Growth of Bacillus cereus in natural and acidified carrot substrates over the temperature range 5–30°C

Minimally processed refrigerated foods have a relevant potential in the food market, although the potential risk posed by sporulated emerging psycrotrophs pathogens has to be evaluated. Bacillus cereus is one of these pathogenic micro-organisms. In this paper, the ability to grow of several strains of B. cereus in nutrient broth and in a carrot-based substrate (broth) was evaluated. All the strains tested grew at 12°C or higher temperatures both in nutrient broth and in carrot substrate. One of the strains was able to grow rapidly even at 5°C. Acidification of the carrot substrate proved to be efficient inhibiting B. cereus. For the two strains tested, growth was not observed at pH below 4·75 acidifiying with citric acid or with lemon juice. When the effect of pH was combined with refrigeration, acidification at pH 5·0 was sufficient to inhibit B. cereus at 12°C or lower temperatures. Therefore, the combination of these two factors could have a great potential to control B. cereus growth in minimally processed foods, such as carrot based products. Additionally, lemon juice can be considered as a more natural alternative to citric acid.     

Application of nonlinear regression analysis to the estimation of kinetic parameters for two enterotoxigenic strains ofBacillus cereus spores

Two enterotoxigenic Bacillus cereus strains, isolated from cooked chilled foods containing vegetables, were characterized in relation to safe food production by studying their heat resistance characteristics. The kinetic parameters of thermal resistance were determined by one-step nonlinear regression and were compared with those calculated by using two linear regressions. Results showed that the new analysis methodology applied in this study can improve the estimation ofD - and z - values. Great differences were found in the thermal resistance of the two strains. TheD_90°C were 4·04 and 39 min for the AV TZ415 and AV Z421 strains respectively. Growth studies indicated that the two strains were able to grow at refrigeration temperatures, but the minimum growth temperature for the AV Z421 strain was 10°C, while the less heat-resistant strain, AV TZ415, grew at 5°C.     

Characterizing asymptotic D-values for Salmonella spp. subjected to different heating rates in sous-vide cooked beef

Inactivation rates of a cocktail of Salmonella spp. in sous vide cooked beef exposed to varied ‘come-up’ heating times of zero (control), and 1–3 h from 10 °C to the processing temperature of 58 °C were examined. The observed survival curves, determined for 58 °C, displayed a slight ‘shoulder’ followed by a non-zero asymptotic D-values. Comparisons of the survival curves confirm that the rate of heating can substantially influence the heat resistance of Salmonella spp. While there was no significant difference between the estimated asymptotic D-values for the control and 1-h come-up heating time survival curves, the estimated D-values were significantly larger for the 2- and 3-h come-up heating times curves. The estimated averages of the asymptotic D-values for the control and 1-h come-up time survival curves are approximately 5.7 min; for the 2-h come-up time curves, 7 min; and for the 3-h come-up time curves, 8 min. These findings could have substantial practical importance to food processors in sous vide cooked beef that are processed by slow heating rate/long come-up times and low heating temperatures.     

Distribution and Characteristics of Bacillus cereus Isolated from Rice in Taiwan

Sixty-six strains of Bacillus cereus were isolated from 12 kinds of rice samples collected in Taiwan by employing polymyxin pyruvate egg yolk mannitol bromothymol blue agar(PEMBA). It was found that in Taiwan B. cereus could be detected in various rice samples (less than 200 CFU per gram). The isolates generally grew poorly under acidic conditions. Spore suspensions prepared from six isolates of B. cereus were used to detect their ability to resist heat. The D_100°C- and _D92°C- values for these six isolates in rice broth ranged from 4.2-6.5 min and 16–36 min, respectively. Most of the isolates were sensitive to chloramphenicol (30 mcg), erythromycin (15 mcg), gentamicin (10 mcg) and streptomycin (10 mcg).     

Use of Dacid-, Dbile-, zacid-, and zbile-Values in Evaluating Bifidobacteria with Regard to Stomach pH and Bile Salt Sensitivity

ABSTRACT: The survival of bifidobacteria in simulated conditions of the gastrointestinal (GI) tract was studied based on the D- and z-value concept. Some Bifidobacterium spp. are probiotics that improve microbial balance in the human GI tract. Because they are sensitive to low pH and bile salt concentrations, their viability in the GI tract is limited. The D- and z-value approach was therefore adopted as a result of observing constant log-cell reduction (90%) when Bifidobacterium spp. were exposed to these 2 different stressing factors. Survivals of one strain each or 4 species of Bifidobacterium was studied at pH between 3.0 and 4.5 and in ox-bile between 0.15% and 0.60% for times up to 41 h. From the D_acid- and D_bile-values, the order of resistance to acid and bile was B. bifidum > B. infantis > B. longum > B. adolescentis. While the former 3 strains retained high cell viability at pH 3.5 (>5.5 log CFU/mL after 5 h) and at elevated bile salt concentration of 0.6% (>4.5 log CFU/mL after 3 h), B. adolescentis was less resistant (<3.4 log CFU/mL). The z_acid- and z_bile-values calculated from the D_acid- and D_bile-values ranged from 1.11 to 1.55 pH units and 0.40% to 0.49%, respectively. The results suggest that the D_acid-, D_bile-, z_acid-, and z_bile-value approach could be more appropriate than the screening and selection method in evaluating survival of probiotic bacteria, and in measuring their tolerance or resistance to gastric acidity and the associated bile salt concentration in the small intestine. Practical Application: The evaluation of the tolerance of bifidobacteria to bile salts and low pH has been made possible by use of D- and z-value concept. The calculated z_acid- and z_bile-values were all fairly similar for the strains used and suggest the effect of increasing the bile salt concentration or decreasing the pH on the D_acid- and D_bile-values. This approach would be useful for predicting the suitability of bifidobacteria and other lactic acid bacteria (LAB) as probiotics for use in real-life situations.     

Performance characteristics of the Duopath® cereus enterotoxins assay for rapid detection of enterotoxinogenic Bacillus cereus strains

The Duopath® Cereus Enterotoxins test (Merck KGaA) is a newly developed gold-labeled lateral flow immunoassay for the detection of Bacillus cereus enterotoxins. The test uses monoclonal antibodies (MAbs) against the L₂ component of hemolysin BL (Hbl) and NheB of the non-hemolytic enterotoxin (Nhe), respectively. The inclusivity and exclusivity of the assay was tested using 44 B. cereus, B. cereus group and Bacillus spp. strains. Apart from the B. mycoides type strain the results were in full agreement with those obtained by other immunological and molecular biological methods. The detection limit of the assay was 6 ng/ml for NheB and 20 ng/ml for the Hbl-L₂-component, respectively. Using artificially and naturally contaminated food samples (n = 76) the assay was positive after 18-24 h enrichment if at least 10² enterotoxin producing B. cereus/g were present. After 30 h enrichment samples contaminated with as low as 1 enterotoxin producing B. cereus/g gave positive results. In addition, testing of suspected colonies for enterotoxin production is possible. The assay is easy to perform and results can be clearly read without instrumentation.     

Enterococci vs non-lactic acid microflora as hygiene indicators for sweetened yoghurt

Coliforms are usually used as a measure of hygiene status in the processing and packaging of dairy products. However, their limited chances of survival have placed a question mark over this role in acid products. Some authors propose Enterococcus as a group for hygienic condition inspections in process lines of fermented dairy products. The aims of this work were, first, to evaluate the viability of enterococcus and non-lactic acid microflora (coliforms,Pseudomonas , Staphylococcus aureus and yeasts) in whole set sweetened yoghurt during refrigerated storage and; second, to evaluate the occurrence of bacterial contaminants in industrial process lines of whole set sweetened yoghurt through a critical control points plan (CCP).Test strains were inoculated at a level of 5·5 log orders. Enterococcus remained viable for 21 days and was still detectable after up to 49 days of cold storage. The viability of Pseudomonas was very poor (D -values lower than 0·69 days). Klebsiella spp., Escherichia coli and Citrobacter spp. showed D -values of 1·61, 1·85 and 2·56 days, respectively, while two S. aureus strains showed D -values of 0·61 and 1·56 days. Kluyveromyces marxianus var. lactis strains tested in this study remained viable for at least 42 days.The in vitro assays performed during this study demonstrated that enterococci could remain viable for a longer period than coliforms. However, from analysis of the industrial reality it became evident that in whole set yoghurt lines, coliforms are the most frequent contaminants. In addition, they can remain viable during the fermentation step and, in some cases, during cold storage of the product. Finally, coliform detection is cheaper and faster than enterococci counts. It can thus be concluded that enterococci have little value as hygiene indicators in the industrial processes of yoghurt. Consequently, coliforms are a suitable hygiene indicator as long as they are determined in the first days after manufacture.     

Resistance of Bacillus cereus and its enterotoxin genes in ready-to-eat foods to γ-irradiation

This study was investigated the resistance of Bacillus cereus ATCC 13752 and its enterotoxin coding genes in cereals, vegetables, and tryptic soy broth to γ-irradiation. Screening for enterotoxin coding genes, viability test, PCR analysis, measurement of DNA concentration, cellular constituents release, SEM, and antibiotic sensitivity tests were performed after irradiation at 0, 1.5, 3, 5, 7, 10, and 15 kGy. Thirty % of strains screened possessed all enterotoxins genes. At 10 kGy B. cereus viable cell count was reduced by 4 logCFU/mL. B. cereus in vegetables and broth demonstrated higher susceptibility than those in cereals. Enterotoxins coding genes could be identified in the γ-irradiated cells, antibiotic sensitivity profile was not affected. Concentration of DNA containing enterotoxin coding genes was reduced and release of cellular constituent was highest at 15 kGy. γ-Irradiation up to 10 kGy did not eliminate B. cereus neither affected their enterotoxins coding genes from ready-to-eat food.     

Contamination of infant powdered milk in use with enterotoxigenic Staphylococcus aureus

One-hundred and ten of the 114 samples of infant milk formulae collected from nursing mothers contained viable staphylococci, with the highest mean count of 1·0 × 10² cfu g⁻¹, from samples collected the day the tin was opened. The highest mean total aerobic plate count was 2·6 × 10³ cfu g⁻¹. Titratable acidity and pH of the reconstituted milk ranged from 0·06 to 0·8 and 6·40 to 6·52 respectively. 52·0% of the 123 isolates were S. aureus, 49·6% produced β-hemolysin and 17·1% produced α-hemolysin. Enterotoxin A was produced by 6·5%, B by 1·6%, C by 2·4%, D by 1·6% and E by 0·8% of the isolates. The total staphylococcal and aerobic plate counts were not affected by either the period elapsed from the opening of tin to sampling or the brand of milk.     

Occurrence,heat and antibiotic resistance profile of Bacillus cereus isolated from raw cow and processed milk in Mezam Division,Cameroon

Bacillus cereus is the aetiologic agent of two distinct forms of food poisoning: the diarrhoeal and emetic syndromes. Little data exist on the prevalence of B. cereus in raw milk and milk products sold in Cameroonian towns. This study was aimed at investigating the occurrence, heat and antibiotic resistance of B. cereus isolated from raw milk and selected milk products in Mezam division, Cameroon. Bacillus cereus was isolated by inoculating samples onto mannitol‐egg yolk‐polymyxin B agar. Isolates were characterised morphologically and biochemically. The occurrence of B. cereus in raw milk (8.22%) was less than that in milk powder (13.33%). Bacillus cereus was not isolated from fermented milk. There was no significant difference (P < 0.05) between the B. cereus load in raw milk (2.6 × 10³ cfu/mL) and milk powder (3.0 × 10² cfu/mL). All the isolates showed haemolysin activity and were sensitive to tetracycline, gentamicin, chloramphenicol and nalidixic acid, but resistant to penicillin, ampicillin and trimethoprim/sulphamethoxazole. The detection of drug‐resistant, haemolysin‐positive isolates should serve as a warning for an impending health hazard following consumption of untreated milk. Heat resistance of isolates was assessed by determining the decimal reduction time; D‐value (time to inactivate 90% of the B. cereus spores); and the heat sensitivity, z (temperature increase leads to a tenfold reduction in the D‐value). The values for D₁₀₀ ranged from 0.5 to 3.5 min, and z‐values ranged from 10.0 to 32.6 °C. These results could be used in the dairy industry to evaluate the importance of heat treatment on B. cereus inactivation and calculation of process efficiency.     

Inactivation of Escherichia coli O157:H7 on surface-uninjured and -injured green pepper (Capsicum annuum L.) by chlorine dioxide gas as demonstrated by confocal laser scanning microscopy

Cells of Escherichia coli O157:H7 on uninjured and injured surfaces of green pepper were inactivated by 0·15–1·2 mg l⁻¹ClO₂gas treatments. A membrane-surface-plating method was used for resuscitation and enumeration of E. coli O157:H7 treated with ClO₂. The location and viability ofE. coli O157:H7 on uninjured and injured green pepper surfaces after ClO₂gas treatments were visualized using confocal laser scanning microscopy (CLSM). Live and dead cells of E. coli O157:H7 on pepper surfaces were labeled with a fluorescein isothiocyanate-labeled antibody and propidium iodide, respectively. A 7·27 log reduction of E. coli O157:H7 on uninjured green pepper surfaces was obtained with a 0·60 mg l⁻¹ClO₂gas treatment for 30 min at 20°C under 90–95% relative humidity. For injured surfaces, a 6·45 log reduction was achieved with a 1·2 mg l⁻¹ClO₂gas treatment. Each ClO₂gas treatment (0·15–1·2 mg l⁻¹ClO₂) for inoculated bacteria on uninjured surfaces showed significantly more reductions (1·23–4·24 log) than for those on injured surfaces (P<0·05). The microphotographs of CLSM showed that bacteria preferentially attached to injured surfaces and those bacteria could be protected from bacterial reduction by the injuries. This study indicates that ClO₂gas treatment can be a potential effective method of pathogen reduction for fresh fruits and vegetables.     

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO₂, ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO₂ in comparison to the other strains of Dekkera. These strains were inoculated (10³–10⁴ cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 °C and 21 °C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10⁵ cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 °C while slower growth was observed at 15 °C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 °C compared to 21 °C.     

Effect of lactic acid on Listeria monocytogenes and Edwardsiella tarda attached to catfish skin

This study evaluated the use of lactic acid to decontaminate Listeria monocytogenes andEdwardsiella tarda attached to catfish skin with or without mucus. At the highest inoculum levels (10⁴–10⁵cfu skin⁻¹), lactic acid (0·5–2·0%) exposure for 10 min reduced counts of L. monocytogenes firmly attached to catfish skin by 0·9–>1·9 log₁₀cfu skin⁻¹and cells loosely attached by 2·7–>3·7 logs. Counts of E. tarda firmly attached to catfish skin were reduced by 0·9–>3·0 logs and cells loosely attached by 1·5–>3·5 logs. Overall bacterial numbers of lactic acid-treated cells that were firmly attached to skin with mucus were higher than on skin without mucus. Firmly attached L. monocytogenes was more resistant to lactic acid than was firmly attached E. tarda. Catfish skin mucus decreased the antimicrobial effect of lactic acid against attached L. monocytogenes and E. tarda.     

The incidence and antibiotic resistance profiles of Salmonella spp. on Irish retail meat products

Irish retail meat (n=74) and poultry samples (n=106) were tested for the presence of naturally occurring Salmonella spp. The pathogen was detected in 28 poultry (n=106), two pork (n=22) and one cooked meat samples (n=20) examined. Salmonella was not isolated from minced beef or lamb samples tested. Initial counts on samples ranged from 0 to log₁₀2·5 cfu g⁻¹. The most commonly isolated serotype was S. bredeney accounting for 48·4%, followed by S. kentucky (35·5%) and S. enteritidis (6·5%). Salmonella spp. (n=31) isolated from food products were also examined for antibiotic resistance. A total of 155 strains (five strains from each isolate) were tested for resistance to 26 antibiotics using the Bauer method. The percentage of samples showing antibiotic resistance amongstSalmonella isolates were as follows: Riampicin (100%); Tetracycline (92·92%); Oxytetracycline (86·26%); Sulphamethoxazole (86·25%) and Streptomycin (80·92%).     

Enterotoxigenic Profiling of Emetic Toxin‐ and Enterotoxin‐Producing Bacillus cereus,Isolated from Food,Environmental, and Clinical Samples by Multiplex PCR

Bacillus cereus comprises the largest group of endospore‐forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin‐ (8 patterns) and enterotoxin‐producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.     

Effects of drying on the gel strength and cation mobility of furcellaran

Thermal stability, by means of air drying a furcellaran powder, and its impact on gel strength and cation mobility were studied. Halogen heating in the temperature range 90–115°C for 15 min resulted in loss on drying (L_D, %). These results can be described by polynom L_D=−9.583+2.989τ−0.249τ²+0.00729τ³+0.1034t (R²=0.9976), indicating a gradual decomposition of carbohydrates. Air-drying induced a decrease in gel strength and the partial removal of potassium, calcium and sodium ions from the matrix. Air drying above 115°C yielded a remarkable destruction of polysaccharides with a total collapse in gelling power.     

Migration of α-tocopherol from an active multilayer film into whole milk powder

Antioxidant active packaging is a promising technology for whole milk powder (WMP) protection. In this study, the migration of α-tocopherol from a multilayer active packaging (made of high density polyethylene, ethylene vinyl alcohol and a layer of low density polyethylene containing the antioxidant) to WMP was studied. A model based on the Fick’s diffusion equation was used to calculate the diffusion coefficients (D) of α-tocopherol as 2.34 × 10⁻¹¹, 3.06 × 10⁻¹¹, and 3.14 × 10⁻¹¹ cm² s⁻¹ at 20, 30 and 40 °C, respectively. The D at 20 °C was different from those at 30 and 40 °C (P < 0.05); but it was similar at 30 and 40 °C. This low influence of temperature on the migration of α-tocopherol from 20 to 40 °C assures the release at real storage and commercialization conditions in regions with warm/hot climate. The antioxidant delivering system delayed the lipid oxidation of WMP and it was more effective at 30 and 40 °C since the rate of oxidative reactions was higher at these temperatures than at 20 °C.     

Effect of carbon dioxide pressure on Listeria monocytogenes in physiological saline and foods

The effect of 15,30 and 60 atm CO₂pressure at 25,35 and 45°C on Listeria monocytogenes was studied. A biphasic curve was observed in the CO₂treatments. A primary curve was characterized with very little killing and a secondary curve was showed straight-line inactivation kinetics. An increase of pressure and/or temperature enhanced the antimicrobial effects of CO₂. Listeria monocytogenes suspended in physiological saline containing 1% brain–heart infusion broth was completely inactivated under 60 atm CO₂treatment in 115,75 and 60 min at 25,35 and 45°C, respectively. A minimum D -value was obtained under 60 atm CO₂pressure at 45°C. High pressure CO₂treatment technique can possibly be applied to reduce microbiol load in whole and skim milk; and carrot, orange, peach, and apple juices.     

Antibiotic resistance and susceptibility to some food preservative measures of spoilage and pathogenic micro-organisms from spices

Antibiogram of 84 strains of Bacillus cereus, 26 strains of Clostridium perfringens, four strains of Staphylococcus aureus, 51 strains of Enterobacteriaceae, two strains of each of Salmonella and Shigella; isolated from spices, were studied against 20 different antibiotics that are commonly used against foodborne diseases, mainly gastroenteritis. All the tested strains of B. cereus, Cl. perfringens, Staph. aureus, Escherichia coli, Salmonella and Shigella were found resistant to at least 3, 4, 7, 6, 10 and 9 antibiotics, respectively. In brain–heart infusion broth supplemented with glucose, the D_100°C-values for B. cereus were 3.5–5.9 min, and the z-values were 17–18°C. The D_100°C-values for Cl. perfringens in fluid thioglycolate medium were higher (10.0–19.8 min) than those of B. cereus. The minimum inhibitory concentrations (MICs) of sodium chloride were 45–80 mg ml⁻¹. While the MIC of benzoic acid for Cl. perfringens, tested on perfringens agar (pH 7.3) plates by incubating anaerobially at 35°C for 24 h, was 1.9–2.2 mg ml⁻¹, for others, tested on nutrient agar (pH 6.8) plates by incubating at 35°C for 18 h in static aerobic condition, it was much less. Similarly, the MIC of sorbic acid for all the tested isolates, excepting Cl. perfringens, was 0.6–1.1 mg ml⁻¹. Of the eight isolates of Cl. perfringens, only three were inhibited at 2.0 mg sorbic acid ml⁻¹, while others were resistant. Sixty percent and 75% of the respective strains of B. cereus and Cl. perfringens were resistant to 5000 IU Nisaplin ml⁻¹, whereas the MIC values of Staph. aureus were between 3000 and 5000 IU ml⁻¹. While studying combined effect of selected hurdles on the growth of enterotoxigenic Cl. perfringens 16-C2, the judicious combination considered was low acid (pH 6.0), 30 mg sodium chloride ml⁻¹ and 1.25 mg benzoic acid ml⁻¹.     

Rheological,textural and sensory properties of gluten-free bread formulations based on rice and buckwheat flour

Mixolab, as the rheological instrument, was utilized to create gluten-free products. According to obtained Mixolab profiles, mixtures of rice flour and husked buckwheat and rice flour and unhusked buckwheat flour expressed rheological properties similar to wheat flour. In both types of mixtures the ratio of rice flour to buckwheat flour was 90:10, 80:20 and 70:30. According to the Mixolab profiles of the investigated systems, gluten-free products containing unhusked buckwheat flour had higher water absorption values, lower stability and weaker protein network structure, as well as lower peak viscosity than those consisted of husked buckwheat flour.Increasing the amount of husked buckwheat flour from 10% to 20% resulted in both G′ and yield stress value increase, but further increase of the husked buckwheat flour on 30% resulted in both G′ and yield stress value decrease. However, increasing the amount of unhusked buckwheat flour from 10% to 20% resulted in significant decrease of G′ and yield stress value having no significant impact with the addition of 30% of unhusked buckwheat flour.Hardness, expressed as a work of compression of the final product, increased with the amount of both types of buckwheat flour. Samples containing UBF expressed not significantly higher values of hardness than those prepared with HBF. According to obtained results of sensory evaluation of the final products it can be concluded that all six combinations of tested gluten-free breads were sensory acceptable.