Antilisterial activity of lactic acid bacteria inoculated on cooked ham

This study was conducted to evaluate the ability of Lactobacillus sakei 1, a bacteriocin-producing (bac⁺) lactic acid bacterium (LAB), isolated from Brazilian fresh pork sausage to inhibit two Listeria monocytogenes strains (serotypes 4b and 1/2a) on cooked, sliced vacuum-packaged ham. L. sakei ATCC 15521 was used as a non-bacteriocin producer (bac⁻). L. monocytogenes (ca. 2 log CFU/mL) and LAB (ca. 6 log CFU/ml) were inoculated on the sterilized ham, vacuum-sealed and incubated at 8 °C for 10 days. A treatment with the bacteriocin Chrisin (UI/ml) was included. Both L. monocytogenes strains were significantly inhibited in the presence of either bac⁺ and bac⁻ LAB in comparison to the control (L. monocytogenes alone). Using a bacteriocinogenic strain of LAB did not offer an additional barrier to listerial growth in the studied meat system. The application of Chrisin did not affect at all the growth of L. monocytogenes.     

Production of cured meat color in nitrite-free Harbin red sausage by Lactobacillus fermentum fermentation

Lactobacillus fermentum was substituted for nitrite to produce cured pink color in a Chinese-style sausage. Treatments included inoculations (10⁴, 10⁶, and 10⁸ CFU/g meat) followed by fermentation at 30 °C for 8 h and then at 4 °C for 16 h. Control sausage (with sodium nitrite, 60 mg/kg meat) was cured at 4 °C for 24 h without L. fermentum. The UV–Vis spectra of pigment extract from L. fermentum-treated sausage were identical to that of nitrosylmyoglobin (NO-Mb) formed in nitrite-treated control. The NO-Mb concentration and the colorimetric a¹ value of sausage treated with 10⁸ CFU/g meat of L. fermentum essentially replicated those in nitrite-cured meat. Free amino acid content in sausage treated with L. fermentum was greater and the pH slightly lower compared with the nitrite-cured control sample. This study showed that L. fermentum has the potential to substitute for nitrite in the sausage production.     

Listeria innocua and aerobic mesophiles during chill storage of inoculated mechanically recovered poultry meat treated with high hydrostatic pressure

Mechanically recovered poultry meat (MRPM) was inoculated with Listeria innocua 910 CECT at a level of approximately 10⁸ CFU g⁻¹. Vacuum-packaged samples were treated by combinations of pressure (350, 400, 450 and 500 MPa), time (5, 10, 15 and 30 min) and temperature (2, 10 and 20°C) and later stored at 2°C for 2 months. Counts of L. innocua and aerobic mesophilic bacteria were determined 1, 4, 7, 15, 30 and 60 days after pressurisation. For mesophiles, in most treatments, pressurization at 2°C gave the significantly best results. High pressure caused a marked bactericidal effect on L. innocua: reductions higher than 7.5 log units were achieved in several cases. Some cells were just sublethally injured by pressure. Samples treated at 500 MPa for 30 min at 2°C had counts of only 2.3 log units after 60 days of chill storage. Noninoculated pressurised MRPM did not show Listeria growth throughout storage. These results suggest that high pressure processing can enhance the microbiological quality of MRPM.     

Pre-selection of protective colloids for enhanced viability of Bifidobacterium bifidum following spray-drying and storage, and evaluation of aguamiel as thermoprotective prebiotic

The objective of this work was to demonstrate that Bifidobacterium bifidum viability to spray-drying and with storage time could be substantially enhanced by pre-selecting protective colloids offering resistance to oxygen diffusion (high activation energy) and adding aguamiel as a thermoprotector prebiotic. Three different protective colloids blends exhibiting relatively high, medium and low activation energies were mixed with B. bifidum harvested in the late log phase, with or without aguamiel, spray-dried at 130, 140 and 155 °C, and stored at 4 °C at a water activity of 0.32. Viability was determined by cell total count in MRS agar. Best viability was achieved when microencapsulating the microorganism in the protective colloids blend exhibiting highest activation energy (40.7 kJ mol⁻¹), with aguamiel, and dried at 140 °C. Viability ranged from 1.26 × 10⁸ cfu g⁻¹ immediately after drying to 6.0 × 10⁶ cfu g⁻¹ after 5 weeks storage time at 4 °C.     

Effect of temperature and growth media on the attachment of Listeria monocytogenes to stainless steel

Listeria monocytogenes ATCC 19111 cultivated in nutrient-rich medium (brain heart infusion, BHI) or starved in minimal medium (10% filter sterilized pond water and 90% sterilized distilled water) were investigated for their initial attachment to austenitic stainless steel No. 4 with satin finish at 4 °C, 20 °C, 30 °C, 37 °C, or 42 °C. A droplet (10 μl) containing  10⁷ CFU/ml of L. monocytogenes suspended in BHI or minimal medium was placed on the stainless steel surface. After holding in saturated humidity for 3 h at the desired temperature the surface was washed and prepared for scanning electron microscopy (SEM). Using SEM, attachment of L. monocytogenes was determined by counting cells remaining on the surface. When L. monocytogenes cultivated in BHI were used, with the exception of the number of attached cells being lower at 42 °C than at 37 °C and 30 °C, the number of attached cells increased with increasing temperature (P < 0.05). When L. monocytogenes starved in minimal medium were used, the number of attached cells also increased with increasing attachment temperature (P < 0.05), but the number of attached cells at 42 °C was lower than that at the other temperatures. The attachment of L. monocytogenes to stainless steel surface was greater when cultivated in rich medium of BHI vs starved in the minimal medium.     

Antilisterial activity of lactic acid bacteria isolated from "Alheiras" (traditional Portuguese fermented sausages): In situ assays

A total of 226 lactic acid bacteria (LAB) isolated from “Alheira”, a traditional Portuguese fermented sausage, were screened for antagonistic activity against some pathogenic microorganisms, including Listeria monocytogenes. The objective was to isolate LAB with antibacterial activity from “Alheiras” and to select strains that could be used in “Alheira” production. Isolates displaying antibacterial activity against Listeria innocua and L. monocytogenes were investigated for the nature of the antibacterial compounds active against these microorganisms. Results showed that two LAB cultures retained activity in the supernatants after neutralization and catalase treatment. These two strains were both identified as Pediococcus pentosaceus. The final aim of this work was to test the antilisterial activity of these two strains during storage of “Alheira mass” (sterilized), at 4 °C. The growth of L. innocua population was significantly suppressed in the paste of “Alheira” when the samples were co-inoculated with the LAB strains, in comparison with the paste only inoculated with L. innocua or co-inoculated with a bacteriocin negative strain of Ped. pentosaceus (ca. 1 × 10⁷ CFU/g after 28 days of incubation).     

Thermal inactivation of the frozen thawed traditional meat starter culture, Pediococcus pentosaceus, in a meat model system

The equation, y_(t) = y₍₀₎e^kt, was fitted (R = 0.9281, 0.9220 and 0.9117, respectively) to thermal inactivation data (55, 60 and 65 °C) of the traditional meat starter culture Pediococcus pentosaceus (10⁷ cfu/ml) in a meat model system. The population reduction constant (‘k’) increased (about 2.5- and 3-fold) with an increase in the treatment temperature (from 55 to 60 °C and from 60 to 65 °C, respectively). The Q₁₀ (55–65 °C) for ‘k’ was 7.63. Thermal treatments of 19.1, 9.0 and 3.1 min (55, 60 and 65 °C, respectively) reduced the population of P. pentosaceus by 2.0 logs. The value of ‘k’ and the duration of the thermal treatment played an important role in the extent of the inactivation of the culture. The “zero inactivation” temperature (T₀) for P. pentosaceus was 49.9 °C. About 5 logs of the culture would be destroyed at 63 and 68 °C within about 15.5 and 6.5 min, respectively.     

Growth of Listeria monocytogenes on vacuum-packaged horsemeat for human consumption

In order to investigate the likelihood of Listeria monocytogenes (serotype 4b, ATCC 19115) growth on vacuum-packaged horsemeat at refrigeration temperature, fourteen horsemeat surface/volume homogeneous 150 g weight pieces were superficially inoculated with serotype 4b L. monocytogenes and vacuum packaged. The samples were stored at 4 ± 1 °C. Two pieces (one for pH determination and one for L. monocytogenes counts) were examined at days 0, 7, 14, 21, 28, 35 and 42. Surface pH did not show significant variations during the experiment. The average L. monocytogenes initial contamination level was 1.77log₁₀ CFU/g. A lag phase of 7 days was recorded. The exponential growth rate between day 7 to day 35 was 0.125log₁₀ CFU/day, corresponding to 3.51log₁₀ CFU/g in 28 days. At the end of the experiment the mean L. monocytogenes log₁₀ CFU/g was 5.78.     

Effect of modified atmospheres on microbiological, color and sensory properties of refrigerated pork

Pork loin samples were stored (4 °C) in nylon polyethylene plastic bags using different modified atmospheres packaging (MAP): vacuum, 100% CO₂ 99% CO₂ + 1% CO, 100% O₂ or 100% CO followed by vacuum. Throughout the storage period Pseudomonas growth was limited in loins packaged in all MAPs evaluated, except for 100% O₂. Psychrotrophs reached 10⁷ CFU g⁻¹ after 20 days of storage except for the loin samples in 100% O₂ MAP that present count above 10⁸ CFU g⁻¹. The 1% CO/99% CO₂ atmosphere was best for preserving the desirable pork loin color and the L* and a* values remained similar to the fresh meat values using this MAP. Pork loins in 99% CO₂/1% CO MAP obtained the highest consumer acceptance scores after 24 h of storage. These samples and those treated with CO and then vacuum packaged received the greatest acceptance scores even after 20 days of storage.     

Prediction of Listeria spp. growth as affected by various levels of chemicals, pH, temperature and storage time in a model broth

The effects of concentration of NaCl (0.5 to 12.5%), methyl paraben (0.0 to 0.2%), sodium propionate (0.3%), sodium benzoate (0.1%), potassium sorbate (0.3%), pH (>5.9) temperature (4 to 30°C), storage time (up to 58 d) and inoculum (>10⁵ to >10⁻² per ml) on the log₁₀ probability percentage of one cell of Listeria spp. to initiate growth in a broth system were evaluated in a factorial design study. At pH 5.96 and temperature ranging from 4 to 30°C the concentrations of sodium propionate, potassium sorbate, and sodium benzoate examined allowed growth of L. monocytogenes with lag phases at 4°C of 18, 27 and 21 days, respectively. For 0.1 and 0.2% methyl paraben growth of all Listeria spp. was initiated at 8°C and 30°C, respectively. At pH 6, concentration of 12% NaCl supported the growth of L. monocytogenes at 8 to 30°C, whereas 12.5% inhibited all Listeria species. Four regression equations were derived relating probability of growth initiation to temperature, concentrations of NaCl and preservatives storage time, and Listeria species specific effects. From these equations, the number of cells needed for growth initiation can be calculated. The impact of this type of quantitative study and its possible application on the development of microbial standards for foods is discussed.     

Influence of various preservatives on the quality of minced beef under modified atmosphere at chilled storage

The effect of preservatives on microbial quality, pH, drip-loss, roasting-loss, colour, and sensorial properties of modified atmosphere packaged (70% O₂ and 30% CO₂) minced beef (M. semimembranosus) stored at (2 ± 0.5 °C) for 12 days was investigated. Beef cubes (approx. 20 × 20 × 20 mm size) were immersed in solutions of 2% and 5% lactic acid, 2% lactic acid combined with 0.5% sodium ascorbate, 20% potassium lactate and 20% potassium sorbate before mincing. Addition of lactic acid was associated with pH drop, which increased drip-loss and roasting-loss. Application of all additives inhibited aerobic micro-organisms (10³–10⁴ CFU g⁻¹ on day 12) compared to reference sample (9 × 10⁵ CFU g⁻¹ on day 12). Lactic acid discoloured samples, while sodium ascorbate seemed to improve colour stability. Despite good visual colour characteristics, potassium sorbate treated samples were organoleptically unacceptable with massive off-flavour.     

Thermophysical properties of processed meat and poultry products

Thermophysical properties of various meat and poultry emulsions were evaluated at four temperatures (20, 40, 60 and 80 °C). Thermal conductivities (0.26–0.48 W m⁻¹ K⁻¹) increased linearly with temperature between 20 and 60 °C. Between 60 and 80 °C, it remained constant for most products except bologna. Curves for thermal conductivity as a function of temperature could be roughly grouped into two different categories: products containing meat particles and those containing meat emulsions. The application of various models was investigated for thermal conductivity prediction. It was found that a three phase structural based Kirscher model had the potential for predicting thermal conductivities with acceptable accuracy. Densities decreased slightly as a function of temperature from 20 to 40 °C. A transition phase was observed from 40 to 60 °C, which was followed by a decrease from 60 to 80 °C. There was a decrease of about 50 kg m⁻³ between the density of a raw product at room temperature (at maximum 1070 kg m⁻³) and the product heated to 80 °C (at minimum 970 kg m⁻³), due to the gelation or setting of the structure. After a transition period from 10 to 30 °C, the heat capacity increased linearly from 30 to 80 °C, and ranged from 2850 to 3380 J kg⁻¹ °C⁻¹, respectively. Densities and heat capacities were strongly influenced by the carbohydrate content (i.e. as the carbohydrate content increased the density decreased). The salt content adversely affected thermal conductivity and thermal diffusivity values. However, these parameters increased with moisture content.     

Selective enumeration and survival of bifidobacteria in fresh cheese

Five species of bifidobacteria (15 strains), two strains of Lactococcus lactis ssp. lactis, two strains of L. lactis ssp. cremoris, and one strain of L. lactis ssp. lactis var diacetylactis were included in a study to develop a selective medium for enumeration of bifidobacteria from fresh cheese. Viable counts of bifidobacteria or lactococci on modified Columbia agar base (CAB with 0.05% cysteine-HCl) plus raffinose (0.5%) containing various selective agents were compared with non-selective media. The mCAB plus raffinose with lithium chloride (2 g L⁻¹) and sodium propionate (3 g L⁻¹) with pH adjusted to 5.1 was used successfully as a selective medium for the enumeration of bifidobacteria from fresh cheese. Using this medium, it was determined that bifidobacteria could survive up to 15 days at a level higher than 10⁶ cfu g⁻¹ in a fresh cheese stored at 4 or 12°C. The decrease in the viable counts of bifidobacteria was faster during storage at 4°C than at 12°C.     

Modeling transfer of Escherichia coli O157:H7 and Staphylococcus aureus during slicing of a cooked meat product

Cross contamination is one of the most important contributing factors in foodborne illnesses originating in household environments. The objective of this research was to determine the transfer coefficients between a contaminated domestic slicing machine and a cooked meat product, during slicing. The microorganisms tested were Staphylococcus aureus (Gram positive) and Escherichia coli O157:H7 (Gram negative). The results showed that both microorganisms were able to transfer to all slices examined (20 successively sliced) and at different inoculum levels on the blade (10⁸, 10⁶ and 10⁴ cfu/blade). The results also showed that the number of log cfu transferred per slice, during slicing, decreased logarithmically for both microorganisms at inoculum levels of 8 and 6 log cfu/blade. The type of microorganism significantly influenced transfer coefficients (p < 0.05) and there was an interaction between inoculum level and transfer coefficient for S. aureus (p < 0.05), but not E. coli O157:H7. Finally, to describe bacterial transfer during slicing, two models (log-linear and Weibull) were fitted to concentration on slice data for both microorganisms (at 6 and 8 log cfu/blade), obtaining a good fit to data (R²  0.73).     

Quantification of Plesiomonas shigelloides in Pure Culture and Clams by Competitive PCR

A quantitative assay for Plesiomonas shigelloides in pure culture and clams based on the competitive polymerase chain reaction was developed. This is the first report for quantitative detection of P. shigelloides by competitive PCR. Forward (PS-F), reverse (PS23RV3), and hybrid primers were designed, and the specificity of the forward and reverse primer for P. shigelloides was proven. An internal standard DNA sequence was synthesized by PCR, with the hybrid primer as the forward primers and PS23RV3 as the reverse primer. A single concentration (0.588 pg/PCR) of internal standard (IS) was used for competitive PCR. The lowest level of detection of P. shigelloides was 80 CFU per PCR in pure culture, 240 CFU/g of clam tissue without enrichment, and 40 CFU/g of clam tissue after 7 hrs. nonselective enrichment at 37°C. There was a linear relationship between the log of the ratio of the relative fluorescent intensities of amplified target DNA bands to the internal standard DNA bands (IS) and the log of the CFU within a certain range in pure cultures and in clam tissue either with or without enrichment. The linear range with cells from a pure culture was the DNA derived from 8.0 × 10¹ to 8.0 × 10⁴ CFU per PCR while with clam tissue, the linear range was the DNA derived from 2.4 × 10² to 2.4 × 10⁵ CFU/g of tissue (1.2 × 10¹ to 1.2 × 10⁴ CFU per PCR) without enrichment, and 4.0 × 10¹ to 1.2 × 10⁴ CFU/g of clam tissue with enrichment, respectively.     

Clostridium perfringens and its toxins in minced meat from Kars, Turkey

The aim of this study was to determine the prevalence of Clostridium perfringens and its toxins in minced meat. A total of 96 minced meat samples were collected from local markets (16) and small butcher's shops (80) in Kars (Turkey). Samples were analysed for the presence of C. perfringens and its toxins using a commercially available ELISA kit. A total of 31 (32%) Clostridium spp. strains were isolated and 17 (55%) of these isolates were identified as C. perfringens. Four (25%) of the samples from local markets and 27 (34%) from small butcher's shops were contaminated with Clostridium spp. Furthermore, C. perfringens was isolated from two (12%) and 15 (19%) samples from local markets and small butcher's shops, respectively. Mean counts of Clostridium spp. were 2.2 ± 0.83 × 10² CFU g^-1 for local markets and 4.35 ± 8.53 × 10² CFU g^-1 for small butcher's shops; mean counts for C. porringers were 2.75 ± 0.21 × 10² and 6.82 ± 10.96 × 10² CFU g^-1 from markets and butcher's shops, respectively. The number of samples contaminated with both Clostridium spp. and C. perfringens was higher in small butcher's shops than in local markets. Moreover, higher numbers of Clostridium spp. and C. perfringens were isolated in samples from small butcher's shops than from local markets. A total of 13 (13%) samples were also positive for toxins produced by the organism, as detected by ELISA. Eleven samples from small butcher's shops and two samples from local markets were positive for the C. perfringens toxins tested. Moreover, two (12%), one (1%), four (4%) and two (2%) samples were contaminated with C. perfringens types A, B, C and D, respectively. In conclusion, some meat samples collected from local markets and small butcher's shops contained C. perfringens and its toxins and, therefore, present a potential risk of food poisoning.     

Modeling the effect of inoculum size and acid adaptation on growth/no growth interface of Escherichia coli O157:H7

The objective of this study was to model with logistic regression the growth/no growth interface of different initial inoculation levels (10¹, 10³ and 10⁵ CFU/ml; study 1), or nonadapted vs acid-adapted (study 2) Escherichia coli O157:H7 as influenced by pH, NaCl concentration and incubation temperature. Study 1 was conducted with a mixture of four E. coli O157:H7 strains grown (35 °C, 24 h) in tryptic soy broth (TSB). Study 2 was conducted with the same mixture of four E. coli O157:H7 strains grown (35 °C, 24 h) in glucose-free TSB with 1% added glucose (final pH 4.83), or in diluted lactic acid meat decontamination runoff fluids (washings; final pH 4.92), or nonadapted cultures prepared in glucose-free TSB (final pH 6.45), or in water washings (final pH 6.87). Parameters included incubation temperature (10–35 °C), pH (3.52–7.32), and NaCl concentration (0–10% w/v). Growth responses were evaluated for 60 days turbidimetrically (610 nm) every 5 days in 160 (study 1) and 360 (study 2) combinations in quadruplicate samples, with a microplate reader. The lower the initial inoculum the higher were the minimum pH and a_w values permitting growth. Differences in the pH and a_w growth limits among inoculum concentrations increased at 15 and 10 °C. Acid-adapted cultures were able to grow at lower pH than nonadapted cultures, while at temperatures below 25 °C, growth initiation of nonadapted cultures stopped at higher a_w compared to acid-adapted cultures for the whole pH range of 3.52 to 7.32. A comparison with available data indicated that our model for acid-adapted E. coli O157:H7 in different environments may provide representative growth probabilities covering both nonadapted and stress-adapted contaminants.     

Behaviour of Listeria monocytogenes in raw sausages (merguez) in presence of a bacteriocin-producing lactococcal strain as a protective culture

The effectiveness of a bacteriocin produced by Lactococcus lactis subsp. lactis M in reducing population level and growth of Listeria monocytogenes ATCC 7644 in fermented merguez sausage was examined. Two different formulas (with or without added nitrites) were assayed and predetermined numbers of Listeria (ca 10⁶ cfu g⁻¹) were added to sausage mixture. The effect of in situ production of the bacteriocin by Lactococcus lactis M on Listeria monocytogenes ATCC 7644 during fermentation and storage of merguez sausages at room (ca 22 °C) or at refrigeration (ca 7 °C) temperature was tested. Results indicated that counts of Listeria monocytogenes were decreased during fermentation of merguez samples fermented with either the bacteriocin-producing Lactococcus lactis M (Bac⁺) or a nonbacteriocin-producing Lactococcus lactis J (Bac⁻). However, reduction in Listeria cfu's was greater in samples fermented with the Bac⁺ than in those fermented with the Bac⁻ starter. In merguez sausage made without nitrites addition, the Bac⁺ starter induced further decrease in Listeria counts by 1.5 log cycles compared with that induced by the Bac⁻ starter. While in merguez samples with added nitrites (0.4%), the effect of the bacteriocin produced in situ was less important than in those made without nitrites addition.     

Microbiological quality of 18 degrees C ready-to-eat food products sold in Taiwan

A total of 164 samples of 18 °C ready-to-eat (RTE) food products, purchased in 1999–2000 from convenience stores and supermarkets in central Taiwan, were examined to determine the microbiological quality of these products. The 18 °C RTE food products, manufactured by 16 factories, were divided into groups based on the type of food and their major ingredients. Aerobic plate count, coliforms, Escherichia coli, Bacillus cereus, Staphylococcus aureus and psychrotrophic Pseudomonas spp. were evaluated. The incidence of E. coli and coliforms in these 18 °C RTE food products was 7.9% and 75.0%, respectively, while 49.8% and 17.9% of the samples were found to contain B. cereus and S. aureus, respectively. Among the samples tested, 1.3% of the food products contained more than 10⁵ CFU g⁻¹ of B. cereus and 0.7% contained more than 10⁵ CFU g⁻¹ of S. aureus. The pH values of the samples were all below 7.0, except for cold noodles, which had pH values ranging from 5.18 to 8.20. Among the five types of 18 °C food products tested, the highest incidence of E. coli (16%) and Pseudomonas spp. (64.0%) were detected in hand-rolled sushi in a cone shape. On the other hand, the highest incidence rate of coliforms, B. cereus, and S. aureus were found in sandwiches (88%), cold noodles (66.7%) and rice balls rolled in seaweed (25.0%), respectively. Food products made of ham contained the highest incidence of coliforms (88.0%) and E. coli (16.0%), while food products containing meat and ham as the major ingredients had the highest incidence rates of B. cereus (62.5%) and S. aureus (26.1%), respectively. For coliforms, E. coli, B. cereus and S. aureus, the percentage of 18 °C RTE food products exceeding the microbiological standards for RTE food accepted by Republic of China was 75.0%, 7.9%, 49.8% and 17.9%, respectively.     

Effects of supercritical carbon dioxide extraction conditions on yields and antioxidant activity of Chlorella pyrenoidosa extracts

Yields and antioxidant activity of Chlorella pyrenoidosa extracts obtained by supercritical carbon dioxide extraction through an orthogonal experiment (L₁₆(4⁵)) were investigated to get the best extraction conditions. The results showed that extraction pressure, temperature and modifier were the main three variables that influenced the yields of extracts. The highest yield was obtained at 32 °C, 40 MPa, 20 L h⁻¹ with dosage of modifier 1 mL ethanol g⁻¹ sample for 3 h. Moreover, increasing pressure and concentrations of modifier led to the increase of extraction yields and antioxidant activity. DPPH radical scavenging method showed that almost all the extracts had significantly higher antiradical activities varying from 29.67 ± 0.29% to 54.16 ± 4.49% comparing to -tocopherol, Trolox, and BHT as references except extracts at 32 °C, 35 MPa and 15 L h⁻¹ without modifier for 1.5 h. These results indicate that supercritical extraction is a promising alternative process for recovering compounds of high antioxidant activity from C. pyrenoidosa.