Induction of pitting corrosion on stainless steel (grades 304 and 316) used in dairy industry by biofilms of common sporeformers

This study hypothesised that there may be induction of pitting corrosion or microbially‐induced corrosion on stainless steel (SS) dairy‐processing surfaces by biofilms of common milk sporeformers such as Bacillus sporothermodurans and Geobacillus stearothermophilus. Scanning electron microscopy (SEM) and micrographs generated from energy‐dispersive spectroscopy were used to observe pitting corrosion and to find the elemental composition and distribution on the control and pitted surfaces. From SEM images and energy‐dispersive spectroscopy micrographs, it was observed that pitting corrosion on SS could be induced by biofilms of both B. sporothermodurans and G. stearothermophilus.     

Efficient removal of spores from skim milk using cold microfiltration: Spore size and surface property considerations

Bacterial spores present in milk can cause quality and shelf-life issues for dairy products. The objectives of this study were to evaluate the effectiveness of microfiltration (MF) in removing Bacillus licheniformis and Geobacillus sp. spores from skim milk using membranes with pore sizes of 1.4 and 1.2 µm, and to investigate the role of spore surface properties in MF removal. Cell sizes were determined by scanning electron microscopy, surface charge by zeta potential analysis, and surface hydrophobicity by contact angle measurements. Commercially pasteurized skim milk was inoculated with a spore suspension at about 10⁶ cfu/mL, and then processed by MF using ceramic membranes at 6°C, a cross-flow velocity of 4.1 m/s, and transmembrane pressure of 69 to 74 kPa. Total aerobic plate and spore counts in the milk were determined before and after MF. All processing runs and surface and product analyses were conducted in triplicate, and data were analyzed statistically. For the same strain, spores were shorter and wider than vegetative cells, averaging 1.37 to 1.59 µm in length and 0.64 to 0.81 µm in width. Reduction of B. licheniformis spores significantly increased with a reduction in MF pore size, from 2.17 log for 1.4-µm pore size, to 4.57 log for 1.2-µm pore size. Both pore sizes resulted in almost complete removal of Geobacillus sp. spores. All spores and the ceramic membrane had a negative surface charge at milk pH, indicating an electrostatic repulsion between them. Bacillus licheniformis spores were hydrophilic, whereas Geobacillus sp. spores were hydrophobic. Consequently, Geobacillus sp. spores had a tendency to cluster in skim milk, preventing their passage even through the 1.4-µm MF membrane, whereas some B. licheniformis spores could still pass through a 1.2-µm membrane. This study demonstrates that efficient removal of spores from skim milk by cold MF may require a smaller membrane pore size than required for removal of vegetative cells of the same species, and that cell surface properties may interfere with the outcome of filtration as would be anticipated based on size alone.     

Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables: cabbage and lettuce

The present study was designed to investigate the ability of six Bacillus cereus strains to attach and form biofilm on cabbage and lettuce surfaces. These six strains were; a reference strain DSMZ 345 and five biofilm-producing strains (aquatic strains; TUB8, TUB30, TUB31, TUB32 and TUB33) isolated from drinking-water distribution network. Hydrophobicity, biofilm formation ability, attachment strength (S_R) of spores and vegetative cells of the six B. cereus strains were also determined. Due to their high hydrophobicity, spores of all strains had high ability to attach polystyrene and did not affect by dilution of tryptone soy broth (TSB, 1:20 v/v) in the in vitro experiment. Significant (p < 0.05) enhancement in vitro biofilm formation by vegetative cells of B. cereus was recorded in the diluted TSB. The highest biofilm formation on cabbage and lettuce surfaces was obtained by spores and vegetative cells of all tested strains on the 4^th hour of the incubation period. These populations were significantly (p < 0.05) increased by elongating incubation time from 4 h to 24 h except DSMZ 345 and TUB8. Biofilm formation behavior obtained by B. cereus spores and vegetative cells on the polystyrene surface was different compared with that recorded on produce surface. The S_R of both spores and vegetative cells of the studied strains to the lettuce surface was higher than that of the cabbage surface. The hydrophobicity, biofilm formation and S_R of spores and vegetative cells of the biofilm-producing strains were higher than that of the reference strain DSMZ 345. Scanning electron microscopy (SEM) exposed random distribution of cells either on the surface or cut edge, without clear obvious affinity for the surface structures. Increasing in the presence of large clusters of cells on leaf surfaces was demonstrated after 4 and 24 h.In conclusion, use of aquatic environmental isolates is more useful for studying biofilm formation than the reference strain. Lettuce surface supported the attachment of B. cereus spores and vegetative cells compared with the cabbage surface. Further investigations are required to improve our knowledge of biofilm formation mechanisms by the human pathogenic microorganisms, especially by using the environmental and clinical isolates. To ensure safety level of green-leafy vegetables, biofilm formation after harvest should be considered as critical control point during handling of these vegetables.     

Characterization of Bacillus sporothermodurans IC4 spores; putative indicator microorganism for optimisation of thermal processes in food sterilisation

Spore-forming bacteria with high heat resistance increasingly challenge industrial sterilisation processes in foods. To ensure stability of manufactured foods, generally worst case scenarios are applied often leading to unwanted over processing of foods. This means bigger requirements of energy and larger emissions of CO₂ into the atmosphere. A heat-resistant spore-forming bacterium, Bacillus sporothermodurans, was tested for its ability to produce consistently highly heat-resistant spore crops to be used to more precisely determine the minimal thermal process that is compatible with microbial stability, thus avoiding the indicated overprocessing. Isothermal survival curves of B. sporothermodurans IC4 spores were determined in distilled water at temperatures up to 125 °C using the capillary tube technique and a thermoresistometer Mastia. B. sporothermodurans consistently produced crops of heat-resistant spores with k_max values at 121 °C up to 0.46 min^?1. After consecutive sporulation cycles, the measured heat resistance of B. sporothermodurans was not reduced. Survival curves showed shoulders that were characterized by means of existing models (Geeraerd, Weibull) that were compared with the classical, log-linear one. Shoulders are a common phenomenon in heat-resistant spore-forming bacteria and may be related to the need of more stringent processing conditions. B. sporothermodurans spores were considered adequate to optimize sterilisation processes of low acid foods under a wide variety of conditions. Establishing precise heat treatments can be a way to guarantee food safety and stability while reducing the use of energy and contaminating emissions.     

Sporulating behavior of Bacillus licheniformis strains influences their population dynamics during raw milk holding

To understand the role of strain variability, population dynamics of 2 strains of Bacillus licheniformis, ATCC 6634 and ATCC 14580, were modeled as a function of temperature (4.0–12.0°C) and duration (0–72 h) using regression analysis. Based on the initial spiking of vegetative cells (approximately 4.0 log cfu/mL) and spores (approximately 2.0 log cfu/mL), regression equations, elucidating B. licheniformis growth behavior during raw milk holding at low temperature, were obtained. Contour plots were developed to determine the time-temperature combinations, keeping the population changes to less than 1.0 log. In vegetative cell spiking study of B. licheniformis ATCC 6634 (S1), cell population changes remained below 1.0 log up to 72 h at 8°C. For B. licheniformis ATCC 14580 (S2), 1.0 log shift was not observed only after 80 h at 8°C, indicating higher multiplication potential of S1 as compared with S2. As S2 was a readily sporulating strain, the vegetative spiking study showed spore formation at different storage temperatures. Evidence of some parallel germination was observed for this strain at 8°C or higher, when raw milk samples were spiked with spores. The experimental values obtained for sporeformers and spore counts were validated with contour plot-generated values. Overall, for raw milk samples predominated by the low sporulating strain, the contour plots suggested holding at 8°C or below for up to 72 h. In the case of the readily sporulating strain (S2), raw milk could be held at 8°C for 80 h, where little or no sporulation is observed. Sporulation behavior, germination and multiplication ability, strain variability, and temperature and duration of holding raw milk influenced the population dynamics of Bacillus species. However, in the presence of equivalent numbers of both types of sporulating strains in raw milk, despite strain variability, holding milk at 8°C for not more than 72 h would keep any cell population changes below 1.0 log. In addition, under these storage conditions, the population would remain as vegetative cells that are likely to be inactivated by pasteurization. The contour plots, so generated, would help predict the population shifts and define optimum holding conditions for raw milk before further processing.     

Potential of yeast isolated from apple juice to adhere to stainless steel surfaces in the apple juice processing industry

The aim of this study was to assess the potential of four yeast strains to initiate a biofilm on stainless steel. The yeasts were isolated from apple juice and were identified as Kluyveromyces marxianus, Candida krusei, Zygosaccharomyces sp. and Rhodotorula rubra. The physiochemical properties of cell surface were determined under different conditions. The adhesion capacity of the yeast strains to stainless steel was assayed in presence of apple juice. Cell surfaces were always negatively charged except for Zygosaccharomyces sp., whose isoelectric point was around 3.0. Neither electrophoretic mobility nor flocculation coefficient correlated with the capacity of attachment to stainless steel. The hydrophobicity expressed by the yeast surfaces at pH 3.0, correlated positively with the rate of adhesion (number of cells/min) of each strain. These results indicated that cell surface hydrophobicity governs the initial attachment of the studied contaminant yeast strains to stainless steel surfaces common to the apple juice processing plant.     

Evaluation of high-intensity ultrasonication for the inactivation of endospores of 3 bacillus species in nonfat milk

Endospores of Bacillus licheniformis [American Type Culture Collection (ATCC) 6634], Bacillus coagulans (ATCC 12245), and Geobacillus stearothermophilus (ATCC 15952) were spiked in sterile nonfat milk, and subjected to high intensity batch ultrasonication treatment at different amplitudes (80 or 100%) and durations (1 to 10 min). Increasing the amplitude from 80 to 100% did not result in enhanced inactivation of G. stearothermophilus endospores. However, an increase in the duration of ultrasonication from 1 to 10 min significantly increased the inactivation of endospores of all 3 species. About 48.96% of the G. stearothermophilus endospores were inactivated by ultrasonication alone, whereas ultrasonication and pasteurization combined increased the inactivation to 65.74%. Inactivation of endospores could be further enhanced to 75.32% by ultrasonication and higher heat (80°C/1 min) combination. Endospores of B. licheniformis and B. coagulans were inactivated to a lesser extent compared with G. stearothermophilus spores. Ultrasonicated B. licheniformis endospores germinated in higher numbers when compared with untreated endospores resulting in their greater inactivation during the combined treatment. During microstructure imaging of ultrasonicated endospores, although no structural damage was noticed, they showed irregular shrinkage and wrinkles with surface coarseness. This may also have contributed to their reduced thermal resistance, in addition to sporulation.     

Role of mechanical vs. chemical action in the removal of adherent Bacillus spores during CIP procedures

This study was designed to evaluate the respective roles of mechanical and chemical effects on the removal of Bacillus spores during cleaning-in-place. This analysis was performed on 12 strains belonging to the Bacillus cereus group (B. cereus, Bacillus anthracis, Bacillus thuringiensis) or to less related Bacillus species (Bacillus pumilus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus subtilis). Adherent spores were subjected to rinsing-in-place (mechanical action) and cleaning-in-place (mechanical and chemical actions) procedures, the latter involving NaOH 0.5% at 60 °C. Results revealed that mechanical action alone only removed between 53 and 89% of the attached spores at a shear stress of 500 Pa. This resistance to shear was not related to spore surface properties. Conversely, in the presence of NaOH at a shear stress of 4 Pa, spores were readily detached, with between 80 and 99% of the adherent spores detached during CIP and the chemical action greatly depended on the strain. This finding suggests that chemical action plays the major role during CIP, whose efficacy is significantly governed by the spore surface chemistry.     

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10² CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.     

Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology

Spores of Bacillus sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. sporothermodurans spores, only d-glucose, l-alanine, and inosine were considered as strong independent germinants. Both inosine and l-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (l-alanine, d-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. sporothermodurans spores was obtained after incubation of spore for 60 min at 35 °C in presence of 9 and 60 mM of d-glucose and l-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors.     

Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia

Spore-forming bacteria, principally Bacillus species, are important contaminants of milk. Because of their high heat resistance, Bacillus species spores are capable of surviving the heat treatment process of milk and lead to spoilage of the final product. To determine the factors influencing the contamination of milk, spore-forming bacteria occurrence throughout the UHT milk production line during winter, spring, and summer was studied. The obtained results confirm that the total viable rate decreases rapidly throughout the production line of UHT milk showing the efficiency of thermal treatments used. However, the persistent high rate of spore-forming bacteria indicates their high heat resistance, especially in spring and summer. In addition, a significant variation of the quality of raw milk according to the location of the collecting centers was revealed. The molecular identification showed a high degree of diversity of heat-resistant Bacillus species, which are isolated from different milk samples. The distribution of Bacillus species in raw milk, stored milk, bactofuged milk, pasteurized milk, and UHT milk were 28, 10, 16, 13, and 33%, respectively. Six Bacillus spp. including Bacillus licheniformis (52.38%), Bacillus pumilus (9.52%), Bacillus sp. (4.76%), Bacillus sporothermodurans (4.76%), Terribacillus aidingensis (4.76%), and Paenibacillus sp. (4.76%) were identified in different milk samples.     

Relevant factors affecting microbial surface decontamination by pulsed light

Pulsed Light (PL) uses intense flashes of white light rich in ultraviolet (UV) light for decontamination. A log-reduction higher than 5 was obtained in one flash and at fluences lower than 1.8 J/cm² on spores of a range of spore-forming bacteria, of vegetative cells of non-spore-forming bacteria and on yeasts spread on agar media. Vegetative cells were more sensitive than spores. The inactivation by PL of Bacillus subtilis, B. atrophaeus, B. cereus, Geobacillus stearothermophilus, and Aspergillus niger spores sprayed on polystyrene was similar. The inactivation by PL of B. subtilis and A. niger spores sprayed on glass was slightly lower than on polystyrene. No alteration of the spore structures was detected by scanning electron microscopy for both PL treated B. subtilis and A. niger spores. The inactivation of spores of B. subtilis, B. atrophaeus, B. cereus and B. pumilus by PL or by continuous UV-C at identical fluences was not different, and was much higher by PL for A. niger spores. The increase in the input voltage of the lamps (which also increases the UV-C %) resulted in a higher inactivation. There was no correlation between the resistance to heat and the resistance to PL. The relative effect of UV-C radiations and light thermal energy on PL inactivation was discussed.     

Inactivation of Alicyclobacillus contaminans spores by dielectric barrier discharge plasma and its biological mechanism

Over the years, as an emerging technology, cold plasma (CP) has been widely used in the preservation of agricultural products. Alicyclobacillus spp. are spore-forming bacteria and difficult to inactivate. They adversely affect the economic value of agricultural products. Nevertheless, there are still few studies related to the inactivation of spores using CP. Herein, the inactivation effect and mechanism of dielectric barrier discharge plasma (DBDP) on Alicyclobacillus contaminans (A. contaminans) spores in phosphate-buffered saline and clarified apple juice were investigated. Plasma treatment at 75 V for 9 min achieved 99% inactivation of spores with the initial inocula of 7.13 and 5.72-log in PBS and apple juice, respectively. And the cell structure of the spores was severely disrupted leading to the leakage of the entocyte. Moreover, the surface properties of the spores were altered, making the adhesion of the spores to the hydrophobic surfaces and the stability of the bacterial suspension decreased leading to more agglomeration of the spores. Further results confirmed the intracellular homeostasis of the spores was also disrupted. Therefore, the inactivation mechanism of DBDP on spores was revealed from different perspectives, and our discoveries promote the theoretical progress in the use of plasma in food sterilization.Industrial relevanceAs an emerging nonthermal preservation technology, cold plasma has been used in food processing. In this study, we confirmed the feasibility of the inactivation of Alicyclobacillus contaminans spores in apple juice by dielectric barrier discharge plasma (DBDP) and investigated the mechanism of plasma inactivation of spores at the molecular biology level. This paper provides preliminary support for the application of low-temperature plasma in food processing such as apple juice.     

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

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

RAPD-based screening for spore-forming bacterial populations in Uruguayan commercial powdered milk

The occurrence of spore-forming bacteria in powdered milk is of concern to the dairy industry due to potential deleterious effects including those resulting from proteolytic and lipolytic activities. Twenty-two powdered milk samples representative of spring and summer production obtained from Uruguayan retail stores were analyzed for type and number of thermophilic and spore-forming bacterial species. Bacillus licheniformis isolates were found to be the most prominent milk powder contaminant followed by Anoxybacillus flavithermus representing 71.5 to 84% of the total microflora. Geobacillus stearothermophilus, however, was not found. B. licheniformis strains F and G were both found in this study but strain F was the prevalent isolate representing 98.9% of the total isolates of this species. A. flavithermus isolates corresponded to strain C in accordance with 16S rRNA gene sequence analysis, however, in contrast with other reports, the RAPD profiles showed three characteristic bands at approximately 650, 1000 and 1650 bp, but lacking a band at 1250 bp. A third group of isolates was identified corresponding to members of a Bacillus subtilis group and Bacillus megaterium. Isolates designated B. licheniformis, A. flavithermus, B. megaterium and the B. subtilis group represented 89.1 to 93.6% of those analyzed, and depended on previous heat treatment and incubation temperatures of the plates. The remaining isolates were Bacillus pumilus and unidentified spore-formers.     

Species-specific real-time PCR assay for enumeration of Anoxybacillus flavithermus and Geobacillus stearothermophilus spores in dairy products

The thermophilic spore-forming bacteria, Anoxybacillus flavithermus and Geobacillus stearothermophilus, grow readily, especially on milk-powder processing lines, and are important for processing-plant hygiene management. We developed a real-time PCR assay using SYBR Green I to monitor the spores of these species in dairy products. We designed new primer pairs specific for each species from the nucleotide sequences of the stage 0 sporulation gene A (spo0A). Ethidium monoazide treatment enabled us to quantify only spores. In dairy products with high protein content, treatment with proteinase K allowed precise quantification of spores. This assay was superior for counting spores, having high linearity (r² = 0.99) and a wide quantification range (10¹ to 10⁶ spores mL⁻¹). This new method can be applied to quantification of spores in samples of milk, skimmed milk, and cream.     

Influence of thermosonication on Geobacillus stearothermophilus inactivation in skim milk

The influence of high intensity ultrasound coupled with thermoprocessing on the inactivation of Geobacillus stearothermophilus vegetative cells and spores in skim milk powder was explored using response surface methodology and two polynomial models were developed. Optimization of cell reduction (4.8 log) was found to be at 19.75% total solids (TS), 45 °C, and 30 s, while optimization of spore reduction (0.45 log) was found to be at 31.5% TS, 67.5 °C, and 17.5 s. Model verification experiments were performed using common milk powder processing conditions. Results showed the inactivation of cells and spores to be most effective before (9.2% TS, 75 °C, and 10 s) and after (50% TS, 60 °C, and 10 s) the evaporator during milk powder processing and may produce an additive effect in microbial reduction when the two locations are combined, resulting in a 5.8 log reduction for vegetative cells and 0.51 log reduction for spores.     

Cell surface hydrophobicity and attachment of pathogenic and spoilage bacteria to meat surfaces

Cell surface hydrophobicity of several pathogenic and spoilage bacteria was determined by hydrocarbon adherence, adhesion to nitrocellulose filter, salt aggregation and adherence to phenyl-sepharose beads. Hydrophobicity of each bacterium was dependent on the method of measurement. Hydrophobicity was compared with the strength of attachment (Sr) of the bacteria to beef muscle surfaces. Bacterial cell surface hydrophobicity, as determined by the bacterial adherence to xylene correlated well with attachment strength (r = 0.800, P < 0.05). Staphylococcus aureus, Clostridium perfringens and Yersinia enterocolitica showed the highest values of attachment strength.     

Occurrence of aerobic bacterial endospores in dried dairy ingredients

The present study investigated types of bacterial endospores in powdered dairy ingredients. For enumeration, the samples were heat-treated at 80°C/12 min for regular spore type, 100°C/30 min for high-heat-resistant spores (HHRS), and 106°C/30 min for specially thermoresistant spores (STS), followed by incubation for 24 h at 37°C (for mesophiles) and 55°C (for thermophiles). Of the three types, the regular spore type (80°C/12 min) was observed to be the most prominent endospore type, and Bacillus licheniformis as the predominant bacterial species in the tested dairy ingredients. This study would be helpful to utilise dry ingredients based on the potential activation of endospores during the product manufacturing processes.