acid chlorogenic equivelent and mg VTME equivelent per 100 mg plant powder. The results showed that extracts from Andrographis paniculata showed antibacterial effects, in which the ethanol extract showed the highest antibacterial effects on bacteria, as it induced inhibitory zones on all testes bacteria, with the largest one was on Escherichia coli ATCC 25922 (18.81±2.22 mm). Extracts with high polyphenol contents and antioxidant activities were methanol, hot water and ethanol, in which the highest polyphenol content belonged to methanol extract (with the value reached to 0.270±0.019 mg acid chlorogenic equivalent per 100 mg plant powder), and the strongest antioxidant activity belong to hot water extract (with the value reached to 0.691±0.115 mg VTME equivalent per 100 mg plant powder).

High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis.

Bacillus licheniformis is a well-known platform strain for production of industrial enzymes. However, the development of genetically stable recombinant B. licheniformis for high-yield enzyme production is still laborious. Here, a pair of plasmids, pUB-MazF and pUB'-EX1, were firstly constructed. pUB-MazF is a thermosensitive, self-replicable plasmid. It was able to efficiently cure from the host cell through induced expression of an endoribonuclease MazF, which is lethal to the host cell. pUB′-EX1 is a nonreplicative and integrative plasmid. Its replication was dependent on the thermosensitive replicase produced by pUB-MazF. Transformation of pUB′-EX1 into the B. licheniformis BL-UBM harboring pUB-MazF resulted in both plasmids coexisting in the host cell. At an elevated temperature, and in the presence of isopropyl-1-thio-β-d-galactopyranoside and kanamycin, curing of the pUB-MazF and multiple-copy integration of pUB′-EX1 occurred, simultaneously. Through this procedure, genetically stable recombinants integrated multiple copies of amyS, from Geobacillus stearothermophilus ATCC 31195 were facilely obtained. The genetic stability of the recombinants was verified by repeated subculturing and shaking flask fermentations. The production of α-amylase by recombinant BLiS-002, harboring five copies of amyS, in a 50-l bioreactor reached 50 753 U/ml after 72 hr fermentation. This strategy therefore has potential for production of other enzymes in B. licheniformis and for genetic modification of other Bacillus species.

Submerged Bioreactor Production of Geobacillus stearothermophilus ATCC 7953 Spores for Use as Bioindicators to Validate Hydrogen Peroxide Inactivation Processes

In the food and pharmaceutical industries, evaluating the sterilization performance preceding aseptic production processes is of central importance. In the case of hydrogen peroxide sterilization of solid surfaces, bioindicators (BI) consisting of spores of Bacillus atrophaeus or Geobacillus stearothermophilus are used to validate the effectiveness and efficiency of the inactivation procedure. Commercial production of G. stearothermophilus is commonly performed on agar plates, where cultivation and sporulation conditions are not well-defined. Therefore, the produced BI can vary in their resistance, which in turn creates unacceptable uncertainties in the evaluation of aseptic processes. Submerged production in the bioreactor would allow more control over sporulation conditions, while reducing production time, resistance variability, and avoidance of false-positive or false-negative test results. In addition, submerged production of G. stearothermophilus so far was a challenge to achieve sufficiently high spore concentrations for BI production. This study reports on the development of a method for submerged production of G. stearothermophilus spores (pH 7.0, 57 °C, 30% pO2) that can achieve 1.6 × 107 spores/mL with a resistance against 35% H2O2 at 25 °C of D25°C,35% H2O2 = 73 s. This resistance ranks within the range of commercially available BI, making the results directly transferable to industrial applications.

Effect of sporulation conditions following solid-state cultivation on the resistance of Geobacillus stearothermophilus spores for use as bioindicators testing inactivation by H2O2

Geobacillus stearothermophilus spores play an essential role in the pharmaceutical and food industries as bioindicators (BI) to evaluate inactivation effects of hydrogen peroxide in processing and packaging lines' sterilization. Unstandardized, inconsistent BI production, as is the case for currently available commercial BI, can lead to variable resistances and incorrect test results. The solution to this problem could be the standardization of BI spore production. This study assessed the effects of sporulation temperature and pH on spore resistance in the production of G. stearothermophilus ATCC 7953 spores on solid-state agar. For this purpose, spores were produced under different sporulation conditions (51–63 °C, pH 6–8), and their resistance was determined by the D-value in 35% liquid hydrogen peroxide at 25 °C. Based on these data, a mathematical equation describing the resistance as a function of the sporulation conditions was derived. The analysis showed a quadratic effect of the sporulation temperature and a linear effect of the examined domain's pH on the resistance, without interactions between these factors. We observed the highest D25C,35%H2O2-value of 309 s at sporulation conditions of 57 °C and pH 6.0.

Development and validation of an extended predictive model for the effect of pH and water activity on the growth kinetics of Geobacillus stearothermophilus in plant-based milk alternatives

The cardinal model for the effect of temperature on Geobacillus stearothermophilus ATCC 7953 growth developed by Kakagianni, Gougouli, & Koutsoumanis, 2016 was expanded for the effect of pH and water activity (aw). The effect of pH (range: 5.7–8.5) and aw (range: 0.985–0.999) on G. stearothermophilus growth rate was studied in tryptone soy broth (TSB) using the Bioscreen C method and further modelled using a Cardinal Model (CM). The estimated values for the cardinal parameters pHmin,pHopt,pHmax,awmin, and awopt were 5.65 ± 0.14, 6.74 ± 0.03, 8.71 ± 0.03, 0.984 ± 0.007 and 0.998 ± 0.001, respectively. The growth behaviour of G. stearothermophilus was investigated in 7 commercial non-refrigerated plant-based milk alternatives under static conditions (62 °C) and the estimated maximum specific growth rates were used to determine the optimum growth rate for each product. The developed model was validated against observed growth of G. stearothermophilus in the 7 products during storage at non-isothermal conditions (testing 4 different temperature profiles). The validation results showed a good performance of the model with overall Bias factor (Bf) = 1.06 and Accuracy factor (Af) = 1.12. The developed model can be used as an effective tool by the food industry in predicting spoilage of plant-based milk alternatives during distribution and storage at retail and domestic levels.

Influence of the incubation temperature and total dissolved solids concentration on the biofilm and spore formation of dairy isolates of Geobacillus stearothermophilus.

Geobacillus species are an important contaminant in the dairy industry and their presence is often considered as an indicator of poor plant hygiene with the potential to cause spoilage. They can form heat resistant spores that adhere to surfaces of processing equipment and germinate to form biofilms. Therefore, strategies aimed towards preventing or controlling biofilm formation in the dairy industry are desirable. In this study we demonstrate that the preferred temperature for biofilm and spore formation among Geobacillus stearothermophilus A1, D1, P3 and ATCC 12980 was 65°C. Increasing the total dissolved milk solids concentration to 20% (w/v) caused an apparent delay in the onset of biofilm and spore formation to detectable concentrations among all the strains at 55°C. Compared to the onset time of the biofilm formation of A1 in 10% (w/v) reconstituted skim milk, addition of milk protein (whey protein and sodium caseinate) caused an apparent delay in the onset of biofilm formation to detectable concentrations by an average of 10 h at 55°C. This study proposes that temperature and total dissolved solids concentration have a cumulative effect on the biofilm and spore formation of G. stearothermophilus A1, D1, P3 and ATCC 12980. In addition, the findings from this study may indicate that preconditioning of stainless-steel surface with adsorbed milk proteins may delay the onset of biofilm and spore formation of thermophilic bacteria during milk powder manufacture.IMPORTANCE The thermophilic bacilli, Geobacillus stearothermophilus is a predominant spoilage bacterium in milk powder manufacturing plants. If their numbers exceed the accepted levels, this may incur financial loses by lowering the price of the end product. Furthermore, they can form heat resistant spores which adhere to processing surfaces and can germinate to form biofilms. Previously conducted research had highlighted the variation in the spore and biofilm formation among three specific strains of G. stearothermophilus isolated from a milk powder manufacturing plant in New Zealand. The significance of our research is demonstrating the effect of two abiotic factors namely temperature and total dissolved solids concentration on the biofilm and spore formation of these three dairy isolates, leading to modifications in the thermal processing steps aimed towards controlling the biofilm and spore formation of G. stearothermophilus in the dairy industry.

A comparison of the spore heat resistance of dairy isolates of Geobacillus stearothermophilus obtained using a CDC biofilm reactor and a sporulating medium

In this study we compared a heat resistance of spores of Geobacillus stearothermophilus A1, D1, P3 and ATCC 12980 obtained using a milk biofilm system under continuous flow with previously reported values obtained by a sporulating medium. The average decimal reduction times of A1, D1, P3 and ATCC 12980 were 4.2, 4.9, 4.1 and 7.9 min, respectively, and were not significantly different (p < 0.05) from the heat resistance of spores obtained by a sporulating medium. In addition, the total calcium content of spores of A1, D1, P3 and ATCC 12980 were 1.9, 2.0, 2.5 and 5.1 ppm, respectively. The dipicolinic acid (DPA) content of spores of A1, D1, P3 and ATCC 12980 were 0.19, 0.26, 0.33, 1.38 pg per spore, respectively. The findings presented here provide new insights which can be used in the design of thermal processing steps in the dairy industry.

Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase).

D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.

The effect of phosphate on the heat resistance of spores of dairy isolates of Geobacillus stearothermophilus

In this study, we show that phosphate decreases the spore heat resistance by accelerating the rate of loss of cations from spores. Heat resistance of spores of Geobacillus stearothermophilus A1, D1, P3 and ATCC 12980 were determined in distilled water containing varying concentrations (0.1, 1 and 2% w/v) of di‑sodium phosphate. The average decimal reduction times (D value) for strains A1, D1, P3 and ATCC 12980 in distilled water were 5.8, 6.8, 5.7 and 9 min at 110 °C respectively. On the addition of 0.1, 1 and 2% w/v of di‑sodium phosphate, the average D110 values of all the strains in distilled water were lowered by 50, 61 and 70% respectively. Addition of 0.05% w/v of Na-EDTA to distilled water resulted in lowering of the average D110 value of all the strains by 55%. Heat resistance of spores of A1, D1, P3 and ATCC 12980 was found to be associated with the Dipicolinic Acid (DPA) content whose concentrations were 0.25, 0.30, 0.27 and 1.6 pg per spore respectively. Analysis by atomic absorption spectroscopy revealed that the phosphate present in the heating medium causes excess release of calcium from spores with 2% w/v phosphate being highly effective, thus confirming the chelating effect of phosphate. This study provides insight into the heat resistance and the increased heat sensitivity of spores of G. stearothermophilus A1, D1 and P3 in the presence of phosphate, which can be used in the design of Cleaning in Place (CIP) systems involving phosphate based cleaning agents to combat biofilms and spores in the dairy industry.

Evaluating the Potential, Applicability, and Effectiveness of Ozone Sterilization Process for Medical Devices

Ozone (O3) can be considered the most potent natural germicide against microorganisms (in vegetative and spore forms) with high efficiency and speed, because of its highly oxidizing activity. Despite this, there are a few studies describing the application of ozone as a sterilizing agent of medical devices. The aim of this communication was to describe the development and validation of a sterilization cycle applied to medical devices. The sterilization process was challenged using Geobacillus stearothermophilus ATCC 7953 spores, which have shown great resistance. The sterilizing effect of ozone was measured using carriers inoculated with 106 CFU/mL spores, introduced into a 3-mL syringe and lumens of tubes of different sizes and diameters simulating hospital medical products, which have undergone a half-cycle or complete cycle. The results of sterilization process studied in active vegetative form of microorganisms showed that the ozone sterilization was effective with a bioburden between 105 to 107 CFU/mL with one pulse sterilizing action. The validation of the process was confirmed by the satisfactory results for the half-cycle, corresponding to a treatment with four pulses allowed sterilizing the material with bioburdens < 106 CFU/mL spores which indicate an appropriate sterility assurance level. The results showed that the ozone may be considered as effective and promising alternative for sterilization of thermosensitive materials and medical devices.

Evaluation of Nanoparticles as Carrier for Bacillus Stearothermophilus Spore Preservation in Bioindicator System

A precise assessment of sterilization processes is very essential in the pharmaceutical and biological industry, because a wrong treatment could result in the spoilage of a product due to microorganism development. Bacillus Stearothermophilus has been established as an industrial bacterium in the production of biological sterilization indicators. The production of biological indicators involving bacterial spore production which is dormant and are extremely resistant to heat stresses. Bacillus Stearothermophilus ATCC 7953 spores are used to monitor the efficacy of steam heat and hydrogen peroxide sterilization processes. In this study, efficacy of replacing nanomaterial as carrier instead of regular paper strip was investigated. For this purpose, the effect of nanomaterial on thermal resistance parameters and spore absorption was assessed and compared with common paper strips of this product.

OPTIMIZATION OF THERMOSTABLE ALPHA-AMYLASE PRODUCTION FROM GEOBACILLUS SP. D413

The qualitative and quantitative α-amylase production capacities of six thermophilic bacilli were screened. Geobacillus sp. D413 was selected for enzyme optimization, as it displayed higher α-amylase activity. The maximum enzyme activities of D413 and G. stearothermophilus ATCC 12980T were observed at the time of 72 h. While the optimal pH of medium for bacterial growth and enzyme production of D413 (pH 7.0) differed from ATCC 12980T (pH 8.0), the optimal temperature for enzyme production was 55°C for both. The effects of various carbon and nitrogen sources were determined by changing their concentrations. The highest bacterial growth and enzyme production were sustained by the starch and maltose containing medium. Both bacterial growth and enzyme production were inhibited by NH4Cl. D413 and ATCC 12980T amylases showed optimal activity at 65ºC, pH 9.0 and at 65ºC, pH 7.5, respectively. They remained active over temperature and pH ranges of 45-75ºC and 4.0-10.5. Their activities retained 65% and 54% when incubated at 75ºC for 10 min and 98-86.5% and 95-84.5% at pH 4.0-10.5 for 15 h at 37ºC. In conclusion, the α-amylase production conditions of D413 have been optimized which can be useful in biotechnological processes such as hydrolysis of starch to glucose.

Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance.

This study was to determine the sources, location and identity of α-glucosidases in dormant/germinating/outgrowing spores and growing cells of Geobacillus stearothermophilus ATCC 7953, an enzymatic activity in spores used in rapid tests of steam sterilization. α-Glucosidase activity in spores and cells was determined measuring methylumbelliferyl-α-d-glucoside (α-MUG) or α-MUG-6-phosphate hydrolysis fluorometrically. While α-MUG-6-phosphate was not hydrolysed by cell or spore extracts, assays with α-MUG showed that: (1) the α-glucosidase activity was inside and outside spores, and the activity outside spores was largely removed by buffer washes or heat activation, whereas α-glucosidase activity was only inside vegetative cells; (2) most α-glucosidase activity in cells and spores was soluble; (3) Western blots and enzyme inhibition using an anti-α-glucosidase antiserum identified ≥2 α-glucosidases in spores and growing cells; (4) α-glucosidase-specific activities were similar in dormant, germinated and outgrowing spore and growing cell extracts; and (5) significant α-glucosidase was synthesized during spore germination and outgrowth and cell growth, this synthesis was not repressed by glucose nor induced by α-MUG, but glucose inhibited α-MUG uptake. α-MUG hydrolysis by G.stearothermophilus is by α-MUG uptake and hydrolysis by ≥2 α-glucosidases associated with dormant spores and synthesized by germinating and outgrowing spores. The enzyme activity observed by sterilization assurance assays appears likely to come from heat-stable enzyme in the spore core and enzyme(s) synthesized in spore outgrowth. The results of this work provide new insight into the science behind a rapid test for steam sterilization assurance.

Development and application of Geobacillus stearothermophilus growth model for predicting spoilage of evaporated milk

The presence of Geobacillus stearothermophilus spores in evaporated milk constitutes an important quality problem for the milk industry. This study was undertaken to provide an approach in modelling the effect of temperature on G. stearothermophilus ATCC 7953 growth and in predicting spoilage of evaporated milk. The growth of G. stearothermophilus was monitored in tryptone soy broth at isothermal conditions (35–67 °C). The data derived were used to model the effect of temperature on G. stearothermophilus growth with a cardinal type model. The cardinal values of the model for the maximum specific growth rate were Tmin = 33.76 °C, Tmax = 68.14 °C, Topt = 61.82 °C and μopt = 2.068/h. The growth of G. stearothermophilus was assessed in evaporated milk at Topt in order to adjust the model to milk. The efficiency of the model in predicting G. stearothermophilus growth at non-isothermal conditions was evaluated by comparing predictions with observed growth under dynamic conditions and the results showed a good performance of the model. The model was further used to predict the time-to-spoilage (tts) of evaporated milk. The spoilage of this product caused by acid coagulation when the pH approached a level around 5.2, eight generations after G. stearothermophilus reached the maximum population density (Nmax). Based on the above, the tts was predicted from the growth model as the sum of the time required for the microorganism to multiply from the initial to the maximum level (tNmax), plus the time required after the tNmax to complete eight generations. The observed tts was very close to the predicted one indicating that the model is able to describe satisfactorily the growth of G. stearothermophilus and to provide realistic predictions for evaporated milk spoilage.

Die another day: Fate of heat-treated Geobacillus stearothermophilus ATCC 12980 spores during storage under growth-preventing conditions

Geobacillus stearothermophilus spores are recognized as one of the most wet-heat resistant among aerobic spore-forming bacteria and are responsible for 35% of canned food spoilage after incubation at 55 °C. The purpose of this study was to investigate and model the fate of heat-treated survivor spores of G. stearothermophilus ATCC 12980 in growth-preventing environment. G. stearothermophilus spores were heat-treated at four different conditions to reach one or two decimal reductions. Heat-treated spores were stored in nutrient broth at different temperatures and pH under growth-preventing conditions. Spore survival during storage was evaluated by count plating over a period of months. Results reveal that G. stearothermophilus spores surviving heat treatment lose their viability during storage under growth-preventing conditions. Two different subpopulations were observed during non-thermal inactivation. They differed according to the level of their resistance to storage stress, and the proportion of each subpopulation can be modulated by heat treatment conditions. Finally, tolerance to storage stress under growth-preventing conditions increases at refrigerated temperature and neutral pH regardless of heat treatment conditions. Such results suggest that spore inactivation due to heat treatment could be completed by storage under growth-preventing conditions.

Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant-Derived, Low-Acid Foods, and Biomaterials.

Cycle threshold (Ct) increase, quantifying plant-derived DNA fragmentation, was evaluated for its utility as a time-temperature integrator. This novel approach to monitoring thermal processing of fresh, plant-based foods represents a paradigm shift. Instead of using quantitative polymerase chain reaction (qPCR) to detect pathogens, identify adulterants, or authenticate ingredients, this rapid technique was used to quantify the fragmentation of an intrinsic plant mitochondrial DNA (mtDNA) gene over time-temperature treatments. Universal primers were developed which amplified a mitochondrial gene common to plants (atp1). These consensus primers produced a robust qPCR signal in 10 vegetables, 6 fruits, 3 types of nuts, and a biofuel precursor. Using sweet potato (Ipomoea batatas) puree as a model low-acid product and simple linear regression, Ct value was highly correlated to time-temperature treatment (R(2) = 0.87); the logarithmic reduction (log CFU/mL) of the spore-forming Clostridium botulinum surrogate, Geobacillus stearothermophilus (R(2) = 0.87); and cumulative F-value (min) in a canned retort process (R(2) = 0.88), all comparisons conducted at 121 °C. D121 and z-values were determined for G. stearothermophilus ATCC 7953 and were 2.71 min and 11.0 °C, respectively. D121 and z-values for a 174-bp universal plant amplicon were 11.3 min and 9.17 °C, respectively, for mtDNA from sweet potato puree. We present these data as proof-of-concept for a molecular tool that can be used as a rapid, presumptive method for monitoring thermal processing in low-acid plant products.

Détection biologique des résidus d’antibiotiques dans le lait et produits laitiers de vache consommés à Ouagadougou, Burkina Faso.

Objective: The traditional breeding is generally associated with bad veterinary practices resulting in anarchical use of antibiotics. The objective of this study is to detect the antibiotic residues in milk and dairy products consumed in Ouagadougou, Burkina Faso. Methods and Results : A total of 201 dairy products samples were collected in open markets and supermarkets then analyzed by the method using Bacillus subtilis ATCC 6633 and Geobacillus stearothermophilus ATCC 10149 for the detection of antibiotic residues in milk. Dairy products were tested

Sterilization Validation of Gas Plasma Exposure Based on ISO Documents(Mainly ISO TC 198 And 194 Documents)

There are so many ISO documents to understand and utilize for validation studies and routine control. A biological indicator (BI) is essential for conducting sterilization validation and routine control. ISO 11138-1 and ISO 14161 are the major documents to follow for BI manufacturers and BI users, respectively. In ISO 11138-1, BI manufacturers must utilize a BI with 106 CFU/carrier as an initial population for validation studies. For routine control, it is approved to use an initial BI population of 105 CFU/carrier according to ISO 11138-1. In ISO 14161 BI users need not imitate BI manufacturers and less than 106 CFU/carrier BI is approved for use in validation studies and routine control. According to ISO 14161 for BI users, the initial population must be identical for validation studies and routine control. For the BI user, there are four types of procedures for sterilization validation and routine control. These are the half-cycle, over-kill, combined BI/bioburden and the absolute bioburden methods. A BI with An initial population of 106 CFU/ carrier must be used for validation studies by BI manufacturers according to ISO 11138-1, but BI users can elect to use a commercially available BI with more than 103 CFU/carrier as the BI for validation studies and routine control as described in ISO 14161. The approved SAL was defined to be 10-6 and this is unchanged for both BI users and BI manufacturers. Therefore, from the initial population of 106 CFU (colony forming unit)/carrier to a SAL of 10-6, the survivor curve must be straight and tailing is not allowed as described below (ISO 11138-1). The BI used for gas plasma sterilization, spores of Geobacillus stearothermophilus ATCC 7953, has characteristics that do not result in a tailing phenomenon. A linear survival curve is obtained from an initial population of 106 CFU/carrier to a SAL 10-6 for all sterilization procedures tested, demonstrating that inactivation kinetics are first order and allowing calculation of the D (decimal reduction) value from the dose or time to decrease 1 log. Chemical indicators (CI) are not approved for use in validation studies; only the use of BI is approved. CIs are only approved for use as a supporting method during routine control according to ISO 11140-1 and 14161. Only a BI is approved for both validation studies and routine control according to ISO 14161.

Inactivation of Endotoxin and Lipid A by Nitrogen Gas Plasma Exposure Running title: Inactivation Endotoxin and lipid A

The gases plasma from several different gases has shown a sporicidal activity. From these gases, nitrogen gas was most difficult to produce atomic nitrogen radicals. However, these radicals have a high energy, indicating that nitrogen gas plasma could be used to sterilize microorganisms and inactivate endotoxins and lipid A. The sterilization mechanism of nitrogen gas plasma may be the synergistic effect of free radicals such as (OH radical and NO radical) and metastables (N2 or O2 metastable). Thus, the target microorganisms such as bioburden were damaged by degradation, which resulted in death. The biological indicator BI used in this study was Geobacillus stearothermophilus ATCC 7953 at an initial population of 106 CFU/carrier. Sterility assurance level (SAL) of 10-6 was confirmed by using the BI. In addition endotoxins were successfully inactivated by nitrogen gas plasma expossure. More than 5 log reduction of endotoxins could be attained within 30 minutes of nitrogen gas plasma exposure. Lipid A degradation by nitrogen gas plasma exposure was confirmed by LC-MS-MS. Material/functional compatibility was also maintained by nitrogen gas plasma exposure due to shallower penetration depth of 10-20 nm. No deterioration of polymers could be observed by nitrogen gas plasma exposure. Residual gases, if any, are non-toxic. From the above description, it can be said that nitrogen gas plasma sterilization is an alternative future sterilization method.

Modeling the behavior of Geobacillus stearothermophilus ATCC 12980 throughout its life cycle as vegetative cells or spores using growth boundaries

Geobacillus stearothermophilus is recognized as one of the most prevalent micro-organism responsible for flat sour in the canned food industry. To control these highly resistant spore-forming bacteria, the heat treatment intensity could be associated with detrimental conditions for germination and outgrowth. The purpose of this work was to study successively the impact of temperature and pH on the growth rate of G. stearothermophilus ATCC 12980, its sporulation ability, its heat resistance in response to various sporulation conditions, and its recovery ability after a heat treatment.The phenotypic investigation was carried out at different temperatures and pHs on nutrient agar and the heat resistance was estimated at 115 °C. The greatest spore production and the highest heat resistances were obtained at conditions of temperature and pH allowing maximal growth rate. The current observations also revealed that growth, sporulation and recovery boundaries are close. Models using growth boundaries as main parameters were extended to describe and quantify the effect of temperature and pH throughout the life cycle of G. stearothermophilus as vegetative cells or as spore after a heat treatment and during recovery.