四种不同接触表面蜡样芽孢杆菌菌膜形成的影响

为了解食源性致病菌蜡样芽孢杆菌在食品加工环境中菌膜形成能力,以玻璃、不锈钢、聚氯乙烯、聚丙烯为接触面,采用超声波平板菌落计数法测定不同环境因素(温度、pH、氯化钠、葡萄糖、苯甲酸钠及山梨酸钾)、不同材料表面蜡样芽孢杆菌(B.cereus)菌膜形成的变化趋势。结果表明:四种材质表面形成B.cereus菌膜能力的大小顺序为:玻璃 > 不锈钢 > 聚氯乙烯 > 聚丙烯。其中,30 ℃,pH7.0时菌膜形成量最大,添加低浓度葡萄糖(4.0%)或氯化钠(0.5%)对B.cereus菌膜形成有显著促进作用(p<0.05),添加0.15%苯甲酸钠、山梨酸钾的菌膜形成量显著高于添加0.10%的菌膜形成量(p<0.05)。本研究为蜡样芽孢杆菌风险评估提供基础数据,为食品工业蜡样芽孢杆菌菌膜的预防和控制奠定基础,为改进蜡样芽孢杆菌的清洗控制措施提供参考。     

Lethality of chlorine, chlorine dioxide, and a commercial produce sanitizer to Bacillus cereus and Pseudomonas in a liquid detergent, on stainless steel, and in biofilm

Many factors that are not fully understood may influence the effectiveness of sanitizer treatments for eliminating pathogens and spoilage microorganisms in food or detergent residues or in biofilms on food contact surfaces. This study was done to determine the sensitivities of Pseudomonas cells and Bacillus cereus cells and spores suspended in a liquid dishwashing detergent and inoculated onto the surface of stainless steel to treatment with chlorine, chlorine dioxide, and a commercial produce sanitizer (Fit). Cells and spores were incubated in a liquid dishwashing detergent for 16 to 18 h before treatment with sanitizers. At 50 microg/ml, chlorine dioxide killed a significantly higher number of Pseudomonas cells (3.82 log CFU/ml) than did chlorine (a reduction of 1.34 log CFU/ml). Stainless steel coupons were spot inoculated with Pseudomonas cells and B. cereus cells and spores, with water and 5% horse serum as carriers. Chlorine was more effective than chlorine dioxide in killing cells and spores of B. cereus suspended in horse serum. B. cereus biofilm on stainless steel coupons that were treated with chlorine dioxide or chlorine at 200 microg/ml had total population reductions (vegetative cells plus spores) of > or = 4.42 log CFU per coupon; the number of spores was reduced by > or = 3.80 log CFU per coupon. Fit (0.5%) was ineffective for killing spot-inoculated B. cereus and B. cereus in biofilm, but treatment with mixtures of Fit and chlorine dioxide caused greater reductions than did treatment with chlorine dioxide alone. In contrast, when chlorine was combined with Fit, the lethality of chlorine was completely lost. This study provides information on the survival and sanitizer sensitivity of Pseudomonas and B. cereus in a liquid dishwashing detergent, on the surface of stainless steel, and in a biofilm. This information will be useful for developing more effective strategies for cleaning and sanitizing contact surfaces in food preparation and processing environments.     

Preservation of aseptic conditions in absorbent pads by using silver nanotechnology

Silver nanoparticles have been formed in fluff pulp and nanostructured Lyocell fibres by immersion in silver nitrate, and a subsequent transformation of the adsorbed silver ions into elementary silver nanoparticles by physical (thermal/UV) or chemical (sodium borohydride) methods. Microscopy revealed that nanoparticles generated by physical methods were regular in shape and efficiently dispersed, while the chemical reduction produced highly aggregated nanoparticles. Nanoparticle size has been found relevant to guarantee high antimicrobial activity, being the samples with big aggregated silver nanoparticles almost inefficient. Indeed a satisfactory correlation between silver ion release and the antimicrobial efficiency against Escherichia coli and Staphylococcus aureus could be confirmed, and furthermore, the highest concentrations tested were efficient to reduce the microbial load in poultry exudates. This work demonstrates that especially designed absorbent materials could be optimised to preserve aseptic conditions during manipulation, leading to feasible applications of a silver based nanotechnology in food technology.     

The role of heat exchangers in the contamination of milk with Bacillus cereus in dairy processing plants

In the dairy industry microbiological contamination may arise from equipment used for handling or processing. In this study it was demonstrated that Bacillus cereus spores could adhere stainless steel, germinate and/or multiply in a tube heat exchanger. The levels of B cereus detect in the tubes, after 24 h at 20°C, varied from less than 1 to 2600 cm^-2. Adhering cells and/or spores were more resistant to cleaning with K500 and sodium hydroxide, determined by laboratory suspension tests and surface tests on stainless steel. This was confirmed in a field trial with a tube heat exchanger after cleaning B cereus could still be isolated from all tubes determined by swab samples of individual tubes. The identity of the isolates was checked by polymerase chain reaction/randomly amplified polymorphic DNA typing to confirm that the organisms found on the surfaces were the same as the bacteria in the ingoing milk.     

Control of Bacillus cereus in foods by Rhodomyrtus tomentosa (Ait.) Hassk. Leaf extract and its purified compound

Edible natural products, either standardized plant extracts or pure compounds, provide unlimited opportunities as safe new antimicrobial agents. This study investigated the antibacterial properties of ethanolic extract from Rhodomyrtus tomentosa (Ait.) Hassk. leaves against Bacillus cereus. Preliminary screening to evaluate the activities of the extract used a paper disc diffusion method against 65 food isolates. The extract produced large inhibition zones in all isolates, ranging from 10 to 18 mm. The results were confirmed by MIC and MBC (16 to 64 and 32 to 256 μg/ml, respectively). Rhodomyrtone, a purified compound, exhibited MIC and MBC at 0.5 and at 2 to 8 μg/ml, respectively. The antimicrobial activity of the extract on vegetative cells and endospores of a representative B. cereus isolate (MIC=32 μg/ml) was assessed by enumerating viable cells at different time intervals up to 24 h. At 2 MICs and 4 MICs, a reduction in the viability of the bacterial cells and endospores was at least 3 log within 6 to 8 h and 2 h after incubation, respectively. Application of the extract in precooked rice and tuna steak demonstrated that after exposure to 16 MICs and 32 MICs, the numbers of viable cells and endospores in both model systems were reduced by at least 2 log within 12 and 6 h, respectively. Since the extract consistently produced remarkable activity against both cells and endospores, it could be used as an alternative food additive for controlling B. cereus without compromising food safety.     

Evaluation of chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer for effectiveness in killing Bacillus cereus and Bacillus thuringiensis spores in suspensions, on the surface of stainless steel, and on apples

Chlorine (10 to 200 microg/ml), chlorine dioxide (10 to 200 microg/ml), and a peroxyacetic acid-based sanitizer (40 and 80 microg/ ml) were evaluated for effectiveness in killing spores of Bacillus cereus and Bacillus thuringiensis in suspensions and on the surface of stainless steel and apples. Water and 5% horse serum were used as carriers for spore inoculum applied to the surface of stainless steel coupons, and 5% horse serum was used as a carrier for inoculum applied to apples. Inocula were dried on stainless steel for 5 h and on apples for 22 to 24 h before treating with sanitizers. At the concentrations of sanitizers tested, sensitivities of planktonic B. cereus and B. thuringiensis spores were similar. A portion of the spores surviving treatment with chlorine and, more markedly, chlorine dioxide had decreased tolerance to heat. Planktonic spores of both species were more sensitive to sanitizers than were spores on the surface of stainless steel or apples. At the same concentrations, chlorine was more effective than chlorine dioxide in killing spores in suspension and on stainless steel. The lethality of chlorine dioxide was markedly reduced when inoculum on stainless steel coupons was suspended in 5% horse serum as a carrier rather than water. Chlorine and chlorine dioxide at concentrations of 10 to 100 microg/ml were equally effective in killing spores on apples. Significant reductions of > or = 3.8 to 4.5 log CFU per apple were achieved by treatment with 100 microg/ml of either of the two sanitizers. The peroxyacetic acid sanitizer (40 and 80 microg/ml) was ineffective in killing Bacillus spores in the test systems investigated. Results provide information on the effectiveness of sanitizers commonly used in the food processing industry in killing Bacillus spores in suspension, on a food-contact surface, and on a ready-to-eat food.     

Aptamer‐Based SERS Detection of Lysozyme on a Food‐Handling Surface

Undeclared food allergens due to cross contamination of processing equipment is a leading cause for food product recalls. Therefore, there is a great need for developing rapid and sensitive methods to detect food allergens. In this paper, an aptamer highly specific to egg white lysozyme was coupled to dendritic silver nanoparticles in order to perform surface enhanced Raman spectroscopy (SERS). The procedure was successfully tested in water and on a stainless steel food‐handling surface. The lowest detectable concentration for lysozyme was 0.5 μg/mL in water and 5 μg/mL on a stainless steel food‐handling surface. Principal component analysis shows a significant change in SERS spectra when lysozyme was present, suggesting the successful capture of lysozyme by the aptamer. Quantification of lysozyme target was also shown from 0 to 6 μg/mL, that is, 0, 0.5, 2, 6 μg/mL. Overall method took less than 40 min. The developed method can be extended to detect other food allergens using specific aptamers.     

Effect of food residues on efficiency of surfactant disinfectants against food related pathogens adhered on polystyrene and ceramic surfaces

The adhesion of pathogenic bacteria on food contact surfaces increases the risk of cross-contamination in the food industry. However, food-borne disease introduced by the production process can be mitigated by surfactant use. This study investigates the effect of food residues (milk, beef gravy and tuna gravy) on the bactericidal efficiency of benzalkonium chloride (BAC) and alkyldiaminoethylglycine hydrochloride (AGH). The test was conducted on pathogenic bacteria (Escherichia coli O26, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and B. cereus spores) dried and adhered to the surfaces of polystyrene and ceramic dishes at room temperature for 1.5 h. The protein and lipid rich food residues protected the bacterial cells from dehydration and from the adverse effects of disinfectants, although bacterial numbers were decreased after drying and the surfaces were clearly sterilized after disinfectant treatment at typical concentrations (0.5 mg/ml–2.0 mg/ml) for 10 min. Following general and proper washing processes, the bactericidal effect of the disinfectants became clearly visible. These results indicate that applying a proper washing process prior to disinfectant treatment can prevent cross-contamination.     

Poly (DL-lactide-co-glycolide) (PLGA) nanoparticles with entrapped trans-cinnamaldehyde and eugenol for antimicrobial delivery applications

Eugenol and trans-cinnamaldehyde are natural compounds known to be highly effective antimicrobials; however, both are hydrophobic molecules, a limitation to their use within the food industry. The goal of this study was to synthesize spherical poly (DL-lactide-co-glycolide) (PLGA) nanoparticles with entrapped eugenol and trans-cinnamaldehyde for future antimicrobial delivery applications. The emulsion evaporation method was used to form the nanoparticles in the presence of poly (vinyl alcohol) (PVA) as a surfactant. The inclusion of antimicrobial compounds into the PLGA nanoparticles was accomplished in the organic phase. Synthesis was followed by ultrafiltration (performed to eliminate the excess of PVA and antimicrobial compound) and freeze-drying. The nanoparticles were characterized by their shape, size, entrapment efficiency, and antimicrobial efficiency. The entrapment efficiency for eugenol and trans-cinnamaldehyde was approximately 98% and 92%, respectively. Controlled release experiments conducted in vitro at 37 °C and 100 rpm for 72 h showed an initial burst followed by a slower rate of release of the antimicrobial entrapped inside the PLGA matrix. All loaded nanoparticles formulations proved to be efficient in inhibiting growth of Salmonella spp. (Gram-negative bacterium) and Listeria spp. (Gram-positive bacterium) with concentrations ranging from 20 to 10 mg/mL. Results suggest that the application of these antimicrobial nanoparticles in food systems may be effective at inhibiting specific pathogens. PRACTICAL APPLICATION: Nanoencapsulation of lipophilic antimicrobial compounds has great potential for improving the effectiveness and efficiency of delivery in food systems. This study consisted of synthesizing PLGA nanoparticles with entrapped eugenol and trans-cinnamaldehyde. By characterizing these new delivery systems, one can understand the controlled-release mechanism and antimicrobial efficiency that provides a foundation that will enable food manufacturers to design smart food systems for future delivery applications, including packaging and processing, capable of ensuring food safety to consumers.     

Evaluation of a novel antimicrobial (lauric arginate ester) substance against biofilm of Escherichia coli O157:H7, Listeria monocytogenes,and Salmonella spp.

The purpose of this study was to evaluate the activity of a novel antimicrobial substance lauric arginate ester (LAE) against selected foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp.) in biofilm. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined and showed that LAE exhibits a strong antimicrobial activity. Biofilms were grown on abiotic stainless steel, rubber, MBEC biofilm device) and biotic (lettuce) surfaces. The efficacy of LAE (50, 100 and 200 ppm) at reducing the biofilm cells on these surfaces was examined by applying LAE for 2 h. Results revealed that LAE exhibited the reduction in biofilm bacteria up to 7 log CFU cm^?2, 3.5 log CFU cm^?2, 4.0 log CFU peg^?1 and 1.5 log CFU cm^?2 on stainless steel, rubber, MBEC and lettuce surfaces, respectively. Overall, these results suggest that LAE has been shown to be a potential alternative to control bacteria in biofilm mode in food industry.     

Atmospheric pressure plasma jet inactivation of Pseudomonas aeruginosa biofilms on stainless steel surfaces

This study established the efficacy of atmospheric pressure plasma jet on Pseudomonas aeruginosa on stainless steel types 316 and 304; with different finishes namely, mirror (MR), hairline (HL) and 2B surfaces. A cocktail of four strains of P. aeruginosa in the mid-stationary growth phase were allowed to attach on the test surfaces, and subjected to atmospheric pressure plasma jet treatment with an air injection rate of 5 l/min, output power of 360 W at 4.22 cm source-to-surface distance. Attachment rates were significantly affected by surface finish, rather than by stainless steel type. The D-values on the 316 stainless steel type ranged from 2.53 s (MR) to 3.16 s (2B); while those on the 304 type ranged from 1.95 s (HL) to 3.27 s (2B). Variations in D-values were observed between surface finishes within a specific stainless steel type. However, significant variations were not observed between the same surface finish of different steel types. The observed antimicrobial efficacy was attributed to the generation of reactive oxygen species, ultraviolet-C rays, and rapid temperature increase (final temperature of 143.42 °C to 174.05 °C) within 15 s of treatment. In the absence of heating, the D-value increased to 16.45 s, but a 5-log (99.999%) reduction in the population was observed in a relatively short treatment time of 90 s.Industrial relevanceThe results obtained in this work demonstrated the potential of using atmospheric pressure plasma jet technology as a non-chemical, non-thermal, and thermal stainless steel food contact surface decontamination against Pseudomonas aeruginosa, a common biofilm-producing bacterium. Such a technology shall help the industry address the challenge of cross contamination in the food manufacturing and food service settings.     

Effect of photocatalytic and hydrophobic coatings on brewery surface microorganisms

The aim of this study was to determine whether process hygiene in the beverage industry could be improved by applying new coating techniques to process surfaces. Photocatalytic titanium dioxide (TiO(2)) and hydrophobic coatings applied to stainless steel with or without added antimicrobial compounds were studied in laboratory attachment tests and in a 15-month process study. No clear reductions in numbers of attached microbes were obtained with photocatalytic coatings, except for coatings to which silver had been added. These TiO(2)+Ag coatings reduced microbial coverage in laboratory studies and in some process samples. Hydrophobic coatings reduced the area coverage of microorganisms in 4-h laboratory studies but did not affect colony counts in laboratory or process studies. The surfaces had changed from hydrophobic into hydrophilic during the process study. The coatings did not mechanically fully withstand process conditions; part of the hydrophobic coatings had peeled off, most of the precipitated Ag had dissolved, and some of the TiO(2) coatings were damaged. In conclusion, functional coatings have potential for reducing microbial loads on beverage industry surfaces, but these coatings need further development.     

The effect of nanometer dimension topographical features on the hygienic status of stainless steel

Wear of food contact surfaces through abrasion may increase the surface roughness and introduce different topographical features. Both of these properties may enhance retention of soil and microorganisms and affect the surface cleanability. To test this hypothesis, stainless steel surfaces with topographical features and surface roughness (Ra) values simulating those of worn in-use surfaces were prepared. Surfaces were imaged and Ra values determined using atomic force microscopy (AFM). These ranged from 23 to 900 nm. Surfaces were sprayed with standardized cell suspensions of Pseudomonas aeruginosa or Staphylococcus aureus and allowed to air dry and were then cleaned using a nonionic detergent delivered via a manual linear cleaning device. There was a 2-log reduction in numbers attached after cleaning, but there was no significant difference (P > 0.05) between the cleanability of the surfaces in terms of the numbers of cells per unit area remaining after cleaning, although cells appeared to be retained within topographical features. Thus, the simulated effect of wear of a hygienic food contact surface did not affect its cleanability after a one-off microbiological soiling event. AFM provided hitherto unavailable information on the topography of worn stainless steel surfaces. In future work, the surfaces will be repeatedly challenged with an organic soil-microorganism mixture after cleaning events, to provide a more rigorous, realistic test.     

Enhancing antimicrobial activity of lysozyme against Listeria monocytogenes using immunonanoparticles

The antimicrobial efficacy of lysozyme may be reduced by undesirable interactions with food components and nontarget bacteria. Immunonanoparticles, i.e., nanoparticles functionalized with pathogen-specific antibodies, may serve as an antimicrobial carrier for improving the stability and activity of antimicrobials in foods. The objective of this research was to study the antimicrobial activity of lysozyme-carrying immunonanoparticles against Listeria monocytogenes. Polystyrene nanoparticles with active carboxyl groups were conjugated with anti-L. monocytogenes antibody through covalent bonding. Enhanced antimicrobial activity of lysozyme-carrying immunonanoparticles was achieved when 0.04 microg/ml anti-L. monocytogenes antibody was used for coating nanoparticles and the resulting immunonanoparticles were then coated with lysozyme for 6 h. Lysozyme-carrying immunonanoparticles with a final concentration of 35 microg/ml reduced L. monocytogenes Scott A populations from ca. 5 log CFU/ml to below the detection limit (< 1 log CFU/ml) within 3 h. However, when 500 microg/ml lysozyme was used, ca. 2 log CFU/ml concentration of L. monocytogenes cells remained culturable after 5 h of treatment. The addition of lysozyme-carrying immunonanoparticles (37 microg/ml) to an L. monocytogenes solution of ca. 7 log CFU/ml for 6 h resulted in 0.9-, 1.0-, and 2.3-log greater reductions of L. monocytogenes cells than that achieved with lysozyme-carrying nanoparticles and lysozyme solutions of 500 and 50 microg/ml, respectively. Overall, lysozyme-carrying immunonanoparticles had significantly more anti-L. monocytogenes activity (P < 0.05) than did lysozyme-carrying nanoparticles and lysozyme solutions at higher concentrations (500 and 50 microg/ml). Our study revealed that the use of lysozyme-carrying immunonanoparticles is more effective than direct addition of lysozyme for inactivating L. monocytogenes in nutrient broth.     

Biofilm formation of Salmonella Typhimurium on stainless steel and acrylic surfaces as affected by temperature and pH level

The effect of temperature (28, 37 and 42 °C) and pH (6 and 7) on the biofilm formation capability of Salmonella Typhimurium on stainless steel and acrylic was investigated. The rate of biofilm formation increased with increasing temperature and pH, while the number of attached cells after 240 h decreased with increasing temperature and was not different between pH 6 and 7. The surface hydrophobicity of bacterial cells was not significantly (p > 0.05) different among tested conditions. Electron-donating/accepting properties changed with pH and temperature, although these changes did not correlate with the ability to form biofilms under respective conditions. Attachment of S. Typhimurium showed a preference for stainless steel compared to acrylic surfaces under all conditions tested. The results suggest that salmonellae were less adherent to acrylic than to stainless steel surfaces; thus, acrylic-type surfaces should be considered for use in the food industry over stainless steel where applicable. The rate of biofilm formation increased at higher temperatures and pH levels within the tested ranges. Hurdle technology using lower temperatures reduced pH may help delay biofilm formation on food contact surfaces contaminated with S. Typhimurium.     

食品无菌包装技术研究进展

随着新型多聚材料和抗菌材料的出现，当今食品行业中将抗菌材料应用于食品无菌包装成为新的研究热点。本文介绍了国际上多种先进的将抗菌多聚物应用于食品包装的方法，以及它们在商业上的应用，并展望未来的无菌包装技术。     

Salmonella enterica Enteritidis biofilm formation and viability on regular and triclosan-impregnated bench cover materials

Contamination of food contact surfaces by microbes such as Salmonella is directly associated with substantial industry costs and severe foodborne disease outbreaks. Several approaches have been developed to control microbial attachment; one approach is the development of food contact materials incorporating antimicrobial compounds. In the present study, Salmonella enterica Enteritidis adhesion and biofilm formation on regular and triclosan-impregnated kitchen bench stones (silestones) were assessed, as was cellular viability within biofilms. Enumeration of adhered cells on granite, marble, stainless steel, and silestones revealed that all materials were prone to bacterial colonization (4 to 5 log CFU/cm(2)), and no significant effect of triclosan was found. Conversely, results concerning biofilm formation highlighted a possible bacteriostatic activity of triclosan; smaller amounts of Salmonella Enteritidis biofilms were formed on impregnated silestones, and significantly lower numbers of viable cells (1 × 10(5) to 1 × 10(6) CFU/cm(2)) were found in these biofilms than in those on the other materials (1 × 10(7) CFU/cm(2)). All surfaces tested failed to promote food safety, and careful utilization with appropriate sanitation of these surfaces is critical in food processing environments. Nevertheless, because of its bacteriostatic activity, triclosan incorporated into silestones confers some advantage for controlling microbial contamination.     

Variable adhesion of Listeria monocytogenes isolates from food-processing facilities and clinical cases to inert surfaces

One hundred one strains of Listeria monocytogenes isolated from seafood and cheese industry samples and from patients with listeriosis were assessed using a microtiter plate method for adhesion to polystyrene and stainless steel surfaces. The adhesion rate for these strains ranged from 3.10 to 35.29% with an inoculum of 8 x 10(8) cells per well. A strong correlation was found between adhesion to polystyrene and stainless steel microtiter plates, indicating that the intrinsic ability of L. monocytogenes to adhere to inert surfaces is stronger than the influence of the surface's physicochemical properties. The clinical strains were less adherent to inert surfaces than were the industrial strains. By integrating other factors such as location of the industrial strains, contamination type of the clinical strains, serotype, and pulsotype into the analysis, some weak but significant differences were noted. For the industrial isolates, the number of cells attached to both surfaces differed significantly depending on whether they were isolated from food or food-processing environments in the seafood and cheese industry. For clinical isolates, sporadic strains exhibited greater adhesion to polystyrene than did epidemic strains. Strains belonging to the pulsed-field gel electrophoretype clusters A and M (lineages II and I, respectively) were less able to adhere to polystyrene and stainless steel than were strains in the more common clusters.     

Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer

Biofilm formation by Bacillus cereus 038-2 on stainless steel coupons, sporulation in the biofilm as affected by nutrient availability, temperature, and relative humidity, and the resistance of vegetative cells and spores in biofilm to sanitizers were investigated. Total counts in biofilm formed on coupons immersed in tryptic soy broth (TSB) at 12 and 22 degrees C consisted of 99.94% of vegetative cells and 0.06% of spores. Coupons on which biofilm had formed were immersed in TSB or exposed to air with 100, 97, 93, or 85% relative humidity. Biofilm on coupons immersed in TSB at 12 degrees C for an additional 6 days or 22 degrees C for an additional 4 days contained 0.30 and 0.02% of spores, respectively, whereas biofilm exposed to air with 100 or 97% relative humidity at 22 degrees C for 4 days contained 10 and 2.5% of spores, respectively. Sporulation did not occur in biofilm exposed to 93 or 85% relative humidity at 22 degrees C. Treatment of biofilm on coupons that had been immersed in TSB at 22 degrees C with chlorine (50 microg/ml), chlorine dioxide (50 microg/ml), and a peroxyacetic acid-based sanitizer (Tsunami 200, 40 microg/ml) for 5 min reduced total cell counts (vegetative cells plus spores) by 4.7, 3.0, and 3.8 log CFU per coupon, respectively; total cell counts in biofilm exposed to air with 100% relative humidity were reduced by 1.5, 2.4, and 1.1 log CFU per coupon, respectively, reflecting the presence of lower numbers of vegetative cells. Spores that survived treatment with chlorine dioxide had reduced resistance to heat. It is concluded that exposure of biofilm formed by B. cereus exposed to air at high relative humidity (> or =97%) promotes the production of spores. Spores and, to a lesser extent, vegetative cells embedded in biofilm are protected against inactivation by sanitizers. Results provide new insights to developing strategies to achieve more effective sanitation programs to minimize risks associated with B. cereus in biofilm formed on food contact surfaces and on foods.     

Physiology of dairy-associated Bacillus spp. over a wide pH range

Bacillus species isolated from alkaline wash solutions used for cleaning in place in South African dairy factories have been suggested to contaminate product contact surfaces of dairy processing equipment and result in post-pasteurization spoilage of milk and milk products. Growth and attachment of such Bacillus isolates under alkaline and acidic conditions have not been previously described. Therefore, the in vitro growth temperature and pH ranges, attachment abilities and hydrophobicity, and enzyme production capabilities of four Bacillus isolates (tentatively identified as B. subtilis115, B. pumilus122, B. licheniformis137 and B. cereus144) previously isolated from the alkaline wash solutions in a South African dairy were examined. Growth pH ranges were determined in buffered Standard One-like Nutrient Broth and in unbuffered 1% Milk Medium at pH values ranging from 3 to 12. Growth and attachment to stainless steel surfaces and production of protease and lipase enzymes were determined in 1% Milk Medium at pH 4, 7 and 10. Colony hydrophobicity of each isolate by the Direction of Spreading Method (DOS) was also determined at pH 4, 7 and 10. In addition, Arrhenius plots were used to examine the growth temperature ranges of the isolates. All isolates grew at pH values ranging from 4.5 to 9.5 in buffered Standard One-like Nutrient Broth, and from pH 4 to 10 in 1% Milk Medium. All isolates also attached to stainless steel at pH 4, 7 and 10 in 1% Milk Medium. Generally the attachment of B. subtilis115, B. pumilus122 and B. lichenformis137 to stainless steel surfaces was enhanced at pH 4 and 10, compared to pH 7. By contrast, the best attachment of B. cereus144 cells to stainless steel surfaces was at pH 7. Planktonic and attached cells of all isolates produced proteolytic enzymes at pH 7 and 10, but not at pH 4. Similarly, planktonic and attached cells of B. subtilis115, B. pumilus122 and B. licheniformis137 produced lipolytic enzymes at pH 7 and 10, and weak lipolysis was observed at pH 4. The Bacillus cereus144 isolate showed no lipolytic activity at pH 10. All isolates exhibited low hydrophobic properties at all pH values even though attachment to stainless steel at the same pH values occurred. None of the isolates grew below 11 degrees C or above 56 degrees C, and optimum growth temperatures were in the high mesophilic range (36-44 degrees C).