MODE OF ACTION OF ETHYLENE OXIDE ON SPORES OF Clostridium botulinum 62A

SUMMARY— Death of spores of Clostridium botulinum when exposed to gaseous ethylene oxide followed first order kinetics. Supplementation of a synthetic medium with the purine and pyrimidine bases of DNA and RNA indicated, as judged by outgrowth from spores which had received sublethal ETO treatments, that the lethal action of ETO on the spores was through alkylation of the guanine and adenine components of DNA. Observed impairment of RNA and protein synthesis was considered an indirect effect resulting from alkylation of DNA components; however, additional evidence bearing on this point is needed to support a more definite conclusion.     

Induced tolerance of Sitophilus oryzae adults to carbon dioxide

Sitophilus oryzae adults were exposed to 40% carbon dioxide (CO₂) in air for seven successive generations and to 75% CO₂ in air for ten successive generations at 26°C and 100% relative humidity (r.h.). The tolerance factor (LT₉₅ selected generation/LT₉₅ non-selected generation) at the 7th generation of insects exposed to 40% CO₂ was 2.15 and at the 10th generation exposed to 75% CO₂, was 3.34. Reduction of the r.h. to 60% in the 75% CO₂ atmosphere caused shortening of exposure times required to obtain the same mortality values, but the tolerance factors remained stable. Removal of selection pressure for five generations of the 40% CO₂ and four generations of the 75% CO₂ selected strains caused significant reduction in tolerance. Augmentation of oxygen concentration to 21% in atmospheres containing 40 and 75% CO₂ did not markedly alter the tolerance factors from those obtained with atmospheres containing the same CO₂ concentrations in air.     

荧光假单胞菌AR4在2-酮基-D-葡萄糖酸工业生产中的应用研究

在噬菌体存在的情况下,采用抗噬菌体菌株荧光假单胞菌(Pseudomonasfluorescens)AR4在200m3发酵罐上进行了2-酮基-D-葡萄糖酸的发酵生产。结果表明,荧光假单胞菌AR4对生产环境中噬菌体的抗性稳定,发酵周期短,发酵转化率高;经过10次传代,荧光假单胞菌AR4对噬菌体的抗性及其发酵生产2-酮基-D-葡萄糖酸的能力没有改变;该菌株的发酵产物可以用于食品抗氧化剂D-异抗坏血酸钠的合成。     

Heat resistance of Bacillus spores when adhered to stainless steel and its relationship to spore hydrophobicity

Twenty-one strains of Bacillus (10 B. stearothermophilus, 3 B. cereus, and 8 B. licheniformis strains) were assayed for spore surface hydrophobicity on the basis of three measures: contact angle measurement (CAM), microbial adhesion to hydrocarbons (MATH), and hydrophobic interaction chromatography (HIC). On the basis of the spore surface characteristics obtained from these assays, along with data on the heat resistance of these spores in water, eight strains of Bacillus (three B. stearothermophilus, three B. cereus, and two B. licheniformis strains) either suspended in water or adhering to stainless steel were exposed to sublethal heat treatments at 90 to 110 degrees C to determine heat resistance (D-value). Significant increases in heat resistance (ranging from 3 to 400%) were observed for the eight strains adhering to stainless steel. No significant correlation was found between these heat resistance increases and spore surface characteristics as determined by the three hydrophobicity assays. There was a significant positive correlation between the hydrophobicity data obtained by the MATH assay and those obtained by the HIC assay, but these data did not correlate with those obtained by the CAM assay.     

紫外线和硫酸二乙酯诱变高产凝乳酶地衣芽孢杆菌的研究

以产凝乳酶地衣芽孢杆菌为出发菌株,通过紫外线诱变和硫酸二乙酯诱变,提高菌株的凝乳活力。经反复诱变筛选获得一株凝乳活力较高且水解活力较低的突变株DES-6,其凝乳活力为184.65SU/mL,比原菌株增加了15.41%,水解活力为23.35U/mL,比原菌株降低了64.80%。传代实验表明,突变株DES-6具有稳定的遗传性。     

2-酮基-D-葡萄糖酸产生菌球状节杆菌K1022抗噬菌体菌株的选育

从 2 酮基 D 葡萄糖酸产生菌球状节杆菌K10 2 2的异常发酵液中分离到 3种噬菌体 ,分别将其命名为KS2 11、KS2 12和KS2 13。采用紫外诱变或自然选育的方法 ,获得了 6株具有抗噬菌体能力的 2 酮基 D 葡萄糖酸高产菌株。研究了抗株C2 2 4和K2 32的遗传稳定性 ,并在 5 0kL的发酵罐上进行了发酵生产试验。结果表明 ,经多次传代 ,菌株C2 2 4和K2 32对噬菌体的抗性及其发酵产酸能力没有改变 ,可以应用于工业生产中     

EFFECT OF EDTA ON THE GERMINATION OF AND OUTGROWTH FROM SPORES OF Clostridium botulinum 62-A

SUMMARY –EDTA, in concentrations above 2.5 mM, was found inhibitory to germination of and outgrowth from spores of C. botulinum Type A and to toxin production in a fish homogenate. Inhibitory action was influenced by pH of the medium in the range pH 6.5–8.1, the action increasing with pH. It was influenced by Mg and Ca concentrations in the medium, equimolar concentrations of added CaCl₂ or MgCl₂, completely erasing the growth inhibitory action. Initial spore concentration also influenced inhibitory efficacy–the higher the spore concentration, the higher the EDTA concentration required for inhibition. There was no evidence that EDTA, in any concentration used, promoted spore germination. Release of Ca, Mg and DPA from incubating spores was suppressed to varying extents by 5.0 and 10 mM EDTA.     

A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH

Bacillus subtilis spore suspensions were subjected to pressure treatments at 100 and 600 MPa at 40 degrees C and over a pH range from 3 to 8. Inactivation of spores under these conditions was maximally 80% and was not increased at low pH. However, higher levels of inactivation were obtained when spores were first pressure treated at neutral pH and then exposed for 1 h to low pH. This large difference in inactivation could be explained by the finding that pressure-induced spore germination, which is known to occur at neutral pH, was inhibited at low pH (< 5). Pressure treatment at low pH made spores more sensitive to heat inactivation, suggesting that demineralized H-spores had been formed. Changes in spore core hydration and pH upon exposure of spores at low pH were studied in a more direct way using green fluorescent protein expressed in recombinant B. subtilis as a reporter protein, and it was confirmed that pressure and heat increase spore permeability for protons. Based on these results, the potential of low temperature, high pressure processes for spore inactivation in acid products is discussed.     

A standard set of testing methods reliably enumerates spores across commercial milk powders

Contamination of dairy powders with sporeforming bacteria is a concern for dairy processors who wish to penetrate markets with stringent spore count specifications (e.g., infant powders). Despite instituted specifications, no standard methodology is used for spore testing across the dairy industry. Instead, a variety of spore enumeration methods are in use, varying primarily by heat-shock treatments, plating method, recovery medium, and incubation temperature. Importantly, testing the same product using different methodologies leads to differences in spore count outcomes, which is a major issue for those required to meet specifications. As such, we set out to identify method(s) to recommend for standardized milk powder spore testing. To this end, 10 commercial milk powders were evaluated using methods varying by (1) heat treatment (e.g., 80°C/12 min), (2) plating method (e.g., spread plating), (3) medium type (e.g., plate count milk agar), and (4) incubation time and temperature combinations (e.g., 32°C for 48 h). The resulting data set included a total of 48 methods. With this data set, we used a stepwise process to identify optimal method(s) that would explain a high proportion of variance in spore count outcomes and would be practical to implement across the dairy industry. Ultimately, spore pasteurized mesophilic spore count (80°C/12 min, incubated at 32°C for 48 h), highly heat resistant thermophilic spore count (100°C/30 min, incubated at 55°C for 48 h), and specially thermoresistant spore enumeration (106°C/30 min, incubated at 55°C for 48 h) spread plating on plate count milk agar were identified as the optimal method set for reliable enumeration of spores in milk powders. Subsequently, we assessed different powder sampling strategies as a way to reduce variation in powder spore testing outcomes using our recommended method set. Results indicated that 33-g composite sampling may reduce variation in spore testing outcomes for highly heat resistant thermophilic spore count over 11-g and 33-g discrete sampling, whereas there was no significant difference across sampling strategies for specially thermoresistant spore enumeration or spore pasteurized mesophilic spore count. Finally, an interlaboratory study using our recommended method set and a modified method set (using tryptic soy agar with 1% starch) among both university and industry laboratories showed increased variation in spore count outcomes within milk powders, which not only was due to natural variation in powders but also was hypothesized to be due to technical errors, highlighting the need for specialized training for technicians who perform spore testing on milk powders. Overall, this study addresses challenges to milk powder spore testing and recommends a method set for standardized spore testing for implementation across the dairy industry.     

Methoprene and synergized pyrethrins as aerosol treatments to control Plodia interpunctella (Hübner), the Indian meal moth (Lepidoptera: Pyralidae)

Aerosol insecticides (also known as ULV or fogging treatments) delivered through an ultra-low-volume application system, are available commercially to control insect pests such as Plodia interpunctella Hübner, the Indian meal moth. However, little is known about the susceptibility of eggs of P. interpunctella to aerosol insecticides applied in active field sites. We conducted several trials by exposing eggs of P. interpunctella to synergized pyrethrins, alone and in combination with the insect growth regulator methoprene. Eggs in diets and packaging materials containing the food products were directly exposed to the aerosols. There was significant variation among the food products, as assessed by adult emergence from exposed eggs, but in general there was no difference in adult emergence from eggs exposed to a 1% versus a 3% pyrethrin formulation when methoprene was included. There was no difference in efficacy between diets and diet packages placed in open areas versus areas that were in some way obstructed to the aerosol. Adult emergence was generally reduced in the treatment combinations compared to untreated controls. A partial budget analysis indicated that the combination treatment of 1% pyrethrins + methoprene represented the lowest risk, lowest cost, and would seem to be the optimum combination. Results show that field applications of aerosols could be used to control P. interpunctella in storage facilities.     

A COMPARISON OF METHODS FOR THE MICROBIOLOGICAL SAMPLING OF COMPRESSED GASES

A study was carried out to evaluate eight methods for microbiological sampling of compressed gases. The devices tested were: Koch exposed plate; exposed honeypot; exposed bottle; Hollaender & Dalla Valle device; Porton impinger; Multistage liquid impinger; ASBC.impinger; membrane filtration method. The devices were compared by presenting a constant number of organisms per unit volume of gas to each device. The organism used was Bacillus stearothermophilus which in its spore form represents the type of organism which is most difficult to entrap. Other aspects of alternative sampling methods which were examined included testing of potential desiccation effects involving vegetative microorganisms on membrane filters, and susceptibility of devices to external contamination. The method recommended was a modification of that of Hollaender & Dalla Valle. This method had the highest collection efficiency and greatest statistical reliability of all those tested. The recommended methods of analysis of the IOB, EBC and ASBC were found to be inferior in a number of respects including susceptibility to external contamination, poor collection efficiency and statistical variability.     

一株芽孢表面稳定展示海藻糖合酶工程菌的构建

以枯草芽孢杆菌(Bacillus subtilis)168-Tres基因组为模板,PCR扩增得到同源臂基因sleB1和cwlJ1,重叠PCR连接sleB1与卡那霉素抗性(kmr )基因,电转获得B. subtilis 168-TresΔsleB菌株;连接cwlJ1与博来霉素抗性(zeor)基因,电转获得B. subtilis 168-TresΔsleBΔcwlJ菌株。结果表明,经kmr、zeor抗性筛选及PCR鉴定,成功获得sleB、cwlJ基因双缺失菌株B. subtilis 168-TresΔsleBΔcwlJ;发酵结果显示,B. subtilis 168-TresΔsleBΔcwlJ与出发菌株的芽孢形成率一致,约为88%;在LB固体培养基和麦芽糖转化生成海藻糖体系中B. subtilis 168-Tres的芽孢萌发数为4.8×108 CFU/mL,B. subtilis 168-TresΔsleBΔcwlJ芽孢未萌发;在麦芽糖转化生成海藻糖体系中,重组菌海藻糖合酶酶活为10.42 U,比原始菌提高了78.7%。敲除sleB、cwlJ基因后,不影响枯草芽孢杆菌生成芽孢的量,但能有效控制芽孢在上述转化体系中的萌发,使芽孢表面稳定展示海藻糖合酶,提高了芽孢的利用率。     

Chemical Induction of Mutation or Variation In Aflatoxin-Producing Cultures of Aspergillus Flavus

SUMMARY– The mutation or variation of a strain of Aspergillus flavus was induced chemically after six successive "generations" of exposures to barium ions. Colony isolates of A. flavus , developing from spores harvested from cultures grown in barium medium, were examined for mutation on the basis of morphology, loss of aflatoxin-producing capacity, and loss of ability to grow in basal medium. The new characteristics exhibited by the mutant of A. flavus are the inability to produce aflatoxins and yellow pigment, accompanied by the loss of fluorescence in the culture under ultraviolet light. These changes did not revert after more than eight successive transfers in a barium-free medium. Mutagenic action of barium ions on the fungus A. flavus was shown by the mutatation of colonies arising from single spores, The characteristics of the mutant are permanent and irreversible.     

Effect of small, acid-soluble proteins on spore resistance and germination under a combination of pressure and heat treatment

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of alpha/beta-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65 degrees C and 100 to 500 MPa) and applied to wild-type and SASP-alpha-/beta- Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65 degrees C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65 degrees C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65 degrees C inactivated wild-type SASP-alpha+/beta+ spores more than mutant SASP-alpha-/beta- spores, and this was attributed to less pressure-induced germination in SASP-alpha-/beta- spores than in wild-type SASP-alpha+/beta+ spores. However, there was no difference in the pressure resistance between SASP-alpha+/beta+ and SASP-alpha-/beta- spores at 100 MPa and ambient temperature (approximately 22 degrees C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that alpha/beta-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.     

Evaluation of resistance development by Tribolium confusum Du Val (Coleoptera: Tenebrionidae) to diatomaceous earth under laboratory selection

Laboratory selection experiments were undertaken in order for the resistance development of Tribolium confusum adults to diatomaceous earth (DE) to be assessed. Adults from an initial laboratory reared population (P) were selected by exposure on hard wheat treated with 800 ppm of DE for 4 days at 25 °C and 65% r.h. The surviving adults and their progeny were maintained on flour and 5% brewer's yeast until the emergence of the next generation of adults. The emerged (F₁) adults were again exposed on treated hard wheat, as described above, and the whole procedure was repeated for 10 consecutive generations. Mortality of the emerged adults was assessed through bioassays that were undertaken in each generation at 25 °C and 65% r.h. In each bioassay, five dose levels of DE (250, 500, 800, 1000 and 1500 ppm) and four exposure intervals (24, 48 h, 7 and 14 days) were tested. Mortality of confused flour beetles generally declined at each dose rate and exposure interval with each generation, and significant differences were observed between the initial population (P) and after five or more (F₅+) generations of selection.     

Occurrence of Bacillus cereus spores with a damaged exosporium: consequences on the spore adhesion on surfaces of food processing lines

This study was designed to evaluate the occurrence of Bacillus cereus spores with a damaged exosporium and the consequences of such damages on spore adhesion. The analysis of nine strains sporulated under optimal conditions (Spo8-agar, 30 degrees C) revealed that damaged exosporia were systematically found in one strain (B. cereus D17) and occasionally in two others (B. cereus ATCC 14579T and B. cereus D6). The prevalence of spores with damaged exosporia increased when sporulation occurred under less favorable conditions (Spo8-broth or high temperature); for example, more than 50% of the B. cereus ATCC 14579T spores were damaged when sporulation occurred at 40 degrees C on Spo8-agar or at 30 degrees C in Spo8-broth. Furthermore, when subjected to shear stresses by circulation of spore suspensions through a peristaltic pump, the exosporium of a significant amount of spores became partially or totally shorn off (for example, 40% of the B. cereus ATCC 14579T spores). The ability of damaged spores to adhere to inert surfaces and to resist cleaning under shear stress was significantly affected when compared with intact spores, resulting in a decreased number of adhering spores (P < or = 0.004) and enhanced resistance to cleaning (P < or = 0.008). This study provides evidence that, under various conditions, the exosporium of B. cereus spores can be partly or wholly damaged, thereby affecting the ability of spores to contaminate the surfaces of food processing lines. The presence of spores devoid of exosporium will be of importance in determining the risk associated with B. cereus spores adherent to food processing line surfaces.     

The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history

While bacterial spores are mostly produced in a continuous process, this study reports a two-step sporulation methodology. Even though spore heat resistance of numerous spore-forming bacteria is known to be dependent on sporulation conditions, this approach enables the distinction between the vegetative cell growth phase in nutrient broth and the sporulation phase in specific buffer. This study aims at investigating whether the conditions of growth of the vegetative cells, prior to sporulation, could affect spore heat resistance. For that purpose, wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores, produced via a two-step sporulation process, was determined from vegetative cells harvested at four different stages of the growth kinetics, i.e. early exponential phase, late exponential phase, transition phase or early stationary phase. To assess the impact of the temperature on spore heat resistance, sporulation was performed at 10 °C, 20 °C and 30 °C from cells grown during a continuous or a discontinuous temperature process, differentiating or not the growth and sporulation temperatures. Induction of sporulation seems possible for a large range of growth stages. Final spore concentration was not significantly affected by the vegetative cell growth stage while it was by the temperature during growing and sporulation steps. The sporulation temperature influences the heat resistance of B. weihenstephanensis KBAB4 spores much more than growth temperature prior to sporulation. Spores produced at 10 °C were up to 3 times less heat resistant than spores produced at 30 °C.     

Delayed Clostridium perfringens growth from a spore inocula by sodium lactate in sous-vide chicken products

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag marinated chicken breast that included 0%, 1.5%, 3%, or 4.8% sodium lactate (NaL; w/w). The packages were processed to an internal temperature of 71.1 degrees C, ice chilled and stored at 4, 19, and 25 degrees C. The total C. perfringens population was determined by plating diluted samples on Tryptose-sulfite-cycloserine agar followed by anaerobic incubation for 48 h at 37 degrees C. At 25 degrees C, addition of 1.5% NaL was effective in delaying growth for 29 h. Increasing the NaL level to 4.8%, C. perfringens growth from a spore inoculum during storage at 25 degrees C for 480 h was not observed. At 19 degrees C, the growth was > 6 log 10 cfu/g by 288 h in control samples. In samples with 3.0% or 4.8% NaL, the growth of C. perfringens from spores was dramatically restricted with little or no growth in 648 h at 19 degrees C. C. perfringens growth was not observed at 4 degrees C regardless of NaL concentration. The D-values at 55 degrees C ranged from 47.40 (no NaL) to 57.58 min (1.5% NaL). Cyclic and static temperature abuse of refrigerated products for 20 h did not permit C. perfringens growth. However, temperature abuse of products for periods 24 h or longer in the absence of NaL led to growth of C. perfringens from a spore inoculum. An extra degree of safety may be assured in such products by supplementation with NaL at 1.5-4.8% NaL level.     

The use of chlorine dioxide to control Alicyclobacillus acidoterrestris spores in aqueous suspension and on apples

Alicyclobacillus acidoterrestris, a thermoacidophilic, spore-forming bacterium, has been identified as a spoilage organism in commercial, pasteurized fruit juices. This study was undertaken to evaluate chlorine dioxide for reducing numbers of A. acidoterrestris spores on laboratory media and on apples. A. acidoterrestris spores in aqueous suspension and on apple surfaces of four different cultivars were treated with several concentrations of chlorine dioxide. Spores in aqueous suspension treated with 40 ppm for 5 min were reduced by more than 4 log. Treatment with 80 ppm for 1 min and 120 ppm for 30 s resulted in about 1.8 log and 4.8 log reductions of spore viability, respectively, and treatment at 80 and 120 ppm for 5 min reduced spore viability to undetectable levels (<0.7 log CFU/ml). When applied to the surfaces of four different apple cultivars ('Red Delicious', 'Golden Delicious', 'Gala', and 'Fuji'), 40 ppm free chlorine dioxide reduced A. acidoterrestris spore numbers by 1.5, 3.2, 4.5, >4.8 log after 1-, 2-, 3-, and 4-min treatments, respectively. Spore numbers were reduced by >4.8 log with 120 ppm free chlorine dioxide after only 1-min treatment. However, there was no significant difference between apple cultivars (P>0.05) on spore reduction. These results show the great effectiveness of chlorine dioxide in controlling A. acidoterrestris spores both in aqueous suspension and on apple surfaces. There was no synergistic effect on spore reduction when chlorine dioxide treatment of aqueous suspension was followed by heat.     

Modeling heat resistance of Bacillus weihenstephanensis and Bacillus licheniformis spores as function of sporulation temperature and pH

Although sporulation environmental factors are known to impact on Bacillus spore heat resistance, they are not integrated into predictive models used to calculate the efficiency of heating processes. This work reports the influence of temperature and pH encountered during sporulation on heat resistance of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 spores. A decrease in heat resistance (δ) was observed for spores produced either at low temperature, at high temperature or at acidic pH. Sporulation temperature and pH maximizing the spore heat resistance were identified. Heat sensitivity (z) was not modified whatever the sporulation environmental factors were. A resistance secondary model inspired by the Rosso model was proposed. Sporulation temperatures and pHs minimizing or maximizing the spore heat resistance (T(min(R)), T(opt(R)), T(max(R)), pH(min(R)) and pH(opt(R))) were estimated. The goodness of the model fit was assessed for both studied strains and literature data. The estimation of the sporulation temperature and pH maximizing the spore heat resistance is of great interest to produce spores assessing the spore inactivation in the heating processes applied by the food industry.