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.     

Potentiation of the lethal effect of peroxygen on Bacillus cereus spores by alkali and enzyme wash

Bacillus cereus present in pipes and heat-exchangers represents a potential quality problem for dairy industry. The peroxygen-containing disinfectants investigated had only negligible sporicidal effect when applied at the recommended in-use temperature and concentration. However, cleaning agents used before disinfection potentiated their lethal activity. Pre-exposure of B. cereus spores to 1% sodium hydroxide at temperatures over 40 degrees C increased the sporicidal effect of the peroxygen-containing disinfectant. The effect was dependent on the alkali concentration and the temperature. Also, a significant potentiating activity of an enzyme-based cleaning agent was obtained, but the effect was smaller than for alkali treatment. The results indicated that disinfectants based on peroxygen can be used to eliminate B. cereus spores at non-corrosive temperatures and concentrations if the surfaces are cleaned with alkali or enzyme-based disinfectants prior to disinfection.     

Germination of Bacillus cereus spores adhered to stainless steel

Adhered spores of Bacillus cereus represent a significant part of the surface-derived contamination in processing equipment used in the dairy industry. As germinated spores lose their resistance capacities instantaneously, efficient germination prior to a cleaning in place treatment could aid to the disinfecting effect of such a treatment. Therefore, spores of B. cereus ATCC 14579 and that of the environmental isolate B. cereus CMCC 3328 were assessed for their germination behaviour when adhered to a stainless steel surface. A mixture of l-alanine and inosine initiated germination of adhered spores efficiently, resulting in 3.2 decimal logarithms of germination. Notably, implementation of a germination-inducing step prior to a representative cleaning in place procedure reduced the number of survivors with over 3 decimal log units, while an alkali treatment alone, as part of the cleaning in place procedure, did not show any effect on B. cereus spore viability. These results show that implementation of a germination step enhances the disinfection effect of currently used cleaning in place procedures.     

Decontamination of fluid milk containing Bacillus spores using commercial household products

Although commercial sanitizers can inactivate bacterial spores in food processing environments, relatively little data exist as to the decontamination of products and surfaces by consumers using commercial household products. Should a large scale bioterrorism incident occur in which consumer food products were contaminated with a pathogenic sporeformer such as Bacillus anthracis, there may be a need to decontaminate these products before disposal as liquid or solid waste. Studies were conducted to test the efficacy of commercial household products for inactivating spores of Bacillus cereus (used as a surrogate for B. anthracis) in vitro and in fluid milk. Validation of the resistance of the B. cereus spores was confirmed with B. anthracis spores. Fifteen commercial products, designed as either disinfectants or sanitizers or as potential sanitizers, were purchased from retail markets. Products selected had one of the following active compounds: NaOCl, HCl, H2O2, acetic acid, quaternary ammonium compounds, ammonium hydroxide, citric acid, isopropanol, NaOH, or pine oil. Compounds were diluted in water (in vitro) or in 2% fat fluid milk, and spores were exposed for up to 6 h. Products containing hypochlorite were most effective against B. cereus spores. Products containing HCl or H2O2 also reduced significant numbers of spores but at a slower rate. The resistance of spores of surrogate B. cereus strains to chlorine-containing compounds was similar to that of B. anthracis spores. Therefore, several household products on the market may be used to decontaminate fluid milk or similar food products contaminated by spores of B. anthracis.     

Promoting Bacillus cereus spore germination for subsequent inactivation by mild heat treatment

Sublethal heat treatment may activate dormant spores and thereby potentiate the conversion of spores to vegetative cells. As the germinated spore is known to possess lower heat resistance than its dormant counterpart, it has been postulated that double heat treatment, i.e., spore heat activation followed by germination and then by heat inactivation, can be used to control spores in foods. Production of refrigerated processed foods of extended durability often includes more than one heat treatment of the food components. This work simulates conventional heat treatment procedures and evaluates double heat treatment as a method to improve spore control in model food matrixes of meat broth and cream sauce. Bacillus cereus NVH 1230-88 spores were supplemented in food model matrixes and heat activated at 70°C and then heat inactivated at 80 or 90°C. The samples were held at 29 to 30°C for 1 h between primary and secondary heat treatments, to allow spore germination. Nutrients naturally present in the food matrixes, e.g., amino acids and inosine, could act as germinants that induce germination. The levels of germinants could be too low to produce effective germination within 1 h. Following primary heat treatment, some samples were therefore supplemented with a combination of L-alanine and inosine, a germinant mixture known to be effective for B. cereus spores. In both matrixes, a combination of double heat treatment (heat activation, germination, and inactivation) and addition of germinants gave a reduction in spore counts equivalent to or greater than that obtained with a single heat treatment for 12 min at 90°C. Addition of germinants was essential to induce effective germination in cream sauce during 1 h at 29 to 30°C, and germinants were therefore a crucial supplement to obtain an effect of double heat treatment in this matrix. These data will be valuable when setting up temperature-time-germinant combinations for an optimized spore reduction in mild-heat-treated foods.     

FACTORS INFLUENCING THE INCIDENCE OF B. CEREUS SPORES IN MILK

The many factors involved in the contamination of milk with B. cereus cells and the importance of minimizing the number of spores at the time of pasteurization are discussed. Methods of controlling the level of B. cereus spores and the use of heat in the cleaning and sterilizing operation are shown, and studies on the level of aerobic spores in raw milk supplies showing a seasonal effect evaluated.     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.     

Synergistic Effect of Electrolyzed Water and Citric Acid Against Bacillus Cereus Cells and Spores on Cereal Grains

ABSTRACT:  The effects of acidic electrolyzed water (AcEW), alkaline electrolyzed water (AlEW), 100 ppm sodium hypochlorite (NaClO), and 1% citric acid (CA) alone, and combinations of AcEW with 1% CA (AcEW + CA) and AlEW with 1% CA (AlEW + CA) against Bacillus cereus vegetative cells and spores was evaluated as a function of temperature (25, 30, 40, 50, or 60 °C) and dipping time (3 or 6 h). A 3-strain cocktail of Bacillus cereus cells or spores of approximately 10⁷ CFU/g was inoculated in various cereal grains (brown rice, Job's tear rice, glutinous rice, and barley rice). B . cereus vegetative cells and spores were more rapidly inactivated at 40 °C than at 25 °C. Regardless of the dipping time, all treatments reduced the numbers of B . cereus vegetative cells and spore by more than 1 log CFU/g, except the deionized water (DIW), which showed approximately 0.7 log reduction. The reductions of B . cereus cells increased with increasing dipping temperature (25 to 60 °C). B . cereus vegetative cells were much more sensitive to the combined treatments than spores. The effectiveness of the combined electrolyzed water (EW) and 1% CA was considerable in inhibiting B . cereus on cereal grains. The application of combined EW and CA for controlling B . cereus cells and spores on cereal grains has not been previously reported. Therefore, the synergistic effect of EW and CA may provide a valuable insight on reducing foodborne pathogens on fruits, vegetables, and cereal grains.     

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.     

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.     

Application of an ultrasound field in chemical cleaning of ceramic tubular membrane fouled with whey proteins

Chemical cleaning of a severe in-pore-fouling may be improved by applying an ultrasound (US) field in a cleaning-in-place (CIP) system, under both the batch and flow conditions. This study is concerned with the cleaning of a 200-nm ceramic membrane, fouled with whey proteins, in an US field of relatively low frequency of 35 kHz, without applying a transmembrane pressure (TMP) and cross-flow velocity. Two types of cleaning agent solutions of different concentrations were applied: alkali (NaOH) and a mixture of commercial detergents (P3-Ultrasil 67 and 69) at a sonication time of 30 min. It was found that the application of US was less effective in the combination with sodium hydroxide than with the mixture of commercial detergents. Using US in a mixture of 0.25% w/w P3-Ultrasil 67 and 0.4% w/w P3-Ultrasil 69 resulted in the highest flux recovery of 86.5 ± 2.9%, after 30 min of sonication, and produced an overall efficiency of 96.3 ± 0.4%, after the second sonication. It was concluded that the application of the US field in a batch mode, combined with the mentioned chemical agents, can significantly improve the cleaning efficiency.     

Efficacy of amphoteric surfactant- and peracetic acid-based disinfectants on spores of bacillus cereus in vitro and on food premises of the German armed forces

In mass catering facilities of the German Armed Forces, foodborne outbreaks are commonly attributed to Bacillus cereus, and spores of this organism are regularly found on equipment surfaces. B. cereus is the causative agent of foodborne emetic or diarrheic disease. Hence, the use of sporicidal disinfectants may provide a starting point for successful risk mitigation of diseases associated with B. cereus. In this study, the amphoteric surfactant-based disinfectant (product A) that has been routinely used in catering facilities of the German Armed Forces and a peracetic acid-based disinfectant (product B) were compared for their efficacy against B. cereus spores in laboratory tests and under field conditions. In a carrier test for B. cereus spores, product A displayed no efficacy against spores of the test strain. In contrast, a substantial reduction in spore concentration (>5-log reduction) was achieved with product B. In a controlled trial conducted in seven Army catering facilities, the reduction in the number of B. cereus-positive samples was significantly greater (P < 0.001) for product B (from 28% initial contamination to 3% after application) than for product A (from 33 to 21%). Product B was estimated to be 6.25 times more efficacious than product A. Field trials confirmed the higher efficacy of the peracetic acid-based disinfectant to reduce B. cereus contamination on surfaces as found for the in vitro experiments. The tests used to assess the sporicidal effects of disinfectants were adequate under practice conditions and could be used for sporicidal testing of disinfectants for catering and food production establishments.     

Effect of chemical dips on unchilled fresh beef inoculated with E. coli, S. aureus, S. faecalis and Cl. perfringens and Stored at 30°C and 20°C

The antimicrobial effect of three chemical dips (A: potassium sorbate, 20g; sodium acetate, 5g; sodium chloride, 5g; water, 100 ml. B: potassium sorbate, 20g; sodium acetate, 5g; sodium chloride, 5g; propylene glycol, 20 ml; glycerol, 10 ml; water, 70 ml and 0·5N NaOH to pH 10. C: potassium sorbate, 10 g; sodium acetate, 10 g; sodium citrate, 10g; sodium chloride, 5g; water, 100 ml and 0·5N NaOH to pH 10) on unchilled f resh beef samples inoculated with E. coli, S. aureus, S. faecalis and Cl. perfringens and stored at 30°C and 20 °C was investigated. Treatments had produced a significant (P < 0·01) inhibitory effect on all the organisms inoculated, with a reduction of about 3 to 5 log units compared with the control. pH changes were comparable with the control at 5°C. No significant differences were found between the treatments and shelf life could be extended up to 44 h at 30°C and 68 h at 20°C whilst, in the control, visual spoilage and high bacterial counts were observed by 20h at 30°C and by 20 to 32h at 20°C. Treatment C, containing potassium sorbate at a low level (10%), was considered better than treatments A and B since potassium sorbate at higher levels had produced slight off-odours without a greater inhibitory effect.     

STUDIES ON FLOWDYNAMICS OF FALLING FILMS OF WATER AND ITS EFFECT ON CLEANING OF STAINLESS STEEL

SUMMARY— Factors such as inclination angle of the test surface, flow rate and temperature and composition of cleaning solutions were examined for their influence on cleaning an 18-in.-wide sheet of No. 4 finish stainless steel by gravity flow of falling films of water and detergent solutions. A suspension of B. subtilis spores was used to contaminate the test surface. After a cleaning treatment, the percentage of spores remaining as measured by a direct surface agar plate method IDSAPI was used to calculate cleaning efficiency. Flows of water and cleaning solutions ranged from 50 to 250 lb/min. After detergent solutions, a post-rinse with water at 100°F for 5 min was used. Another setup with a smaller plate and a constant flow rate of 45 lb/min was used to study the effect on cleaning of varying plate angle between the limits of 60 degrees either side of vertical. Washing time was standardized at 10 min for the large plate and 15 min for the smaller one. Results indicated that cleaning improved with temperature, flow rate and with the presence of acid or alkaline cleaning agents. Cleaning was most effective when the plates were in the vertical position.     

Life cycle assessment (LCA) of cleaning-in-place processes in dairies

Four Cleaning-In-Place (CIP) methods for dairies were compared using life cycle assessment (LCA). The methods were conventional alkaline/acid cleaning with hot water disinfection, one-phase alkaline cleaning with acid chemical disinfection, enzyme-based cleaning with acid chemical disinfection and the conventional method with disinfection by cold nitric acid at pH 2. Production of detergents, transport, the user phase in the dairy and waste management of containers were included. The user phase was found to be the most important part of the life cycle. The CIP methods with small volumes and low temperatures, such as enzyme-based cleaning and one-phase alkaline cleaning, turned out to be the best alternatives for the impact categories energy use, global warming, acidification, eutrophication and photo-oxidant formation. Milk residues flushed out in the rinsing phase were the main contributor to eutrophication, but the phosphorus and nitrogen in the detergents also influenced the results. Evaluation of toxic substances poses a methodological problem in LCA. In this study, detergents partly composed of toxic substances were included, and the overall assessment was that the one-phase alkaline cleaning method was preferable from an environmental point of view. A qualitative assessment of toxicity was performed.     

烛式过滤机过滤元件清洗方法研究

采用化学方法对烛式过滤机滤片的清洗进行研究,通过单因素和正交试验对清洗工艺进行优化,结果表明:NaOH清洗效果较好,其最佳工艺为NaOH浓度50 g/L,清洗温度60℃,清洗时间2 h。     

A response surface investigation of the effects of sodium chloride and tripolyphosphate on the thermal properties of beef muscle

A response surface investigation, utilizing a central composite rotatable design, measured the thermal response of beef muscle samples treated with sodium chloride (NaCl, 0·5-2%) and sodium tripolyphosphate (TPP, 0·2-0·6%). Low temperature transitions were reduced in enthalpy and transition temperature by increased salt concentration. Enthalpy was raised by increased TPP levels. Total enthalpy, over the temperature range 45-90°C, demonstrated the strong stabilizing effect of TPP at salt levels below 1%, and the lack of effect of TPP concentrations over 0·5% with salt levels greater than 1·25%. NaCl and TPP concentrations in excess of 1·5 and 0·4%, respectively, show little additional effect on thermal response.     

Evaluation of the direct epifluorescent filter technique for assessing the hygienic condition of milking equipment

The hygienic condition of 6 milking installations, 3 sanitized by circulation cleaning (CC) with chlorine-based chemicals and 3 by flushing with acidified boiling water (ABW), was tested using rinses of quarter strength Ringer's solution. The bacterial content of the rinses was determined using both colony counts and the direct epifluorescent filter technique (DEFT). A comparison of testing methods gave correlation coefficients between colony count and DEFT of 0.82 for plants using CC and 0.46 for plants using ABW. Five strains of bacteria belonging to different genera and commonly found on milking equipment were exposed to various degrees of heat and to various concentrations of chlorine. The effects of such treatments on the staining characteristics of the organisms were studied. It was observed that Staphylococcus aureus and Streptococcus lactis, although killed by heat treatment, stained a bright orange when treated with acridine orange dye. Pseudomonas fluorescens, Escherichia coli and vegetative cells of Bacillus cereus did not take up the orange stain after heat treatment, nor did any of the 5 strains stain orange after treatment with NaOCl. It is suggested that the DEFT is a useful and rapid means of counting bacteria in rinses of equipment where sterilization is due primarily to chlorination, but in the absence of a stain which can differentiate more accurately between dead and living organisms its use is limited where sterilization is carried out solely by heat.     

REMOVAL KINETICS OF BACILLUS CEREUS SPORES FROM STAINLESS STEEL PIPES UNDER CIP PROCEDURE: INFLUENCE OF SOILING AND CLEANING CONDITIONS

Cleaning efficiency is of prime importance for food industries to ensure both the quality and safety of the products. The removal kinetics of Bacillus cereus spores adhering to unheated stainless steel pipes was studied under turbulent flow conditions (Reynolds’number of 77500 and 116300) in order to be close to those encountered in industrial practice. The experimental data was fitted using a hyperbolic tangent model. Variance analysis was then performed to underline any potential effects on the kinetics of the processing parameters, such as soiling conditions, soiling media and mean walls shear stress during cleaning. A significant influence of the adhesion medium (milk or saline) is shown at the level of spore removal (P < 0.001). This trend could probably be explained by the change in the surface properties of spores and stainless steel surfaces when covered by milk macromolecules. After milk soiling in turbulent flow conditions, removal efficiency was enhanced by a factor of 2.5 to remove 50% of the initial spore contamination and by a factor of 2 for the remaining spores after 30 min of cleaning. No effect of the two mean wall shear stresses (9.4 and 19.1 Pa) has been identified. The removal kinetic model proposed here could now permit the effect on the cleaning efficiency of a wide range of CIP conditions to be tested.     

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

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