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.     

Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces

One source of foodborne listeriosis is related to a virulent strain established in the food-processing environment. We used direct epifluorescence microscopy (DEM) to evaluate the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces, in wet conditions at room temperature. Biofilms that were allowed to develop for 72 h were subsequently treated with different concentrations of phage (5, 6, 7, and 8 log PFU/ml). L. monocytogenes was monitored up to 48 h, using both DEM and cultivation method. When L. monocytogenes was monitored using a cultivation method, the first significant reduction was observed after phage treatment with 7 and 8 log PFU/ml at 8 h. Subsequently, these treatments achieved undetectable levels of pathogen at 48 h, with a mean reduction of 5.29 log CFU/cm². Conversely, when samples were evaluated using DEM in treatments with 6, 7, and 8 log PFU/ml, although disaggregation of biofilms could be observed after 8 h, viable cells were still present up to 48 h (maximal reduction 1.5 log units). The phage titer remained stable or increased up to 2.59 log units during the study period. In conclusion, phage P100 may provide an adjuvant measure to control L. monocytogenes biofilm on stainless steel surfaces. However, phage treatment must be used in combination with other hygienization measures to increase efficacy. In this study, DEM was a good tool to quickly and accurately assess the real effect of phage P100 on L. monocytogenes biofilms.     

Characterization of a novel bacteriocin produced by Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish

A novel bacteriocin, sakacin LSJ618, produced by the strain Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish, was studied. L. sakei LSJ618 was identified by both phenotypical and physiological tests combined with 16S rDNA sequence analysis. Sakacin LSJ618 is sensitive to hydrolytic enzymes including lipase, is stable between pH 2-8, and is heat resistant (30 min at 121 °C). Sakacin LSJ618 exhibits inhibitory activity against food-spoiling bacteria and food-borne pathogens, including the Gram-positive Listeria monocytogenes, Staphylococcus aureus, Sarcina sp., Micrococcus luteus, and the Gram-negative Proteus sp. and Escherichia coli, but not against most of the lactic acid bacteria tested. Maximal production of bacteriocin was reached in the late stationary phase, and inhibitory activity declined within 26 h. The mode of action of sakacin LSJ618 was determined to be bactericidal, as evidenced by its action upon Micrococcus tetragenus. After partial purification by ammonium sulfate precipitation and Sephadex G-25 chromatography, the molecular weight of sakacin LSJ618 was determined to be 5.2 kDa by Tricine-SDS-PAGE. The identified properties of sakacin LSJ618 indicate that it is a novel bacteriocin with potential application as a biopreservative in the food industry.     

Induction of plantaricin MG under co-culture with certain lactic acid bacterial strains and identification of LuxS mediated quorum sensing system in Lactobacillus plantarum KLDS1.0391

A bacteriocin named plantaricin MG was produced by Lactobacillus plantarum KLDS1.0391 which was isolated from “jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China. Bacteriocin production was increased significantly when L. plantarum KLDS1.0391 co-cultured with certain lactic acid bacteria (LAB) strains. Among 76 strains belonging to the genera Lactobacillus, Lactococcus, Leuconostoc and Streptococcus, four strains namely Lactobacillus helveticus KLDS1.9207, Enterococcus faecium KLDS4.0352, Lactobacillus reuteri KLDS1.0737 and Enterococcus faecalis KLDS4.0313 were shown to induce bacteriocin production of L. plantarum KLDS1.0391. Cell numbers of L. plantarum KLDS1.0391 were greatly enhanced when co-cultured with four bacteriocin-inducing strains. Bacteriocin production was not induced by autoclaved cultures and cell-free supernatants (CFS) of inducing strains, indicating that living cells of inducing strains might be necessary for enhancement of bacteriocin production. The presence of plnD and luxS genes was detected by polymerase chain reaction (PCR), and the existence of plNC8HK gene was detected by single oligonucleotide nested PCR (SON-PCR).     

Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juice by PCR against hilA gene

A novel PCR based assay was devised to specifically detect contamination of any Salmonella serovar in milk, fruit juice and ice-cream without pre-enrichment. This method utilizes primers against hilA gene which is conserved in all Salmonella serovars and absent from the close relatives of Salmonella. An optimized protocol, in terms time and money, is provided for the reduction of PCR contaminants from milk, ice-cream and juice through the use of routine laboratory chemicals. The simplicity, efficiency (time taken 3-4 h) and sensitivity (to about 5-10 CFU/ml) of this technique confers a unique advantage over other previously used time consuming detection techniques. This technique does not involve pre-enrichment of the samples or extensive sample processing, which was a pre-requisite in most of the other reported studies. Hence, this assay can be ideal for adoption, after further fine tuning, by food quality control for timely detection of Salmonella contamination as well as other food-borne pathogens (with species specific primers) in food especially milk, ice-cream and fruit juice.     

Aflatoxin B1 degradation by Bacillus subtilis UTBSP1 isolated from pistachio nuts of Iran

Pistachio nuts are among the commodities with the highest risk of aflatoxin contamination in Iran. Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for humans and livestock. In nature, there are microorganisms which are capable of reducing aflatoxins contamination in food and feed products. In this study, Bacillus subtilis strain UTBSP1 was isolated from pistachio nuts and studied for the degradation of AFB1. The AFB1 contents were determined by the use of HPTLC and HPLC as well as multiple reactions monitoring (MRM) method in LC-MS/MS. The results indicated B. subtilis UTBSP1 could considerably remediate AFB1 from nutrient broth culture and pistachio nut by 85.66% and 95%, respectively. Cell free supernatant fluid caused an apparent 78.39% decrease in AFB1 content. The optimal conditions for AFB1 degradation by cell free supernatant appeared at 35-40 °C, during 24 h. Furthermore, the results indicated that AFB1 degradation is enzymatic and responsible enzymes are extracellular and constitutively produced. The destructive AFB1 differed from standard AFB1 chemically, and lost a fluorescence property.     

Influence of temperature and surface kind on biofilm formation by Staphylococcus aureus from food-contact surfaces and sensitivity to sanitizers

This study aimed to assess the adhesion, detachment kinetic and biofilm formation of Staphylococcus aureus isolates from food services surfaces on stainless steel and polypropylene surfaces when cultivated in a vegetable-based broth at 7 and 28 °C, and the efficacy of peracetic acid (30 mg/L) and sodium hypochlorite (250 mg/L) in removing the bacterial cells from the matrix of the preformed biofilm. The isolates adhered over 4 Log cfu/cm² regardless the surface kind and incubation temperature. Cell detachment was around 3 Log cfu/cm² over the first six contacts with agar characterizing a high persistence of cells on the tested surfaces. Number of cells (5-7 Log cfu/cm²) needed for biofilm formation was noted at all experimental systems already after 3 days of incubation. A range of 2.0-3.3 and 1.5 to 2.1 Log cfu/cm² was observed in the reduction of cells in biofilm matrix caused by peracetic acid and sodium hypochlorite, respectively. The isolates of S. aureus revealed high capability to adhere and form biofilm on the tested surfaces in both assayed incubation temperature.     

Effect of Hibiscus sabdariffa extract and Nigella sativa oil on the growth and aflatoxin B1 production of Aspergillus flavus and Aspergillus parasiticus strains

This study was undertaken to evaluate the inhibitory effect of Hibiscus sabdariffa calyx extract at concentrations of 5, 7.5, 10 and 12.5 g/100 ml and Nigella sativa oil at concentrations of 1, 2 and 3 ml/100 ml on the growth and aflatoxin B₁ production by Aspergillus parasiticus (CBS 921.7) and Aspergillus flavus (SQU 21) strains. The inhibition of aflatoxin B₁ production by the different concentrations of H. sabdariffa calyx ranged between 91.5-97.9% and 87.1-93.3% for A. flavus and A. parasiticus strains, respectively, whereas the inhibition by different concentrations of N. sativa oil ranged between 47.9 and 58.3% for A. flavus and 32-48% for A. parasiticus strains. The different concentrations of H. sabdariffa calyx and N. sativa oil had no significant effect on the growth of either Aspergillus species. Neither H. sabdariffa calyx nor N. sativa oil detoxified pure aqueous aflatoxin. Our results suggest that H. sabdariffa calyx and N. sativa oil extracted from seeds had metabolic effects on aflatoxin biosynthesis pathway of both Aspergillus species and can be used as an effective biocontrol and non-toxic biopreservatives in food industry against aflatoxin contamination.     

Resistances to UV-C irradiation of Salmonella Typhimurium and Staphylococcus aureus in wet and dried suspensions on surface with egg residues

To clarify the effects of food sediments on ultraviolet-C (254 nm) sanitation in food-related environments, we examined the resistance of pathogenic bacteria (Salmonella Typhimurium and Staphylococcus aureus) cells, in wet and dried suspensions adhered with 1.5-15% w/v egg albumen, 1.5-15% yolk or 3.0-30% whole egg solutions, against UV-C irradiation. Bacterial suspensions (0.1 ml of 8 log CFU/ml) were put on 47 mmφ glass dishes and dried at room temperature (20-24 °C) for 180 min in a bio safety cabinet with ventilation. Viable S. Typhimurium and S. aureus cells in distilled water decreased during the drying period from 7.2 to 3.2 and from 8.0 to 6.5 log CFU/dish, respectively,. On the other hand, the bacteria cells were protected from drying by egg compounds, even by the lowest concentration. The UV-C treatment (0.16 mW/cm² for 10 min) showed a clear bactericidal effect in the absence of egg compounds. However, the bactericidal effect was inhibited by 15% yolk and 30% whole egg. Results in this study suggested that the small food sediment protect bacteria on the surfaces from dryness and UV-C irradiation and it might introduce cross contamination.     

Prediction of Escherichia coli O157:H7 adhesion and potential to form biofilm under experimental conditions

Escherichia coli O:157:H7 adhesion and potential to form biofilm on three different surfaces commonly used in the food industry was evaluated using probabilistic models; the surfaces tested were stainless steel 304 (SS304), poly(vinyl chloride) film covered with thick cloth (PVC1) and poly(vinyl chloride) film covered with thin cloth (PVC2). Using a Central Composite Rotational Design (CCRD), the effect of contact time (0 h, 7 h, 24 h, 41 h and 48 h) and temperature (12 °C, 17 °C, 28 °C, 39 °C and 44 °C) on the probability of achieving a particular adherent cell count (Log₁₀ CFU cm⁻²) was determined. By analyzing response surface plots and their corresponding contour plots and by determining quadratic equations for each surface, experimental values were shown to be significant in accordance with predicted values in all cases. The adjusted determination coefficient (R_adj²) was 90.5%, 97.2% and 98.9% for SS304, PVC1 and PVC2, respectively, and the level of significance was P ≤ 0.001. The bias factor (B_f) and accuracy factor (A_f) both approached 1.0 for the three surfaces evaluated. The model equations for predicting optimum response values were verified effectively by a validation data set for all surfaces evaluated. Therefore, an RSM provides a useful and accurate method for predicting E. coli O157:H7 adhesion and potential to form biofilm on SS304, PVC1 and PVC2 and could be considered to be a standard way to ensure food safety with respect to E. coli O157:H7 contamination through adhesion and biofilm formation.     

Impact of Pediococcus pentosaceus strain L006 and its metabolites on fumonisin biosynthesis by Fusarium verticillioides

The aim of this work was to study the effectiveness of a Pediococcus pentosaceus strain L006, isolated from maize leaf and previously characterised for its high antifungal efficiency, on fumonisin biosynthesis by Fusarium verticillioides. Studies performed in GYEP medium supplemented with amylopectin showed a significant increase in fumonisin production when the F. verticillioides strain was simultaneously co-inoculated with the P. pentosaceus strain or inoculated in a three-day-old culture of this lactic acid bacteria. Our studies also demonstrated that some extracellular metabolites produced in MRS medium by the P. pentosaceus strain L006 were able to significantly reduce fumonisin production in liquid medium as well as on maize kernels. Fumonisin yields by F. verticillioides inoculated on autoclaved maize kernels were reduced by a factor ranging from 75% to 80% after 20 days of incubation. Our results illustrate the potential risk linked to the use of an antagonistic bacterial agent to manage fumonisin contamination, while emphasizing the potential use of bacterial metabolites to counteract fumonisin accumulation in kernels.     

Effects of spray volume, type of surface tissue and inoculum level on the survival of Escherichia coli on beef sprayed with 5% lactic acid

Membrane, fat and cut muscle surfaces of beef were inoculated with Escherichia coli at numbers about 4, 1 or −1 log cfu/cm². The inoculated meat was sprayed with water or 5% lactic acid at volumes of 0.5, 0.1 or 0.02 ml/cm². Spraying with water reduced the numbers of E. coli on membrane surfaces by up to 1 log unit, but had little effect on the numbers of E. coli on fat or cut muscle surfaces. Spraying with 5% lactic acid reduced the highest numbers of E. coli on membrane surfaces by up to 4 log units; but those numbers on fat or cut muscle surfaces were reduced by ≤1.5 log unit, and the reductions declined with decreasing volumes of 5% lactic acid. With inocula of 1 log cfu/cm², spraying lactic acid in any volume reduced the numbers of E. coli on membrane or fat surfaces by about 1 log unit, and the numbers on cut muscle surfaces by between 0.8 and 0.2 log unit. E. coli were detected in enrichment cultures of samples from all surfaces inoculated with E. coli at −1 log cfu/cm² and sprayed with 5% lactic acid at 0.5 ml/cm². The findings indicate that spraying relatively heavily contaminated cuts or trimmings with 5% lactic acid at ≥0.1 ml/cm² can be expected to reduce numbers of E. coli and, presumably, associated pathogens by between 0.5 and 1 log unit. However, such a treatment is likely to be at best marginally effective for reduce the numbers of these organisms on lightly contaminated product.     

Effect of compression and decompression rates during high hydrostatic pressure processing on inactivation kinetics of bacterial spores at different temperatures

We investigated the effect of changing compression and decompression rates of High Hydrostatic Pressure (HHP) treatments on inactivation of spores. Bacillus subtilis (PS832) spores were inoculated in Tris buffer, skimmed milk and orange juice. The samples were subjected to HHP treatments of 600 MPa for 3 min at 60 °C and 70 °C. Microbiological analyses were carried out at 0, 1, 7 and 15 days of refrigeration storage (4-5 °C). Flow cytometery technique was used for the estimation of sublethally injured population. After 15 days, all pressure treated matrices at 70 °C showed higher spore inactivation caused by slower compression rates as compared to faster ones. However, at 60 °C, the inactivation caused by slower compression was not significantly different from faster rates. Slow decompression was found to be more lethal in 60 °C and 70 °C HHP treated samples. It is concluded that slow compression combined with slow decompression has a greater impact on inactivation of B. subtilis spores than any combination of fast compression and fast decompression at 60 °C and 70 °C processing temperatures. However the population of sub-lethally injured cells was found to be higher with fast compression and slow decompression rates.     

Effects of green tea extract on reducing Vibrio parahaemolyticus and increasing shelf life of oyster meats

This study investigated effects of tea extract on growth of pathogenic Vibrio parahaemolyticus and potential utilization in post-harvest treatment to extend shelf life of Pacific oysters (Crassostrea gigas). Longjing Tea, which exhibited strong bactericidal activity against V. parahaemolyticus, was selected to use in this study. Tea extract containing equal or higher than 4.6 g/l total phenolic contents (TPC) as gallic acid equivalents (GAE) determined by Folin-Ciocalteau method could reduce a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl from 4.5 log CFU/ml to non-detectable level (<1 log CFU/ml) within 8 h. A treatment of shucked oysters with tea extract containing 9.1 g/l TPC as GAE for 2 h at 23 ± 1 °C with oyster/tea extract ratio of approximate 0.9 g/ml resulted in greater (p < 0.05) V. parahaemolyticus reductions (0.8 log MPN/g) compared to controls (0.2 log MPN/g). The following shelf life study indicated that green tea treatment at oyster/tea extract ratio of approximate 0.7 g/ml could enhance reducing V. parahaemolyticus while retarding the growth of total bacteria in oysters during 5 ± 1 °C storage. Therefore, green tea might be utilized as a natural antimicrobial agent to inactivate V. parahaemolyticus in oysters and extend the shelf life during refrigeration storage.     

Effect of seasonal variations and lactation times on aflatoxin M1 contamination in milk of different species from Punjab, Pakistan

During October 2009 to September 2010, aflatoxin M₁ (AFM₁) levels were analyzed by HPLC-FLD in 356 milk samples of different lactating species (buffalo, cow, goat, sheep and camel) from Punjab (Pakistan). Recoveries of AFM₁ ranged from 92 to 97% and the limit of detection (LOD) was 0.004 μg/L. For all lactating species the mean concentration of AFM₁ was significantly higher in winter season than in summer (p < 0.05). The results showed that 55, 56, 32, 58 and 27% of winter milk samples of buffalo, cow, goat, sheep and camel exceeded the EU maximum limit (0.05 μg/kg), compared with 38, 33, 21, 36 and 14% of summer milk samples, respectively. For all lactating species the mean concentration of AFM₁ was significantly higher in morning milks than in evening milks (p < 0.05). The percentage of morning milk samples exceeding the EU maximum limit was 72, 67, 69, 71 and 44% for buffalo, cow, goat, sheep and camel, while for evening milks percent non compliant rates were 39, 30, 18, 33 and 25%, respectively. The level of AFM₁ tended to be higher in animal species fed mainly on concentrate mixtures (buffalo and cow) than in other species grazing on fresh greens.     

Influence of sunflower oil based nanoemulsion (AUSN-4) on the shelf life and quality of Indo-Pacific king mackerel (Scomberomorus guttatus) steaks stored at 20 °C

The influence of nanoemulsion (AUSN-4) on the microbiological, proximal, chemical, and sensory qualities of Indo-Pacific king mackerel (Scomberomorus guttatus) steaks stored at 20 °C was studied for a time period of 72 h. AUSN-4 treatment showed initial reduction (P > 0.05) in the heterotrophic, H₂S and lactic acid bacterial populations in 12 h, followed by a gradual increase in their respective populations. Irrespective of treatments, reduction in total carbohydrate, protein, and fat contents were observed in all samples with an increase in storage time (h), with AUSN-4 treated steaks having the lowest reduction. AUSN-4 treatment significantly (P < 0.05) decreased the values of chemical indicators of spoilage throughout the storage period. Organoleptic evaluation revealed that AUSN-4 treated steaks showed an extension of shelf life of 48 h, when compared with control and antibiotic treated samples, respectively. Based on the results obtained in our present study we conclude that sunflower oil based nanoemulsion preservative technique is able to extend the shelf life and maintain the quality of S. guttatus steaks during storage.