Substratum attachment location and biofilm formation by Bacillus cereus strains isolated from different sources: Effect on total biomass production and sporulation in different growth conditions

Bacillus cereus, an endospore forming human pathogen associated with foodborne diseases, can form biofilms and attach to surfaces of processing equipment in the food industry. It is a consistent source of contamination and/or cross contamination of processed food products. The objective of this study was to understand substratum attachment location and biofilm formation behavior of B. cereus strains under different growth conditions. A total of 60 strains isolated from food, human, or farm and a number of reference strains were used in this study. Substratum attachment locations of these strains in 96-well microtiter plates were highly diversified among these strains. Strains isolated from food showed higher preference to attach at the air-liquid interface during early stage of biofilm formation. To the best of our knowledge, this is the first report showing the level of substratum attachment location and biofilm formation of B. cereus strains isolated from different sources. Substratum properties did not affect biofilm formation location when a number of selected strains were grown on stainless steel coupon, indicating that biofilm formation location might be independent of the type of substratum. Substratum attachment location and biofilm formation related phenotypes such as total biomass production, number of sessile cells, and sporulation were closely correlated. Substratum attachment location and sporulation behavior were strongly affected during biofilm formation under nutrient stress condition. The number of spores was significantly increased in biofilms grown under nutrient stress condition even though total biomass formation was lower. Our results on substratum attachment location and related biofilm formation behavior are substantially important for food industries where different surfaces are prone to B. cereus attachment, particularly for setting up and implementing clean in place (CIP) protocols.     

Rutin inhibits mono and multi-species biofilm formation by foodborne drug resistant Escherichia coli and Staphylococcus aureus

Considering the role of biofilm in food spoilage and the food industry, inhibition of biofilm formation by natural agents is expected to be safe and could also enhance the efficacy of other antimicrobial strategies for controlling microbial food spoilage. Plant flavonoids are known for their diverse biological activity including antimicrobial. Therefore, rutin was investigated for its biofilm inhibitory activity at sub-minimum inhibitory concentrations (sub-MICs) values against common foodborne pathogens (Escherichia coli and Staphylococcus aureus). Minimum inhibitory concentrations (MIC) ranged from 400 to 1600 μg/ml against the selected strains. Sub-MICs (1/16 × MIC to 1/2 × MIC) were used to assess the inhibition of biofilm formed by E. coli and S. aureus in microtitre plate assay. Mono strain biofilm formation by Escherichia coli and Staphylococcus aureus was greatly reduced by rutin at their respective 1/2 × MIC. For multi-species (E. coli: and S. aureus) biofilm formation, the reduction in biofilm production was concentration dependent. No significant bacteria mass reduction was recorded for any sub-MIC. SEM images of biofilm inhibition on steel chips confirmed the reduction in number of microcolonies. Exopolysaccharide production responsible for adherence and maturation of biofilms was also significantly (p ≤ 0.05) reduced at respective concentrations of rutin in tested strains. To the best of our knowledge, this is the first study describing the effect of flavonoid, rutin on multi-species biofilms consisting of S. aureus and E. coli. Findings of the study indicate a potential application of rutin in the prevention of biofilm on industrial equipment and food contact surfaces and prevent food contamination and spoilage.     

Nucleolytic enzymes from the marine bacterium Cobetia amphilecti KMM 296 with antibiofilm activity and biopreservative effect on meat products

Cultivation of Pseudomonas aeruginosa (P. aeruginosa), Bacillus subtilis (B.subtilis), Salmonella enterica (S. enterica), and Staphylococcus aureus (S. aureus) isolated from meat products together with the marine bacterium Cobetia amphilecti KMM 296 (Cobetia marina) resulted in inhibition of their cell growth and complete degradation of biofilms of P. aeruginosa and B. subtilis. The degradation patterns of their mature biofilms treated with Cobetia amphilecti (C. amphilecti) extracellular nucleolytic enzymes revealed that the highly active alkaline phosphatase CmAP could be a significant antibiofilm factor. Moreover, CmAP possessed strong dose-dependent inhibition effect on de novo biofilm formation by bacterial cells regardless of the species. The concentration of CmAP for exhibition of maximal effect on microbial growth and biofilms was 1.1 μg/ml with activity of 2.5 units/ml that diminished total aerobic mesophilic and lactic acid counts in sausage shells by 3.5 log units for 5 days and 2.5 log units for 6 days, respectively. The stabilized antifungal effect continued the same period of storage. The nuclease-like enzymes CmEEP and CmNUC were species-independent and largely degraded biofilms at lowered pH. The biopreservative effect on meat products at low concentration and psychrophilicity of CmAP can be used for enzymatic cleaning of surfaces in food industry.     

The effect of polyesterurethane belt surface roughness on Listeria monocytogenes biofilm formation and its cleaning efficiency

Contamination of surfaces in direct contact with food in the processing line is the major cause of finished product cross-contamination, especially conveyor belt surfaces that transfer food products. Polyesterurethane (PSU) is one of the materials widely used in conveyor belt systems; therefore, the objective of this study was to examine the effect of PSU conveyor belt surface roughness on the ability of biofilm formation by two strains of Listeria monocytogenes, i.e. ATCC19114 and ATCC51782. Additionally, the efficiency of a cleaning and sanitizing procedure adopted by a chicken meat plant in Thailand in eliminating biofilm formation on belt surfaces was assessed. The roughness values (Ra) were 0.05 ± 0.00 μm and 1.44 ± 0.01 μm for new (unused) and old conveyor belts (in use for 5 years), respectively. It was found that at 30 °C, both strains of L. monocytogenes formed robust biofilms regardless of differences in surface roughness. Conversely, at 15 °C, the ability of L. monocytogenes to form biofilms decreased with the lower Ra value. Under a simulated processing environment, cleaning and sanitizing reduced L. monocytogenes biofilms from both old and new conveyor belt surfaces to certain levels; however, a total reduction could not be achieved. In addition, more highly concentrated cleaning and sanitizing agents resulted in greater reduction. Results from this study clearly illustrate that surface roughness of material is also an important factor which may hinder effective cleaning and sanitizing. Therefore, the roughness value may be used as an indicator for evaluating the life of conveyor belts being used in the food industry.     

Occurrence and diversity of Bacillus cereus and moulds in spices and herbs

Spices and herbs can contain toxin-producing bacteria and moulds, which can cause health problems for consumers and contribute to food spoilage and shelf-life reduction. The aims of the present work were (i) to determine the occurence and levels of B. cereus and moulds; (ii) to charactize the incidence and diversity of B. cereus emetic toxin (ces, CER), and diarrhoeal toxin-encoding genes (entFM, nheA, hblC, cytK) and toxigenic potential of Hbl toxin-producing B. cereus strains. Black ground pepper samples showed the most contamination with the highest concentration of B. cereus 2.49 log₁₀ CFU/g. Moreover, cumin contained the most prominent mould concentration level of 3.6 log₁₀ CFU/g. The most common moulds were Aspergillus and Penicillium spp. Compared to packaging type, all products acquired from the local market, except curry for B. cereus, exchibited high concentrations of B. cereus and moulds. Four genes were identified – 96% of B. cereus strains contained entFM, 94% nheA, 56% hblC, 42% cytK. None of the samples contained emetic toxin-encoding genes (ces, CER). Toxigenic potential of Hbl toxin was found in 72% of B. cereus strains. Different temperature, moisture levels and hygiene practices were observed at places of sale in local markets thus facilitating contamination and development of moulds. Moreover, the presence of B. cereus and its ability to produce toxins in spices and herbs, may suggest the need to establish microbiological criteria for mould and spore-forming bacteria in spices and herbs.     

The specific anti-biofilm effect of gallic acid on Staphylococcus aureus by regulating the expression of the ica operon

Staphylococcus aureus (S. aureus) biofilms are of considerable interest in food safety because biofilms can increase the risk of food contamination and enhance the pathogenicity of bacteria. The ica-encoded polysaccharide intercellular adhesin (PIA) plays an important role in biofilm formation. In this study, the MIC of gallic acid against S. aureus in suspension and in biofilms was 2 mg/mL and 4 mg/mL, respectively. Quantitative crystal violet staining of biofilms showed that 2 mg/mL gallic acid can effectively inhibit biofilm formation and the ESEM images clearly showed the three-dimensional biofilm morphology of the S. aureus and the resulting anti-biofilm effect. The determination of viable bacteria in the biofilm revealed that gallic acid penetrated the biofilm to kill S. aureus, the bactericidal effect on the biofilm bacteria was comparable to that of planktonic bacteria. We further explored the influence of gallic acid on ica family gene expression and polysaccharide slime formation in S. aureus biofilm formation. The results showed that icaR was significantly activated that; icaA and icaD were downregulated in a dose-dependent manner with increasing concentrations of gallic acid; however, the expression of icaB and icaC was not significantly affected. The polysaccharide slime formation was reduced as well. Based on these results, gallic acid, as a natural substance, may play an important role in the food industry.     

Removing Staphylococcus aureus and Escherichia coli biofilms on stainless steel by cleaning-in-place (CIP) cleaning agents

We investigated the effects of cleaning-in-place (CIP) cleaning agents, food additives and other compounds (EDTA, Tween20 and SDS) on the removal of Staphylococcus aureus and Escherichia coli biofilm from stainless steel plates. S. aureus formed thick biofilm and was more resistant to cleaning than E. coli biofilm. Strong acidic and strong alkaline CIP cleaning agents showed a significant cleaning effect. Strong alkaline CIP cleaning agents were especially effective for the removal of S. aureus biofilm.     

Sporulation dynamics and spore heat resistance in wet and dry biofilms of Bacillus cereus

Environmental conditions and growth history can affect the sporulation process as well as subsequent properties of formed spores. The sporulation dynamics was studied in wet and air-dried biofilms formed on stainless steel (SS) and polystyrene (PS) for Bacillus cereus ATCC 10987 and the undomesticated food isolate B. cereus NIZO 4080. After harvesting and maturation, the wet heat resistance of spores obtained from these biofilms was tested and compared to planktonic and agar plate-derived spores. Drying/air exposure of the preformed 24 h old biofilms accelerated spore formation for both strains and resulted in higher final spore percentages. Prolonged dry incubation of more than three days triggered germination of spores in the biofilms of ATCC 10987. Spores harvested from wet biofilms on SS displayed the highest heat resistance compared to liquid, agar plate and PS biofilm derived spores. The D_95 °C values for these spores were 17 and 22 min for NIZO 4080 and ATCC 10987, respectively, which was 2 and 1.2 fold higher compared to planktonic spores of these strains. Spores obtained from dried biofilms of ATCC 10987 displayed reduced heat resistance compared to wet biofilm spores. The results indicate that environmental conditions encountered by biofilms affect sporulation dynamics and spore heat resistance, thus affecting subsequent quality issues and safety risks related to these biofilms.     

Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria

Biofilms represent a great concern for food industry, since they can be a source of persistent contamination leading to food spoilage and to the transmission of diseases. To avoid the adhesion of bacteria and the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants, microbial compounds that can modify the physicochemical properties of surfaces changing bacterial interactions and consequently adhesion. Different concentrations of the biosurfactants, surfactin from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated to reduce the adhesion and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Staphylococcus aureus, Listeria monocytogenes and Salmonella Enteritidis were studied using polystyrene as the model surface. The pre-conditioning with surfactin 0.25% reduced by 42.0% the adhesion of L. monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1.0% reduced by 57.8% adhesion of L. monocytogenes and by 67.8% adhesion of S. aureus to polystyrene.Biosurfactants were less effective to avoid adhesion of mixed cultures of the bacteria when compared with individual cultures. After 2 h contact with surfactin at 0.1% concentration, the pre-formed biofilms of S. aureus were reduced by 63.7%, L. monocytogenesby 95.9%, S. Enteritidis by 35.5% and the mixed culture biofilm by 58.5%. The rhamnolipids at 0.25% concentration removed 58.5% the biofilm of S. aureus, 26.5% of L. monocytogenes, 23.0% of S. Enteritidis and 24.0% the mixed culture after 2 h contact. In general, the increase in concentration of biosurfactants and in the time of contact decreased biofilm removal percentage. These results suggest that surfactin and rhamnolipids can be explored to control the attachment and to disrupt biofilms of individual and mixed cultures of the food-borne pathogens.     

Evaluation of electrolyzed water as cleaning and disinfection agent on stainless steel as a model surface in the dairy industry

In the dairy industry, cleaning and disinfection of surfaces are important issues and development of innovative strategies may improve food safety. This study was aimed to optimize the combined effect of alkaline electrolyzed water (AEW) and neutral electrolyzed water (NEW) as alternative cleaning and disinfection procedure on stainless steel plates (SSP) with and without electropolishing. NEW at 10 ppm total available chlorine (TAC), achieved a ˃5 log CFU/mL reduction of milk spoilage bacterial suspension, grown in trypticase soy broth (8.7 log CFU/mL of each bacterial strain: Pseudomonas aeruginosa, Enterococcus faecalis and Micrococcus luteus) contacted for 30 s. An optimal design of experiments was used to assess the combined effect of cleaning with AEW, followed by disinfection with NEW (40 ppm TAC, contact time 3 min). Tested factors were contact time (10, 20 and 30 min), concentration of AEW (100, 200 and 300 mg NaOH/L), temperature (30, 40 and 50 °C), and surface type (304-2B SSP with or without electropolishing), using sixteen treatments with two replicates. The response variable was bacterial cells removal (log CFU/cm²). All main effects, two factors interactions and a quadratic term significantly influenced cells removal, and were modeled using a second order polynomial. Best cleaning procedures were significantly affected by surface roughness; electropolished SSP required 10 min, 100 mg/L AEW at 30 °C, whereas SSP without modification required 30 min, 300 mg/L AEW at 30 °C. From confirmatory tests cells removed were 3.90 ± 0.25 log CFU/cm² for electropolished SSP, and 3.20 ± 0.20 log CFU/cm² for SSP without modification. NEW is non-corrosive, and can be advantageously used for environmentally friendly cleaning and disinfection processes.     

Effectiveness of a phage cocktail as a biocontrol agent against L. monocytogenes biofilms

Listeria monocytogenes can persist and form biofilms in a food environment which are difficult to eradicate because biofilms are inherently resistant to a variety of antimicrobial treatments. Therefore, alternative approaches such as bacteriophages have been suggested as a promising biocontrol agent against biofilms. The aim of this study was to evaluate the efficacy of a cocktail bacteriophage product (ListShield™) against L. monocytogenes biofilms. These biofilms were established on lettuce, stainless steel, rubber, and a MBEC biofilm device and exposed to the ListShield™ phage preparation (1 × 10⁸ PFU/mL) for 2 h. ListShield™ had sufficient potency to significantly reduce the biofilm (P < 0.05) in all cases. Biofilm reduction achieved after ListShield™ treatment on the stainless steel coupon was 1.9–2.4 log CFU/cm² and on the rubber surface approximately 1.0 log CFU/cm². Phage application on lettuce inactivated biofilm bacteria up to 0.7 log CFU/cm². These results suggest that bacteriophage preparation ListShield™ is an effective tool for the inactivation of L. monocytogenes biofilms in the food industry.     

Antimicrobial peptides derived from hen egg lysozyme with inhibitory effect against Bacillus species

In food industry, Bacillus species are encountered in deteriorating many food products thus shortening their shelf-life. Moreover, Bacillus cereus and the subtilis group (Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus) have been recognized as food poisoning agents. Lysozyme peptides preparation (LzP) is a commercially available as a natural food preservative. Although, LzP derived from lysozyme yet it showed only 11% of the lysozyme lytic activity. LzP at a concentration of 100 μg ml⁻¹ completely inhibited B. subtilis, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. coagulans, B. amyloliquefaciens, B. polymexa and B. macerans. However, B. cereus and B. stearothermophilus showed a slightly higher resistance. Interestingly, LzP at concentration ⩾10 μg ml⁻¹ showed inhibitory effect on both vegetative and spore forms of B. subtilis. Moreover, LzP was stable at 95 °C for 30 min and at different pH values (4.5–7). In conclusion, LzP may be useful to control growth of Bacillus spoilage organisms.     

Antioxidant properties of coriander essential oil and linalool and their potential to control Campylobacter spp.

Foodborne diseases remain common around the world with Campylobacteriosis being the most commonly reported zoonosis in the European Union in 2013. Campylobacter jejuni and Campylobacter coli are the main species associated with human illness. Furthermore, Campylobacter can develop biofilms which is becoming a major problem within the food industry. In addition to foodborne pathogens, oxidation is a non-microbial cause of deterioration of food causing loss of quality and safety. Thus, there is an urgent need in the food industry for new and effective strategies that can help prevent food contamination, spoilage and consequently, foodborne illnesses. Essential oils are known for their antimicrobial and antioxidant properties and are already widely used in the food industry. So, the aim of this work was to study the antimicrobial and anti-biofilm activity of coriander essential oil and its major compound linalool against C. jejuni and C. coli strains, as well as their effect in the quorum sensing (QS) system and their potential as antioxidants. Our results, demonstrated that both compounds have anti-Campylobacter activity, inhibited in vitro biofilm formation and promoted biofilm dispersion even at sub-MIC concentrations and interfered with the QS system through the inhibition of violacein production. Moreover, the essential oil and linalool were shown to have radical scavenging properties and lipid peroxidation inhibition ability which could make them potential alternatives to synthetic antioxidants. In sum, our results demonstrated the antibacterial, anti-biofilm, anti-QS and antioxidant potentials of the coriander essential oil and its major compound, linalool, suggesting that they could be used in the food industry to enhance shelf life of food products and increase food safety without requiring chemical additives or preservatives.     

Short communication: Fate of major foodborne pathogens and Bacillus cereus spores in sterilized and non-sterilized Korean turbid rice wine (Makgeolli)

The objective of this study was to examine the fate of foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus) and B. cereus spores in Korean turbid rice wine (Makgeolli). Samples of sterilized and non-sterilized turbid rice wine were inoculated with each of the vegetative bacteria or B. cereus spores at 3–4 log CFU/ml. The samples were stored at 5 °C or 22 °C, and bacterial survival was monitored over 28 days. Despite the harsh environment (alcohol content: 6–7% and pH: 3.43–3.98), long-term survival of pathogens was observed. Survival time was different depending on the type of beverage (pathogens survived longer in sterilized wine than in non-sterilized wine), cellular state (spores survived longer than vegetative cells), species (B. cereus survived longer than other species), and storage temperature (pathogens survived longer at 5 °C then at 22 °C). The number of B. cereus spores remained constant at both temperatures. The vegetative B. cereus population declined rapidly within 1 day, but then remained steady for up to 28 days (1.20–1.55 log CFU/ml in sterile wine). These results indicate that B. cereus formed spores that survived for a long time; therefore, it is possible that B. cereus may exist as spores in turbid rice wine. E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus survived for up to 28, 14, 14, and 14 days, respectively, in sterilized wine at 5 °C. Thus, the health implications of the long-term survival of pathogens in alcoholic beverages should be carefully considered. The results provide new information that may be useful in predicting the potential microbiological hazards associated with turbid rice wine.     

Inhibition of spoilage and toxigenic Bacillus species in dough from wheat flour by the cyclic peptide enterocin AS-48

Enterocin AS-48 was tested against rope-forming Bacillus subtilis CECT 4002 and Bacillus licheniformis CECT 20, as well as on Bacillus cereus and Bacillus pumilus strains in broth and in experimental dough from wheat flour. Vegetative B. subtilis cells in liquid broth were rapidly inactivated by AS-48 (7 AU/ml). In wheat dough, higher bacteriocin concentrations of 14 and 23 AU/g were required for inactivation of B. subtilis vegetative cells and endospores activated to germinate, respectively. B. cereus LWL1 and B. licheniformis CECT 20 were inactivated by AS-48 (14 AU/g) in doughs stored at 22 °C much faster compared to doughs stored at 10 °C. Strains of Bacillus pumilus were partially inactivated in dough by bacteriocin addition (14 AU/g). Results from this study indicate that enterocin AS-48 can reduce the populations of spoilage and potentially-toxigenic bacilli in wheat dough, decreasing the risks for spoilage and food intoxication.     

Adherence kinetics, resistance to benzalkonium chloride and microscopic analysis of mixed biofilms formed by Listeria monocytogenes and Pseudomonas putida

Comparison between the resistance to BAC and the microscopic structure between mixed-species biofilms formed by different strains of Listeria monocytogenes and Pseudomonas putida CECT 845 under different scenarios and that obtained by the corresponding monospecies L. monocytogenes biofilm was carried out. The association of P. putida with L. monocytogenes quickens biofilm formation and increases significantly (p < 0.05) the BAC-resistance of the biofilm after 4 days of incubation at 25 °C respecting to that formed by monospecies biofilms. According with the adherence profiles of P. putida, two different patterns of association between both species (A and B) were identified, being type A pattern found in the mixed biofilms much more resistant to BAC. After 11 days of incubation, a destructuration of mixed biofilms occurred in all experimental assays, being in 2 out of 5 experimental cases (4032 and BAC-adapted 5873 on polypropylene) accompanied by a sharp decrease in the number of adhered cells. Microscopic analyses demonstrated that complex three-dimensional microscopic structure showed the highest resistance to BAC (4032-SS). Obtained results clearly highlight that to improve disinfection protocols for assuring food safety, it is necessary to mimick those bacterial association that occur in nature.     

Production of biofilm by Listeria monocytogenes in different materials and temperatures

Listeria monocytogenes, considered as one of the most important foodborne pathogens, is easily found on surfaces, particularly in the form of a biofilm. Biofilms are aggregates of cells that facilitate the persistence of these pathogens in food processing environments conferring resistance to the processes of cleaning and may cause contamination of food during processing, thus, representing a danger to public health. Little is known about the dynamics of the formation and regulation of biofilm production in L. monocytogenes, but several authors reported that the luxS gene may be a precursor in this process. In addition, the product of the inlA gene is responsible for facilitating the entry of the microorganism into epithelial cells that express the receptor E-cadherin, also participates in surface attachment. Thus, 32 strains of L. monocytogenes isolated from different foods (milk and vegetables) and from food processing environments were analyzed for the presence of these genes and their ability to form biofilms on three different surfaces often used in the food industry and retail (polystyrene, glass and stainless steel) at different temperatures (4, 20 and 30 °C). All strains had the ilnA gene and 25 out of 32 strains (78.1%) were positive for the presence of the luxS gene, but all strains produced biofilm in at least one of the temperatures and materials tested. This suggests that genes in addition to luxS may participate in this process, but were not the decisive factors for biofilm formation. The bacteria adhered better to hydrophilic surfaces (stainless steel and glass) than to hydrophobic ones (polystyrene), since at 20 °C for 24 h, 30 (93.8%) and 26 (81.3%) produced biofilm in stainless steel and glass, respectively, and just 2 (6.2%) in polystyrene. The incubation time seemed to be an important factor in the process of biofilm formation, mainly at 35 °C for 48 h, because the results showed a decrease from 30 (93.8%) to 20 (62.5%) and from 27 (84.4%) to 12 (37.5%), on stainless steel and glass, respectively, although this was not significant (p = 0.3847). We conclude that L. monocytogenes is capable of forming biofilm on different surfaces independent of temperature, but the surface composition may be important factor for a faster development of biofilm.     

The sanitizing action of essential oil-based solutions against Salmonella enterica serotype Enteritidis S64 biofilm formation on AISI 304 stainless steel

Bacterial adhesion and biofilm formation by Salmonella enterica serovar Enteritidis is a serious concern in the food processing industry; organism persistence in biofilms represents a continual source of contamination. Due to unsuccessful disinfection processes and emerging resistance, conventional control methods are rapidly becoming ineffective, necessitating the development of new control strategies. The following study evaluated the anti-biofilm effect of disinfectant solutions formulated with essential oils (EOs) of peppermint (Mentha piperita) and lemongrass (Cymbopogon citratus) against biofilm formation by S. enterica serotype Enteritidis S64 on stainless steel surface AISI 304 (#4) after 10, 20 and 40 min of contact. A minimum inhibitory concentration (MIC) of 7.8 μL/mL was found for both EOs and disinfectant solutions were formulated based on these MIC values. Ten minutes of sanitizing solution contact significantly reduced (p ≤ 0.05) adhered bacterial populations for both EOs tested. After 20 and 40 min of treatment, cell counts were not detected. Thus, M. piperita and C. citratus EOs can be considered convenient, quality alternatives to the application of conventional sanitizing agents in the food industry; further, use of these EOs addresses the increasing consumer demand for natural products.     

A new risk analysis of Clean-In-Place milk processing

A new risk analysis of Clean-In-Place (CIP) removal of proteinaceous milk deposits is presented and compared with traditional approaches. The aim was to gain insight into random (stochastic) errors that may lead to unexpected failure of an otherwise well-operated CIP plant. CIP failure is defined as failure to remove proteinaceous deposits on wet surfaces in an auto-set cleaning time. The method is based on the two-stage, unit-operations model and experimental data of Bird (1992) and Bird and Fryer, 1991a, Bird and Fryer, 1991b. A risk factor (p) together with a practical tolerance is defined in terms of the auto-set CIP time to remove a specified deposit and the actual cleaning time as affected by stochastic changes (t_T′). This is computationally convenient as it can be articulated so that all values p > 0 highlight an unwanted outcome i.e. a CIP failure. Process behaviour is simulated using a refined Monte Carlo sampling of the temperature distribution of the alkali cleaning fluid (1% w/v NaOH) on a (0.00015 m) proteinaceous deposit together with a specified range of tolerances (5%) on the auto-set clean time. Results reveal that with a cleaning fluid at a nominal mid-range temperature of 50 °C CIP will fail in 4.2% of all operations. On average, this equates to 15 failures per annum. This information cannot be obtained from traditional single point simulation (with or without sensitivity analyses). This new (Friday 13th) risk analysis represents a significant advance because all practical scenarios that could exist are computed. Quantitative evidence reveals that CIP is actually a mix of successful cleaning operations together with failed ones. The insight provided can be used quantitatively to reduce cleaning failures and improve safety through changes to the plant including improved temperature control.     

Adhesion of Staphylococcus aureus on stainless steel treated with three types of milk

Staphylococcus aureus has the ability to adhere and to form biofilm on inert surface such as stainless steel commonly used in food industry. The biofilm formed on the surface of milk processing equipments could be a source of dairy products contamination. This contamination causes a food poisoning. In this paper the S. aureus adhesion on stainless steel treated by three types of milk (ultrahigh-temperature (UHT)-treated milk; UHT skimmed milk, UHT semi-skimmed milk) was investigated.Stainless steel was exposed to three types of milk with a different amount of fat component. Contact angles measurements were used to determine the surface physicochemical properties of substratum treated with the three milk products. The hydrophobicity and electron acceptor properties of stainless steel seem to be decreasing with the amount of fat component present in milk but its electron donor property increase with this component. The ability of S. aureus to adhere to stainless steel treated and untreated with milk was also examined. Treatment with the three types of milk reduces bacterial attachment. On treated substratum, the adhesion extent was affected by the type of milk and consequently by the amount of fat component. The lower and the higher adhesion were obtained when the steel was treated by the UHT semi-skimmed milk and UHT skimmed milk respectively. The correlation between physicochemical properties and S. aureus adhesion show that this latter was controlled by hydrophobicity and electron donor properties.The findings of this work can contribute to develop strategies for prevent S. aureus adhesion on stainless steel and biofilm formation. Also they could be taken into account in cleaning and disinfection procedures.