Dual-enzymatic synthesis of lactulose in organic-aqueous two-phase media

Lactulose has been successfully synthesized by dual-enzymatic method in organic-aqueous two-phase media using lactose and fructose as the raw materials. Cyclohexane–buffer system C₆H₁₂:buffer = 95:5 (v/v) was employed as the organic-aqueous media for the reaction. The dual-enzymatic system was consisted of immobilized lactase (IL) and immobilized glucose isomerase (IGI). Immobilized lactase was prepared by cross-linking the free lactase into Fe₃O₄-chitosan magnetic microspheres. The main enzymatic reaction parameters were investigated, including reaction temperature (T), pH value and reaction time (t). Under the optimum reaction conditions, i.e., lactose 0.8 g mL^?1, fructose 0.1 g mL^?1, IL 0.1 g mL^?1, IGI 0.05 g mL^?1, T = 30 °C, pH = 8.0 and t = 2 h, the obtained highest lactulose yield was approximately 151 g L^?1 and the corresponding productivity was 75.5 g L^?1 h^?1. Experimental results indicated that the organic-aqueous media can significantly promoted the transglycosidation activity of lactase and therefore improve the lactulose yield. The possible reaction mechanism of the synthesis of lactulose using IL and IGI in two-phase system was also proposed.     

Radio-sensitivity of pathogens in inoculated prepared foods of animal origin

The effectiveness of irradiation for inactivating Staphylococcus aureus, Listeria ivanovii, Salmonella Typhimurium, and Escherichia coli in the prepared foods of animal origin was investigated. Commercially available seasoned and cooked beef, fried egg, and ham were purchased, radiation-sterilized, and inoculated at 10⁶–10⁷ CFU/g with each of the four pathogens and stored at three storage conditions at 10 °C, 20 °C, and 30 °C. D₁₀-values of S. aureus, L. ivanovii, Salmonella Typhimurium, and E. coli were 0.34±0.01, 0.24±0.02, 0.24±0.01, and 0.27±0.01 kGy, respectively. No viable cells were detected at 3 kGy of irradiation. Salmonella mutagenicity assay (Ames test) indicated that the 10 kGy irradiated samples were statistically similar to non-irradiated control samples in the Salmonella mutagenicity assay (Ames test). These studies demonstrate that irradiation can be used as an additional safety tool to produce microbiologically safe and wholesome prepared foods of animal origin.     

Comparison of palladium–magnesium nitrate and ammonium dihydrogenphosphate modifiers for lead determination in honey by electrothermal atomic absorption spectrometry

The aim of the present work was to develop and optimize two procedures for the determination of Pb content in honeys by electrothermal atomic absorption spectrometry without any sample pretreatment. Palladium–magnesium nitrate and ammonium dihydrogenphosphate were used as chemical modifiers. Honey was diluted in water, and hydrogen peroxide, nitric acid, and Triton X-100 were added to minimize the matrix effect. RSD (lower than 10%) and the analytical recovery (98–101%) were acceptable for both methods. Pd–Mg(NO₃)₂ was the method selected for further direct Pb determinations in honey samples because it presented the best limit of detection (LOD = 1.6 ng g⁻¹). Moreover, this method allowed Pb determination using a calibration graph, instead of an addition graph, which is an important advantage. The direct proposed methods have been applied to the determination of Pb content in representative honey samples from Galicia (NW Spain). The lead concentrations found in the analyzed samples were in the range 1.71–75.0 ng g⁻¹.     

Synergistic effect of sonication and high osmotic pressure enhances membrane damage and viability loss of Salmonella in orange juice

The efficacy of using sonication (50 ± 0.2 W, 20 kHz), combined with subsequent concentration and storage at high osmotic pressure, has been evaluated to reduce levels of Salmonella bacteria in different solutions (PBS, sucrose and orange juice) at varying concentrations. To visualize the impact on cell membranes, we used a staining protocol (propidium iodide [PI] and 4′,6′-diamidino-2-phenylindole [DAPI]). Sonication alone did not cause significant membrane damage. Storage alone, for 48 h and at high osmotic pressure (10.9 MPa), affected membrane permeability in 20% of cells. However, sonication, combined with storage, considerably increased loss of membrane integrity, resulting in a significant logarithmic reduction of microorganisms. When the combination was applied to contaminated orange juice, a 5 log₁₀ cfu ml^?1 reduction of Salmonella spp. was obtained. “Osmosonication”—the synergistic combination of sonication and subsequent storage at high osmotic pressure—is an innovative alternative for the non-thermal decontamination of liquid foods.     

Biochemical changes throughout the ripening of a traditional Spanish goat cheese variety (Babia-Laciana)

The physico-chemical characteristics, proteolysis (classical nitrogen fractions, caseins and their degradation products and free amino acids), and lipolysis (fat acidity and free fatty acids) were studied throughout the ripening of three batches of Babia-Laciana cheese, a Spanish traditional variety made from raw goats’ milk. The main compositional characteristics of this cheese at the end of the ripening are its high content of total solids (TS) (78.0±2.4 g 100 g⁻¹ of cheese) and fat (61.1±1.2 g 100 g⁻¹ of TS), the presence of residual lactose (1.6±0.8 g 100 g⁻¹ of TS) and its low content of sodium chloride (1.1±0.7 g 100 g⁻¹ of TS) and ash (2.8±0.5 g 100 g⁻¹ of TS). Its pH values (4.44±0.72) are extraordinarily low. The evolution and final values of the different nitrogen fractions show that this cheese undergoes a very slight proteolysis, a fact which was corroborated when the caseins and their degradation products were quantified: β-casein did not undergo any modification throughout ripening, while only 21% of the α_s-caseins were degraded. Free amino acids content increased by a factor of about 7 throughout ripening, resulting in a high content of γ-amino butyric acid and a low content of glutamic acid at the end of the process. Fat acidity increased very slightly, approximately 4.5 times, during ripening, reaching final values of 3.5±2.2 mg KOH g⁻¹ of fat. The total free fatty acids content showed a similar evolution to fat acidity. At the end of the ripening process, the main free fatty acid was C_18:1, followed by C₁₆ and C₁₀.     

Determination of moisture,solids-not-fat and fat-by-difference in butter using routine methods according to ISO 8851/IDF 191—an international collaborative study and a meta-analysis

An international collaborative study of routine methods for the determination of moisture, solids-not-fat (SNF) and fat-by-difference (i.e. fat (g 100 g^–1 product) =100−moisture (g 100 g^–1 product)−SNF (g 100 g^–1 product)) in butter was carried out to obtain estimates of the precision parameters repeatability (2.8s_r) and reproducibility (2.8s_R). Ten laboratories tested blind duplicate samples of eight different butters according to standard operating procedures jointly drafted by the International Standards Organisation (ISO) and the International Dairy Federation (IDF) (draft ISO standard 8851, parts 1–3; draft IDF standard 191, parts 1–3). Estimates of repeatability and reproducibility respectively were: moisture, 0.28 g 100 g^–1 product and 0.44 g 100 g^–1 product; SNF, 0.20 g 100 g^–1 product and 0.34 g 100 g^–1 product; fat-by-difference, 0.30 g 100 g^–1 product and 0.51 g 100 g^–1 product. These estimates are similar to those found previously in a New Zealand study. A meta-analysis, pooling the results of the present study and the New Zealand study, yielded overall estimates of repeatability and reproducibility as follows: moisture, 0.31 g 100 g^–1 product and 0.42 g 100 g^–1 product; SNF, 0.20 g 100 g^–1 product and 0.39 g 100 g^–1 product; fat-by-difference, 0.35 g 100 g^–1 product and 0.54 g 100 g^–1 product, and these are recommended for adoption in the respective methods. This study completes the validation work for the respective methods, which may now be progressed for final adoption as international standard routine methods. A protocol is proposed for dealing with situations where multiple precision estimates are available.     

Optimization of the associative growth of novel yoghurt cultures in the production of biomass, β-galactosidase and lactic acid using response surface methodology

The associative growth of Streptococcus thermophilus 95/2 (St 95/2) and Lactobacillus delbrueckii ssp. bulgaricus 77 (Lb 77) isolated from the Toros mountain region of Turkey was investigated with respect to lactic acid, biomass and β-galactosidase enzyme production using response surface methodology (RSM). The ratio (St 95/2:Lb 77) of the strains and media formulation had significant effect on all responses (p < 0.001). The predicted enzyme activity (2.14 U mL^?1), lactic acid (22.50 g L^?1) and biomass (7.11 g L^?1) production at optimum conditions were very close to the actual experimental values (2.14 U mL^?1, 22.94 g L^?1 and 7.86 g L^?1, respectively). The optimum conditions were to use these cultures in a ratio of 1.66:1.62 (St 95/2:Lb 77) in a medium containing whey (5%), corn steep liquor (4%), potassium phosphate (2%) and peptone (2%) at 43 °C for 8 h. The associative growth provided 6.4% and 39% more β-galactosidase activity and 8.73% and 44% more lactic acid compared with the results obtained using pure St 95/2 and Lb 77 strains, respectively.     

Migration of α-tocopherol from LDPE films to corn oil and its effect on the oxidative stability

The migration of α-tocopherol (α-T) from low density polyethylene (LDPE) films, added with 20 (film A) and 40 mg g^?1 (film B) to corn oil for 12 weeks at 5, 20 and 30 °C was determined. A LDPE film added with no α-T was used as control (film C). Diffusion coefficient (D) values for the film A system were 1.4 × 10^?11, 7.1 × 10^?11 and 30.3 × 10^?11 cm² s^?1 at 5, 20 and 30 °C, respectively. Meanwhile, D values for the film B system were 1.3 × 10^?11, 9.6 × 10^?11 and 51.1 × 10^?11 cm² s^?1 at the same temperatures. The activation energy (E_a) for the diffusion of α-T was 126.5 (film A) and 105.9 kJ mol^?1 (film B). The effect of the migration of α-T on the oxidative stability of corn oil was evaluated by monitoring hexanal content by solid phase micro-extraction (SPME) and gas chromatography. The hexanal content in the oil showed that both films added with α-T resulted suitable to maintain the oxidative stability of the oil for about 16 weeks at 30 °C, compared to 12 weeks for the oil in contact with the film C.     

Optimization of solid–liquid extraction of phytochemicals from Garcinia indica Choisy by response surface methodology

A central composite rotatable design was employed to study the effect of ultrasound assisted extraction conditions namely sonication amplitude (10–90%), sonication cycle (0.1–1.0 s^?1), solid–liquid ratio (2–10) and extraction time (5–35 min) on the total anthocyanin extraction from Garcinia indica Choisy. Overall extractions of total anthocyanin, acidity and antioxidant activity were considered as response variables. The significant (p < 0.05) response surface models with high coefficients of determination values (R²) ranging from 0.91 to 0.93 were fitted for the experimental data, which indicated that the polynomial response models fitted well for describing the extraction efficiencies of anthocyanin, acidity and antioxidant activity. Based on the design, the optimal conditions for obtaining higher extraction were extraction time 35 min, cycle ranging from 0.44 to 0.48 s^?1, percentage amplitude ranging from 10 to 14%, and solid–liquid ratio 10. The graphical optimization of superimposed contour plots fulfilled the conditions to obtain total anthocyanin (Y₁) ≥ 135 mg/100 g DW, total titratable acidity (Y₂) ≤ 25 g/100 g DW and antioxidant activity (Y₃) ≥ 14.5 M Trolox/100 g DW. The study demonstrated that response surface methodology can be utilized for deriving the optimum conditions for extraction of anthocyanin from G. indica Choisy.     

Development and validation of a liquid chromatography/linear ion trap mass spectrometry method for the quantitative determination of deoxynivalenol-3-glucoside in processed cereal-derived products

Cereal-based food can be frequently contaminated by the presence of mycotoxins derived from Fusarium fungus, and, in particular, by deoxynivalenol (DON). Nowadays, analytical strategies for the detection of DON are well developed, but there are gaps for what concerns a correct identification, quantification and toxicological evaluation of the respective metabolites, mainly related to detoxifying actions via plant metabolism or to processing technologies and also referred to as “masked” mycotoxins.Here, we report the development of a liquid chromatography/linear ion trap mass spectrometry method capable of determining deoxynivalenol-3-glucoside (DON-3G), which is the main known DON metabolite, in different processed cereal-derived products. Samples were extracted with a mixture of methanol/water (80:20; v/v) and cleaned up using immunoaffinity columns. Chromatographic separation was performed using a core–shell C₁₈ column with an aqueous acetic acid/methanol mixture as the mobile phase under gradient conditions. The method was in-house validated on a bread matrix as follows: matrix-matched linearity (r² > 0.99) was established in the range of 10–200 μg kg^?1; trueness expressed as recovery was close to 90%; good intermediate precision (overall RSD < 9%) and adequate detection quantitation limits (4 and 11 μg kg^?1, respectively) were achieved. Furthermore, applying a metrology approach based on intralaboratory data, the estimated measurement expanded uncertainty was determined to be equal to 29%. The reliability of the method was finally demonstrated in bread, cracker, biscuit and minicake commodities, resulting in relatively low levels of DON-3G, which were not higher than 30 μg kg^?1.     

Effect of tomato by-products in the diet of Comisana sheep on composition and conjugated linoleic acid content of milk fat

To evaluate the effect of supplementing the diet of Comisana sheep with by-products from industrial tomato manufacture on the fat composition and conjugated linoleic acid (CLA) content of milk fat, two groups of 50 ewes each were fed either total mixed ration standard (TMRS) or total mixed ration with added tomato by-products (TMRA). Milk fat composition was determined by high-resolution gas chromatography (HRGC). The milk fat content for the animals fed the TMRA diet increased by 6.41% (P < 0.01) after six weeks, compared with the animals fed the TMRS diet. The CLA content in the milk fat for the group of animals fed the TMRA diet was 19.8% (P < 0.05) higher than for those fed the TMRS diet, and reached 1.33 g 100 g^?1 fat, while the polyunsaturated fatty acid (PUFA) content increased by a 6.43% (P < 0.05) and reached 7.12 g 100 g^?1 fat. The fatty acid composition showed an increase in the amount of polyunsaturated fatty acids. The n ? 3:n ? 6 ratio increased by 13% in the milk from sheep fed with the TMRA diet. These observations were confirmed by triglyceride analysis, which showed a decrease in the amount of short-chain (C₂₈–C₃₂) and medium-chain (C₃₄–C₄₂) triglycerides after six weeks, while the opposite was observed for the long-chain triglycerides (C₄₄–C₅₄).     

A survey of dry-salted natural casings for the presence of Salmonella spp., Listeria monocytogenes and sulphite-reducing Clostridium spores

A monitoring study was performed for the presence of Salmonella spp., Listeria monocytogenes and sulphite-reducing Clostridium spores in (internationally) traded dry-salted natural hog and sheep casings. Two hundred and fourteen consignments were examined for Salmonella spp. and L. monocytogenes, and 138 for the Clostridium spores.None of the 214 sampled consignments (25 g amounts investigated) yielded Salmonella spp., or L. monocytogenes.Differential reinforced clostridial medium was effective in detecting the presence of sulphite-reducing clostridia. Iron sulphite agar (ISA) overall showed higher clostridia counts as compared to differential reinforced clostridial agar (DRCA). The maximum spore counts obtained on DRCA and ISA were 17.5 and 2500 cfu g⁻¹, respectively. From the casings from China, 3 (of 35 hog consignments) and 7 (of 22 sheep) showed spore counts above 100 cfu g⁻¹. From the remaining 81 samples, originating from Netherlands, New Zealand and UK, none showed a count above 100 cfu g⁻¹.The relevance of the presence of sulphite-reducing Clostridium spores for the manufacture of various meat products is discussed. It is recommended that determination of the Clostridium strains present is carried out and their properties investigated in relation to the manufacture of meat products, since some of the strains may be potentially pathogenic and/or able to spoil products.     

Routine and sensitive SPE-HPLC method for quantitative determination of pheophytin a and pyropheophytin a in olive oils

A sensitive and specific HPLC method with tandem diode array-fluorescence detection (DAD-FL) has been developed and validated for the simultaneous determination of pheophytin a (phy a) and pyropheophytin a (pyrophy a) in olive oils. Pigments were extracted with reverse phase solid phase extraction (RP-SPE) and subsequently analysed by HPLC-DAD-FL. The chromatographic analysis was carried out isocratically on ODS2 RP column using methanol–acetone (1:1 v/v) at flow-rate of 2.0 ml min⁻¹. Specificity of the method was assured by the simultaneous detection by UV–visible (410 nm) and FL (λ_Ex: 410 nm; λ_Em: 672 nm). Both compounds could be baseline separated within 7 min. The method was validated and applied in olive oil samples recently extracted as well as stored during 12 months. The limit of detection (LOD) defined at a signal-to-noise ratio of about 3 was ∼21.6 ng g⁻¹ for pyrophy a and ∼24.6 ng g⁻¹ for phy a under FL detection, and ∼148.0 ng g⁻¹ for both analytes under UV–visible detection. The calibration graphs were linear (r² > 0.9999; p < 0.01) between 0.25–14.00 ng μl⁻¹ for pyrophy a and 0.25–19.00 ng μl⁻¹ for phy a, under both fluorescence and UV–visible detection conditions. Recoveries of phy a and pyrophy a were over 94% as estimated by the standard addition method. Relative standard deviation for the intra-day and inter-day determination of phy a and pyrophy a were lower than 3.7% and 8.0%, respectively.     

Water activity change at elevated temperatures and thermal resistance of Salmonella in all purpose wheat flour and peanut butter

Water activity (a_w) is a major factor affecting pathogen heat resistance in low-moisture foods. However, there is a lack of data for a_w at elevated temperatures that occur during actual thermal processing conditions, and its influence on thermal tolerance of pathogens. The objective of this study was to gain an in-depth understanding of the relationship between temperature-induced changes in a_w and thermal resistance of Salmonella in all purpose flour and peanut butter at elevated temperatures (80 ^oC). Equilibrium water sorption isotherms (water content vs. water activity) for all purpose wheat flour and peanut butter over the range of 20 to 80 °C were generated using a vapor sorption analyzer and a newly developed thermal cell. The thermal resistance (D₈₀-values) of Salmonella in all purpose wheat flour and peanut butter with initial a_w of 0.45 (measured at room temperature, ~ 20 °C) was determined via isothermal treatment of small (< 1 g) samples. When increasing sample temperature from 20 to 80 °C in sealed cells, the a_w of all purpose flour increased from 0.45 to 0.80, but the a_w of peanut butter decreased from 0.45 to 0.04. The corresponding estimated D₈₀-values of Salmonella in all purpose flour and peanut butter with 20 ^oC a_w of 0.45 were 6.9 ± 0.7 min and 17.0 ± 0.9 min, respectively. The significantly (P < 0.05) higher D₈₀-value of Salmonella in peanut butter than in all purpose flour may be partially attributed to the reduced a_w in peanut butter in comparison to the increased a_w in all purpose flour at 80 °C. The improved understanding of temperature-induced changes in a_w of low-moisture products of different composition provides a new insight into seemly unpredictable results, when using heat treatments to control Salmonella in such food systems.     

Total anthocyanin content determination in intact açaí (Euterpe oleracea Mart.) and palmitero-juçara (Euterpe edulis Mart.) fruit using near infrared spectroscopy (NIR) and multivariate calibration

The aim of this study was to evaluate near-infrared reflectance spectroscopy (NIR), and multivariate calibration potential as a rapid method to determinate anthocyanin content in intact fruit (açaí and palmitero-juçara). Several multivariate calibration techniques, including partial least squares (PLS), interval partial least squares, genetic algorithm, successive projections algorithm, and net analyte signal were compared and validated by establishing figures of merit. Suitable results were obtained with the PLS model (four latent variables and 5-point smoothing) with a detection limit of 6.2 g kg^?1, limit of quantification of 20.7 g kg^?1, accuracy estimated as root mean square error of prediction of 4.8 g kg^?1, mean selectivity of 0.79 g kg^?1, sensitivity of 5.04 × 10^?3 g kg^?1, precision of 27.8 g kg^?1, and signal-to-noise ratio of 1.04 × 10^?3 g kg^?1. These results suggest NIR spectroscopy and multivariate calibration can be effectively used to determine anthocyanin content in intact açaí and palmitero-juçara fruit.     

Rapid determination of safranal in the quality control of saffron spice (Crocus sativus L.)

A method to determine safranal content based on non-polar solvent extraction followed by UV–Vis analysis available in the industry for quality control of saffron spice has been studied. Ultrasound-assisted extraction of safranal was carried out and optimised with respect to the solvent: diethyl ether, hexane and chloroform; the time of extraction; and the concentration of saffron in each organic solvent. Best extraction conditions were obtained when 20 g L^?1 of saffron was extracted with chloroform for 15 min. Intra-laboratory validation of the optimised conditions and analysis by UV–Vis spectrophotometry showed satisfactory results in linearity, repeatability, intermediate precision and recovery. The limit of detection was 1 mg safranal kg^?1 saffron and the limit of quantification was 3 mg safranal kg^?1 saffron.     

Simultaneous determination of anthocyanins,coumarins and phenolic acids in fruits,kernels and liqueur of Prunus mahaleb L

In the fresh tissues of Prunus mahaleb L., three classes of phenolics were characterised: phenolic acid derivatives (main compound being o-coumaric acid glucoside), quercetin glycosides, and anthocyanins (cyanidin 3,5-diglucoside, cyanidin 3-sambubioside, cyanidin 3-xylosyl-rutinoside and cyanidin 3-rutinoside). Coumarin was also identified. The kernels showed a high content of coumarin (0.87 mg g^?1) which is the main class of metabolites in this sample, but present in pitted berries as well (0.63 mg g^?1). Flavonoids are mainly concentrated in the skin and pulp (0.55 mg g^?1). In ‘Mirinello di Torremaggiore’ liqueur, produced from P. mahaleb L. in accordance with traditional procedures, anthocyanins make up 16.5%, phenolic acids 43.3%, coumarin 36.2% and flavonoids 4% of total compounds. Anthocyanins are the main class in solid residues from liqueur production (70%). These findings point out that solid residues of P. mahaleb can be considered an interesting and innovative source of appreciable amounts of cyanidin glycosides (3.3 mg g^?1).     

Activation energy measurements in rheological analysis of cheese

Activation energy of flow (E_a) between 30 and 44 °C was calculated from temperature sweeps of cheeses with contrasting characteristics to determine its usefulness in predicting rheological behavior upon heating. Cheddar, Colby, whole milk Mozzarella, low-moisture part-skim Mozzarella, Parmesan, soft goat, and Queso Fresco cheeses were heated from 22 to 70 °C, and E_a was calculated from the resulting Arrhenius plots. Protein and moisture content were highly correlated with E_a. The E_a values for goat cheese and Queso Fresco, which did not flow when heated, were between 30 and 60 kJ mol^?1. Cheddar, Colby, and the Mozzarellas did flow upon heating, and their E_a values were between 100 and 150 kJ mol^?1. Parmesan, the hardest cheese, flowed rapidly with heat and had an E_a > 180 kJ mol^?1. E_a provides an objective means of quantitating the flow of cheese, and together with elastic modulus and viscous modulus provides a picture of the behavior of cheese as it is heated.     

Probiotic properties of folate producing Streptococcus thermophilus strains

This study aimed at assessing the probiotic potential of two high folate producing Streptococcus thermophilus strains (RD102 and RD104) isolated from Indian fermented milk products by both in vitro and in vivo tests. These strains were able to survive at pH 2.5 and 2% bile with a good bile salt hydrolase activity, cell surface hydrophobicity and sensitivity to most of the clinically important antibiotics. On evaluation for gastrointestinal transit tolerance these showed a viable count of 5 log cfu mL^?1 and 7 log cfu mL^?1, respectively in simulated gastrointestinal juice of pH 2.0 and 2% bile. During the in vivo feeding trial in mice the strains showed a viable count of about 7 log cfu g^?1 faeces and 6 log cfu g^?1 of large intestine, respectively. These strains were hence observed to possess favorable strain specific probiotic properties and have the potential to be a source of novel probiotics.     

A risk characterization model of Salmonella Typhimurium in Irish fresh pork sausages

A second-order simulation model was built to estimate the risk of Salmonella Typhimurium associated with the consumption of Irish fresh pork sausages. To select appropriate hazard characterization models, an initial appraisal of the current dose-response models was conducted. The cooking modality of grilling was associated with a higher mean risk of infection per serving (1.399 × 10^? 6; 95% CI: 7.54 × 10^? 7–2.65 × 10^? 6) than frying (6.246 × 10^? 7, 95% CI: 2.78 × 10^? 7–1.17 × 10^? 6). When the risk was extrapolated over the consumption in a year period, the mean risk of infection increased considerably to 8.541 × 10^? 5 with an expected number of infections and illnesses of 184.3 (95% CI: 26–664) and 17 (95% CI: 2–63), respectively. Results highlighted the importance of consumer education, as scenario analysis predicted that, for the current level of Salmonella in pork sausage, decreasing the product's cold storage by approximately 8 h and cooking for an additional half minute can reduce the current risk level by ～ 50%.