In-milk inactivation of Escherichia coli O157:H7 by the environmental lytic bacteriophage ECPS-6

Shiga toxin-producing Escherichia coli is a common foodborne pathogen which transmission includes dairy products. In the search for novel biocontrol methods, bacteriophages have become important candidates for the eradication of foodborne pathogens. The aim of this study was to evaluate the bacteriophage-mediated reduction of E. coli O157:H7 in raw and filtered milk. Laboratory-scale tests showed that the bacteriophage ECPS-6 efficiently adsorbed to E. coli O157: H7 cells. Furthermore, ECPS-6 remained stable when heated at 70°C for 20 min and in a wide pH range from 3.0 to 11.0. The trials on contaminated milk were performed using filtered and unfiltered raw milk contaminated with 1 × 10⁵ CFU × ml⁻¹ of E. coli O157: H7. Bacteriophage was added at multiplicity of infection (MOI) 5 and 50. The ECPS-6 reached the highest lytic activity at MOI = 5 (25°C) which resulted in 4.74 Log₁₀ CFU × ml⁻¹ and 7.3 Log₁₀ CFU × ml⁻¹ reduction after 10 days for both tested strains, respectively. Under refrigerated conditions (4°C) the quantity of E. coli decreased to 1.5 Log₁₀ CFU × ml⁻¹ and 3.04 Log₁₀ CFU × ml⁻¹ for these strains, respectively. Usage of MOI = 50 for the treatment unfiltered milk led to the reduction of E. coli O157:H7 A-2 below the detection limit after 6 hr.     

Shelf Life of Shrimp (Penaeus merguiensis) Stored at Different Temperatures

The sensory, microbiological and biochemical changes were determined in shrimp (Penaeus merguiensis) stored at 0° to 35°C. Mean aerobic plate count of fresh shrimp, initially 5.0 × 10⁵ CFU/g, increased with time and temperature to 6.4 × 10⁹ CFU/g at 35°C after 24 hr. A total of 560 different bacteria were isolated and identified. In addition, the dominant organisms were tested for ability to reduce TMAO, produce indole and hydrolyze protein. The initial bacteria were 30% Gram + organisms but changed to predominantly Grampsychrotrophs at lower temperatures and to mesophiles at higher temperatures. Odor, texture and color qualities decreased; TMA, TVB, pH and Indole increased with time and temperature. Shelf life of shrimp ranged from 7 hr at 35°C to 13 days at 0°C.     

Physiological Activity of Deep-Frozen Concentrates of Leuconostoc Strains

11 strains of Leuconostoc cremoris and 1 strain of Leuconostoc lactis were the subject of investigations. Basing on the evaluation of growth parameters in the different cultivation media and on the diacetyl production ability in milk 2 strains of Leuconostoc cremoris and 1 strain of Leuconostoc lactis were chosen for preparing frozen concentrated biomass. Sterile milk or cream (18% fat) were used as protecting agents. Concentrated biomass was frozen at ?70 °C and stored at ?30 °C for 3 months. The prepared concentrates contained from 1.6 × 10¹¹ to 2.8 × 10¹¹ colony forming units (CFU)/g. Except for one (strain “S”) the strains retained 100% survival. All the strains showed high growth activity after their recovery in milk (3.3 × 10⁸ - 8.4 × 10⁸ CFU/ml). Both protecting agents applied proved to be equally good for preserving viability of the cells and for their acidifying activity. In spite of the protecting agent a decrease in the aroma activity by 25–50% was observed after one month of the biomass storage. Nevertheless, concentrated frozen multicomponent starters containing Leuconostoc strains retained the ability to produce diacetyl and acetoin on the unchanged level during the three months storage period.     

Reproducibility of Microbiological Counts on Frozen Cod: A Collaborative Study

Reproducibility of aerobic plate count (APC), coliform and fecal coliform counts in frozen cod was examined by 14 laboratories (3 government, 4 university and 7 industry) using primarily FDA recommended procedures. In order to assure homogeneity of samples, the fish was comminuted and thoroughly mixed. The 35°C and the “room temperature” (26°C) APC on Sample A (a good quality product) were 1.3 × 10⁵/g and 2.6 × 10⁵/g, respectively, and for Sample B (a questionable quality product) the counts were 2.9 × 10⁵/g and 6.4 × 10⁵/g, respectively. Standard deviations among counts (log₁₀ values) were 0.26 and 0.32 for the 35°C counts and 0.24 and 0.40 for the 26°C counts of Sample A and B, respectively. The std. dev. were reduced to 0.19, 0.22, 0.17 and 0.25, respectively, when counts that did not show significant overlap (based on Dur can's New Multiple Range Test) were excluded from the calculations. Coliform counts on Sample A (not inoculated) ranged from <30 to 4.6 × 10⁵/100g. Coliform and fecal coliform counts for Sample B (inoculated at estimated level of 1 × 10⁴/100g) were 5.8 × 10³/100g and 5.5 × 10³/100g, with a std. dev. of 0.43 and (.37, respectively. The std. dev. for the coliform counts was reduced to 0.30 when counts from one laboratory were excluded, based on Duncan's New Multiple Range Test.     

Effect of temperature on initial bacteria of prawn

This study was carried out to evaluate the effect of heat and cold on the microorganisms existing in prawn. Roasting for 5 min, boiling for 10 min and storage at ? 18 °C for 10, 20 and 30 days were applied on raw prawn to study the effect of each treatment on the bacterial load. The average counts of APC (aerobic plate count) at 25 °C and 0 °C, Enterobacteriaceae, Coliforms and Staphylococci were 1.3 × 10⁷, 6 × 10⁴, 3 × 10⁴, 2 × 10² and 3 × 10³ organisms per gram raw prawn sample, respectively, then reduced to 10⁴, 8 × 10², 3 × 10³, < 3 and 3 × 10² organisms per gram roasted prawn in shell, respectively. Such counts were more reduced in roasted peeled samples. Boiling reduced the average counts of APC at 25 °C and 0 °C, Enterobacteriaceae, Coliforms and Staphylococci from 8 × 10⁸, 2 × 10⁵ 2 × 10⁷, 3 × 10 and 3 × 10⁴ organisms per gram raw prawn sample to 2 × 10⁵, 3 × 10³, 2 × 10², < 3 and 4 × 10² organisms, respectively. Concerning freezing storage, it could be observed that the average counts of APC at 25 °C and 0 °C, Enterobacteriaceae, Coliforms and Staphylococci were reduced from 4 × 10⁷, 5 × 10⁵, 3 × 10⁵, 10³ and 3 × 10² organisms per gram raw sample to 5 × 10⁴, 10³, 3 × 10², 10 and < 2 × 10² organisms per gram after freezing storage for 30 days.     

A Study on the Growth Potential of Staphylococcus aureus In Boletus edulis, A Wild Edible Mushroom, Prompted by a Food Poisoning Outbreak

A dish of Boletus edulis, a wild edible mushroom, in vinegar caused staphylococcal food poisoning in 13 of 35 diners in a restaurant. Enterotoxicosis was confirmed by detection of toxins A and D in the dish. Staphylococcus aureus growth potential in B. edulis was studied by inoculating fresh and frozen and thawed bolete with S. aureus strains VTTE 530, 757, 793 and 805 and storing for 3 days at 15 and/or 21°C. Essentially no staphylococcal growth was observed in frozen and thawed mushrooms contaminated with strains 530, 793 or 805 and stored at 15 or 21°C. In fresh B. edulis the same strains showed slight growth at 21°C. Frozen and thawed bolete inoculated with strains 530 and 757 (isolated from mushroom soup, nonenterotoxigenic) supported staphylococcal growth in 2 days at 21°C from a level of 4.8±10⁴ and 5.4±10³ to 2.0±10⁶ and 7.0±10₇ cfu/g, respectively. Enterotoxin was not detected in these samples.     

Fate of Listeria on various food contact and noncontact surfaces when treated with bacteriophage

Study objective was to determine efficacy of a bacteriophage suspension against Listeria spp. when applied to three common types of materials used in food manufacturing facilities. Materials included two food contact materials (stainless steel and polyurethane thermoplastic belting) and one noncontact material (epoxy flooring). Coupons of each material were inoculated with a cocktail containing L. monocytogenes and L. innocua (4 to 5-log₁₀ CFU/cm²). Two phage concentrations and a control, 0, 2 × 10⁷ and 1 × 10⁸ PFU/cm² were evaluated. Treated samples were held at 4 or 20°C for 1 and 3 hr to determine the effect of temperature and treatment time. Reductions in Listeria populations ranged from 1.27 to 3.33 log₁₀ CFU/cm² on stainless steel, from 1.17 to 2.76 log₁₀ CFU/cm² on polyurethane thermoplastic belting, and from 1.19 to 1.76 log₁₀ CFU/cm² on epoxy resin flooring. Higher phage concentration (1 × 10⁸ PFU/cm²), longer treatment time (3 hr), and processing area temperature of 20°C showed a greater (p ≤ .05) reduction of Listeria on the stainless-steel and polyurethane thermoplastic belting coupons. Overall, Listeria reduction by phage treatment occurred on all three materials tested, under all conditions.     

Efficiency of modified skimmed milk base media to achieve high exopolysaccharide/cell ratios by Lactobacillus delbrueckii subsp. bulgaricus SZ2 in optimised conditions defined by the response surface methodology

Exopolysaccharide (EPS) production by Lactobacillus delbrueckii subsp. bulgaricus SZ2 was optimised in modified MRS (M‐MRS) using the response surface methodology (RSM). Maximum EPS production was 74.3 ± 2 mg/L, and the optimised values of the three variables predicted for maximum EPS production included a temperature of 38.7 °C, Bacto‐casitone and glucose concentrations of 24.5 and 29.6 g/L, respectively. To compare EPS production in MRS and skimmed milk (SM), the kinetics of EPS formation and growth were monitored in M‐MRS, SM, skimmed milk plus 2% additional sucrose (Suc‐SM) and skimmed milk containing Bacto‐casitone (20 g/L) and yeast nitrogen base (5 g/L) (BY‐SM). EPS production in all the media tested seemed to be growth‐related. The EPS/cell ratios were determined to be 3.12 × 10^?10, 1.43 × 10^?10, 4.42 × 10^?11 and 3.16 × 10^?11 mg/cell, in Suc‐SM, SM, M‐MRS and BY‐SM, respectively, clearly indicating the greater effect of C/N ratio when cell behaviour in EPS production is considered.     

Heat resistance of Byssochlamys nivea, Byssochlamys fulva and Neosartorya fischeri isolated from canned tomato paste

Cultures of heat resistant molds (20) were isolated from spoiled canned tomato paste in order to estimate the pasteurization efficiency applied to commercially canned products. Ascospores of nine strains grown on malt extract agar for 30 days at 30°C, survived heating at 85°C for 20 min when initial numbers were near 10⁵/mL. Of these heat resistant strains were identified: two Byssochlamys nivea, three Byssochlamys fulva and four Neosartorya fischeri strains. Ascospores of all cultures were more heat resistant in tomato juice than in phosphate buffer. Thermal death rate curves were nonlogarithmic but approached logarithmic death rates at higher temperatures. The thermal destruction time for 1 log₁₀ at 90°C was 1.5 min for a Byssochlamys nivea strain, 8.1 min for a Byssochlamys fulva strain and 4.4 to 6.6 min for Neosartorya fischeri strains.     

Comparative physiology and fermentation performance of Saaz and Frohberg lager yeast strains and the parental species Saccharomyces eubayanus

Two distinct genetic groups (Saaz and Frohberg) exist within the hybrid Saccharomyces pastorianus (S. cerevisiae × S. eubayanus) taxon. However, physiological/technological differences that exist between the two groups are not known. Fermentative capability of the parental S. eubayanus has likewise never been studied. Here, 58 lager strains were screened to determine which hybrid group they belonged to, and selected strains were characterized to determine salient characteristics. In 15 °P all‐malt wort fermentations at 22 °C, Frohberg strains showed greater growth and superior fermentation (80% apparent attenuation, 6.5% alcohol by volume in 3–4 days) compared to all other strains and maintained highest viability values (>93%). Fermentation with S. eubayanus was poor at the same temperature (33% apparent attenuation, 2.7% alcohol by volume at 6 days and viability reduced to 75%). Saaz strains and S. eubayanus were the least sensitive to cold (10 °C), though this did not translate to greater fermentation performance. Fermentation with S. eubayanus was poor at 10 °C but equal to or greater than that of the Saaz strains. Performance of Saaz yeast/S. eubayanus was limited by an inability to use wort maltotriose. [¹⁴C]‐Maltotriose transport assays also showed negligible activity in these strains (≤0.5 µmol min^?1 g^?1 dry yeast). Beers from Saaz fermentations were characterized by two‐ to sixfold lower production of the flavour compounds methyl butanol, ethyl acetate and 3‐methylbutyl acetate compared to Frohberg strains. Higher alcohol and ester production by S. eubayanus was similar to that of Frohberg strains. Copyright © 2013 John Wiley & Sons, Ltd.     

Prevention of Clostridium botulinum Type A, Proteolytic B and E Toxin Formation in Refrigerated Pea Soup by Lactobacillus plantarum ATCC 8014

The ability of Lactobacillus plantarum ATCC 8014 to inhibit Clostridium botulinum toxin production in pea soup was investigated. Soup containing C. botulinum spores (10³/g) with and without L. plantarum (10⁶/g) were evaluated. Soup containing only type A spores was toxic on days 1 and 2 when incubated at 35°C and 25°C, respectively. Soup containing only proteolytic type B spores was toxic on days 2 and 5 at 35°C and 25°C, respectively. Soup containing only type E spores was toxic at 25°C, 15°C, and 5°C in 7, 7, and 63 days respectively. No toxin was found in soup containing C. botulinum spores plus L. plantarum at any temperature studied.     

Gamma irradiation as a means to eliminate Listeria monocytogenes from frozen chicken meat

Cells of Listeria monocytogenes ATCC 35152 were sensitive to gamma irradiation in phosphate buffer, pH 7.00 (D₁₀, dose required for 10% survival—0.15 kGy) at 0–5°C. The cells showed higher radiation survival when irradiated under frozen condition, with a D₁₀ of 0.3 kGy. The protection offered by shrimp/chicken/kheema homogenates (100 g litre^?1) was evidenced by even higher D₁₀ values (0.5 kGy) at both 0–5°C and cryogenic temperature. Boneless chicken meat samples were artificially inoculated with L monocytogenes ATCC 35152 cells at low (5 × 10³) colony-forming unit (cfu) g^?1 and high (5 × 10⁶ cfu g^?1) concentrations and irradiated at 1, 3, 4, 6 kGy doses under cryogenic conditions. The efficacy of the radiation process was evaluated by detecting L monocytogenes during storage at 2–4°C in the irradiated samples. These studies, when repeated with three other serotypes of L monocytogenes, clearly suggested the need for a dose of 3 kGy for elimination of 10³ cfu cells of L monocytogenes g^?1 from air-packed frozen chicken meat.     

Bacteria and food poisoning organisms in milk

Individual milk samples of 50 goats, 50 ewes and 50 cows were examined for the total viable count, coliform bacteria, staphylococci and salmonellae. Growth of enterotoxin A producing Staphylococcus aureus NCTC 10 652 in the milk of the three animal species was also studied. The average total count was 1.9 × 10⁷ cells/ml for cow's, 7.7 × 10⁶ for goat's and 2.7 × 10⁶ for ewe's milk with micrococci staphylococci, rods and streptococci being the predominant organisms in the three milks, respectively. Goat's milk contained the lowest numbers of coliforms and ewe's milk the highest numbers. Staphylococcus aureus could not be detected in goat's milk, whilst 16 and 26% of the ewe's and cow's milk samples contained 100 and 80 cells/ml, respectively. Out of 39 coagulase positive staphylococci, 27 were thermonuclease positive, 18 produced lecithinase and 15 fermented mannitol. Red blood cells of sheep origin were much more resistant to lysis by ewe's strains compared to bovine strains. Growth curves of Staphylococcus aureus were nearly linear at 17°C but exponential at 31°C without lag phase. Hazardous numbers of about 10⁶ cells/ml were readily reached at 31°C after 6 h and at 17°C after 18 h. Salmonellae could not be detected in any of the samples examined. Out of 19 enterobacteria suspected to be salmonellae 11 proved to be Proteus and 8 Citrobacter.     

Neutral and acidic ascorbate oxidases from satsuma mandarin (Citrus unshiu Marc): Isolation and properties

The neutral and acidic forms of ascorbate oxidase (AAO) were isolated and purified from young fruits of satsuma mandarin by a combination of (NH₄)₂SO₄ fractionation, ion-exchange and hydrophobic chromatography, and gel filtration. The neutral and acidic AAO were both diamers of two subunits with native molecular weights of 133 and 136 kDa, respectively. Neutral AAO was stable in the range of pH 5 and 8 and exhibited optimal activity at pH 5.5 and 45°C. In contrast, acidic AAO was stable between pH 5 and 10 and exhibited optimal activity at pH 5.5 and 50°C. When L-ascorbate and D-isoascorbate were used as substrates, K_m values of neutral AAO were 2.98 × 10^?4 M and 3.36 × 10^?4 M and those of acidic AAO were 1.99 × 10^?4 M and 1.86 × 10^?3 M, respectively. The activities of the two forms of AAO were totally inhibited by metabisulphite and cyanide, substantially suppressed by higher concentrations of diethyldithiocarbamate and fluoride, and not inhibited at all by monovalent and divalent metal ions tested.     

Survival of Lactobacillus bulgaricus During Freezing and Freeze-Drying After Growth in the Presence of Calcium

Two morphologically distinct isolates of L. bulgaricus 1243 (designated F and O), were examined for stability during freezing and freeze-diying following growth in a nutrient broth medium supplemented with varying concentrations of calcium. While cellular morphology did not influence survival during ?20°C frozen storage, calcium prevented freezing death in both isolates. Maximum survival occurred at calcium concentrations ranging between 5.4 × 10^?3 and 6.7 × 10^?3M. Calcium supplemented L. bulgaricus 1243-F displayed a five-fold decline in viable count (3.2 × 10⁸ to 6.1 × 10⁷) due to ?20°C freezing, compared with cells propagated in broth without calcium, which declined 200-fold (1.7 × 10⁸ to 6.3 × 10⁵). Mg⁺⁺ and Mn⁺⁺ salts failed to exert protective effects. Calcium was ineffective in preventing freeze-injury, since cells displayed decreased acid production in milk following ?20°C storage.     

ELASTIC MODULUS of ORANGE JUICE ICE

The elastic modulus of orange juice ice as well as pure water ice was measured at temperatures between ? 10°C and ? 196°C by compression tests. the elastic modulus of pure water ice was almost constant in the whole range of the tested temperatures: 5.6 × 10⁹ N/m² at ? 196°C and 4.0 × 10⁹ N/m² at ? 10°C. In contrast, the elastic modulus of orange juice ice changed greatly with temperature: 11 × 10⁹ N/m² at ? 196°C and 0.2 × 10⁹ N/m² at ? 10°C. Orange juice ice was considered to consist of pure water ice particles which are dispersed in a matrix of concentrated amorphous solution (CAS). the elastic modulus of CAS was calculated using an equation for a disperse system.     

Lemon (Citrus limon, Burm.f.) essential oil enhances the trans-epidermal release of lipid-(A, E) and water-(B6, C) soluble vitamins from topical emulsions in reconstructed human epidermis

Topical bioavailability of lipid‐ and water‐soluble vitamins is a critical issue for protecting or anti‐ageing formulations. Using 17‐day‐old SkinEthic^® reconstructed human epidermis, we investigated (at 34°C) the role of lemon EO in enhancing the penetration of α‐tocopherol (E) and retinyl acetate (A), pyridoxine (B₆) and ascorbic acid (C), released from O/W or W/O emulsions. D‐limonene, α‐pinene and p‐cymene (65.9, 2.2 and 0.5%w/w of the oil) had skin permeability coefficients Ps (10^?3 cm h^?1) of 0.56 ± 0.03 (or 0.73 ± 0.02), 0.72 ± 0.05 (or 0.98 ± 0.05) and 0.84 ± 0.04 (or 1.14 ± 0.04), respectively, when incorporated in a W/O (or O/W) emulsion. Vitamins B6, C and A had Ps values of (3.0 ± 0.4) × 10^?3, (7.9 ± 0.6) × 10^?3 and (0.37 ± 0.02) × 10^?5 cm h^?1, respectively, and their flux through the skin was enhanced by a factor of 4.1, 3.4 and 5.8, respectively, in the presence of lemon EO. The penetration of vitamin E was nine‐fold enhanced. Lemon EO produced only reversible modification of TEWL, and it is a safe and effective penetration enhancer for topical administration of lipid‐ and water‐soluble vitamins.     

Microwave Processing to Destroy Salmonellae in Corn-Soy-Milk Blends and Effect on Product Quality

Enriched corn-soy-milk (CSM) blends were inoculated with Salmonella senftenberg and packed in 50-lb (22.7 kg) bags. The inoculated product and CSM bags containing natural salmonellae contamination were heated from 21° to 56.7°C through 82.2°C in 3.9 – 10 min, respectively, using a 60 Kw (2450 MHz) continuous microwave tunnel, and then palletized with and without 4-cm spacers between rows. Spacer palletized bags cooled to 43°C after 9.7 hr with moving air and after 23.3 hr without moving air, but 60 hr was required with no spacers. Initial most probable numbers (MPN) of 4 × 10² cells/g for S. senftenberg were reduced 10²-fold to 10⁵-fold after processing at 56.7° through 82.2°C, respectively. Nutritional damage occurred at process temperatures of 77.8° and 82.2°C. At 61.1° and 67.2°C, MPN were reduced 2 × 10³-fold with no significant change in rheology, moisture, color, vitamins A and B₁, available lysine, and protein efficiency ratios.     

Influence of ultrasound on mass transport during cheese brining

 The effect of ultrasound on mass transfer during cheese brining has been investigated. The rate of water removal and NaCl gain increased when ultrasound was applied in comparison with brining performed under static or dynamic conditions, suggesting that ultrasound improves both external and internal mass transfer. A simple diffusional model was developed to simulate mass transport during acoustic brining. Model parameters were estimated using experimental data from acoustic brining experiments carried out on cheese cylinders of 1.7×10^–2m radius and 3×10^–2 m height at different temperatures (5, 15 and 20  °C). Effective water (D _W) and NaCl (D _S) diffusivities estimated using the proposed model ranged from 5.0×10^–10m²/s and 8.0×10^–10 m²/s at 5  °C to 1.3×10^–9m²/s and 1.2×10^–9 m²/s at 20  °C. Both D _W and D _S varied with temperature according to the Arrhenius equation. Through the proposed model, water losses and NaCl gains of the experiments used in the parameter identification were accurately simulated (average %var=98.2%) and also of two additional acoustic experiments carried out under different conditions of temperature (10  °C) and sample size and geometry [parallelepiped of (6×2.5×1.25)×10^–2 m] to those used in the parameter identification (average %var=98.4%). Received: 22 September 1998 / Revised version: 20 November 1998     

Ultraviolet Treatment of Orange Juice to Inactivate <Emphasis Type="Italic">E. coli</Emphasis> O157:H7 as Affected by Native Microflora

The effect of yeast concentration on ultraviolet (UV) inactivation of five strains of Escherichia coli O157:H7 from different sources, inoculated both individually and simultaneously in orange juice, was analyzed and mathematically modeled. The presence of yeast cells in orange juice decreases the performance of UV radiation on E. coli inactivation. UV absorption coefficients in the juice increased with increasing yeast concentration, and higher UV doses were necessary to inactivate bacterial strains. UV intensities of I = 3.00 ± 0.3 mW/cm² and exposure times (t) between 0 and 10 min were applied; radiation doses (energy, E = I × t) ranging between 0 and 2 J/cm² were measured using a UV digital radiometer. All the tested individual strains showed higher resistance to the treatment when UV radiation was applied at 4 °C in comparison to 20 °C. UV inactivation of E. coli O157:H7 individual strain was satisfactory fitted with a first order kinetic model. A linear relationship was found between UV absorptivities and D values (radiation doses required to decrease microbial population by 90%) for each strain. The dose required to reach 5-log reduction for the most unfavorable conditions that is the most UV resistant strain, and maximum background yeast concentration was 2.19 J/cm² at 4 °C (corresponding to 11 min of UV treatment) and 2.09 J/cm² at 20 °C (corresponding to 10.55 min of UV treatment). When a cocktail of strains was inoculated in orange juice, the logistic equation was the best model that fits the experimental results due to the deviation from the log-linear kinetics. The UV resistance between strain cocktail and single strain were mathematically compared. Slopes of the decline curves for strain cocktail at high UV doses were lower than the slopes of the log-linear equation calculated for the individual strains, even for the most resistant one. Therefore, microbial inactivation tests using a cocktail of strains are particularly important to determine the performance of the UV inactivation treatment.