Characterization and identification of lactic acid bacteria in "morcilla de Burgos"

A total of 176 lactic acid bacteria (LAB) isolated from a typical Spanish blood sausage called "morcilla de Burgos" were identified by means of phenotypic characteristics and 16S rDNA RFLP (ribotyping). LAB were isolated from "morcilla" of different producers and in different storage periods, which includes unpackaged, vacuum and modified atmosphere packaged "morcilla" and vacuum packed and pasteurised "morcilla". The knowledge of specific spoilage bacteria of "morcilla de Burgos" will be useful to design new preservation methods to extend the shelf-life of this product. Identification made according to phenotypic and biochemical characteristics shows the majority of the isolates were heterofermentative LAB (93.2%) and eight different bacterial groups could be distinguished (A-G). Weisella viridescens was the main species detected (42%). In addition, Leuconostoc spp. (23.9%), Weissella confusa (11.4%) and Lactobacillus fructosus (5.7%) species were found. Few strains were phenotypically misidentified as Lactobacillus sanfrancisco, Pediococcus spp., Lactobacillus sakei/curvatus and Carnobacterium spp. and 11 strains remained unknown. Most of the leuconostocs were identified as Leuconostoc mesenteroides and Leuconostoc carnosum species. Ribotyping shows a quite good correlation with phenotypic methods, although it has been possible to identify 15 different clusters. W. viridescens and leuconostocs were also the predominant LAB. Strains identified as W. confusa by phenotypic characteristics were resolved in W. confusa and Weissella cibaria by ribotyping. Neither Carnobacterium piscicola nor Lb. sanfrancisco were identified by means of genotypic method. All Lb. fructosus strains and some more included in different phenotypic groups (17 strains in total) could not be associated with any reference strain (cluster VII).     

Physical properties of surimi sausages subjected to high hydrostatic pressure treatment

Food Science and Biotechnology - We aimed to investigate the effect of high pressure treatment (HPT) on the physical properties of surimi sausages. For protein gelation, high hydrostatic pressure...     

Assessment of safe enterococci as bioprotective cultures in low-acid fermented sausages combined with high hydrostatic pressure

Three bacteriocinogenic, non-aminogenic and non-virulent Enterococcus strains (Enterococcus faecium CTC8005, Enterococcus devriesei CTC8006 and Enterococcus casseliflavus CTC8003) were used as starter cultures in low-acid fermented sausages to assess their competitiveness and their bioprotective potential against Listeria monocytogenes and Staphylococcus aureus. The inoculated strains were successfully monitored by RAPD-PCR. All the strains were able to grow, survive and dominate the endogenous enterococci population and avoided the growth of Enterobacteriaceae. E. devriesei CTC8006 and E. faecium CTC8005 particularly inhibited the growth of L. monocytogenes during the whole ripening process. S. aureus was not affected by the inoculated bacteriocinogenic enterococci strains.     

Physiological damages of Listeria monocytogenes treated by high hydrostatic pressure

A fluorescent glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), which had been developed previously for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeasts. Fluorescence intensities of the yeast population were measured by fluorescence spectrophotometry upon exposure to antifungal agents after staining with 2-NBDG and were compared to the number of colony forming units (CFU). A good correlation was obtained between the yeast viability, determined by the CFU, and the accumulation of 2-NBDG by yeast cells (correlation constant: r=0.98). Susceptibility testing of amphotericin B and miconazole against yeast strains by plate count and 2-NBDG fluorescence method yielded corresponding results. In conclusion, we found that staining with 2-NBDG is a rapid and sensitive method for the assessment of yeast cell viability.     

Assessment of high hydrostatic pressure and starter culture on the quality properties of low-acid fermented sausages

The addition of starter culture and high pressure processing after ripening improved the microbial quality of low-acid fermented sausages (fuet and chorizo). The use of Lactobacillus sakei CTC6626 and Staphylococcus xylosus CTC6013 as starter culture significantly reduced Enterobacteriaceae and Enterococcus levels in the finished sausages. Moreover, the addition of starter culture produced sausages of similar quality to traditional low-acid fermented sausages. Slightly lower pH values and higher cohesiveness were obtained for both fuet and chorizo with starter culture. Sensory analysis showed no differences between lots of chorizo whereas starter fuet was more acid and gummy. High pressure induced an additional reduction of Enterobacteriaceae in non-starter sausages. An increase of textural properties was observed after pressurization. No other differences were observed between non-treated and pressurized sausages.     

Behavior of Yersinia enterocolitica strains inoculated in model cheese treated with high hydrostatic pressure

The effects of high hydrostatic pressure treatment and the ability for survival, repair, and growth of three human pathogenic serotypes (O:1, O:3, O:8) of Yersinia enterocolitica were investigated in washed-curd model cheese made with pasteurized bovine milk. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed at 0, 1, 7, and 15 days to assess the viability of the Yersinia population. A long-term study (up to 60 days of ripening after high hydrostatic pressure treatment) was also undertaken. Treatments at 400 and 500 MPa caused maximum lethality, and only the treatment at 300 MPa showed significant differences (P < 0.05) between serotypes; the most baroresistant was O:3. Ability to repair and grow was not observed after 15 days of storage at 8 degrees C. Yersinia counts in untreated cheese samples also decreased below the detection limit at day 45 in the long-term study. These results suggest that the cheese environment did not allow recovery of injured cells or growth. A primary contributing factor to this effect seemed to be the low pH resulting from the production of lactic acid during cheese ripening.     

Microbiological quality of mechanically recovered poultry meat treated with high hydrostatic pressure and nisin

The combined effect of high hydrostatic pressure processing, addition of nisin and acidification on aerobic mesophilic and psychrotrophic bacterial populations of mechanically recovered poultry meat (MRPM) kept under refrigeration (2°C) was evaluated 1, 15 and 30 days after pressurization. Nisin (0, 12.055, 100 and 200 ppm) and glucono-delta-lactone (GdL; 0 and 1%) were added to MRPM. Vacuum-packaged samples were treated at 350 or 450 MPa and 2°C for 15 min using both continuous pressurization and three-cycle oscillatory pressurization for 5 min per cycle. In some samples a reduction of mesophile counts between 3.0544 and 5.0538 log cfu g⁻¹was found. Psychrotrophes seemed to be more sensitive; in samples with 100 ppm of nisin and GdL treated at 450 MPa with cycles they were reduced to undetectable levels (a lethality of approximately 7.055 log units). Cycle pressurization showed slightly better results than continuous pressurization. The combination of 350 MPa, 100 ppm of nisin and 1% of GdL was enough to extend the shelf life of MRPM during the 30 day chilled storage.     

Synergistic effect of carbon dioxide atmospheres and high hydrostatic pressure to reduce spoilage bacteria on poultry sausages

Synergistic effect of combining high hydrostatic pressure (HHP) and CO₂ atmospheres has been studied against Leuconostoc carnosum, Brochothrix thermosphacta, Salmonella enteritidis, Campylobacter jejuni and Listeria innocua separately inoculated in poultry sausages. The microbial counts of the HHP treated samples (350 MPa 10 min at room temperature) and CO₂ atmosphere packaged were compared with non pressure treated and air packaged ones, analyzed at 20 h and 7 days after the treatment. The results showed a synergistic effect of the combination of these two preserving technologies against all the microorganisms studied, except in S. enteritidis which showed a greater resistance under CO₂ atmospheres, and C. jejuni, that is especially sensitive at high pressures. It seems that cell damage produced by high pressure facilitates the penetration of carbon dioxide into the microorganisms' cells, affecting their metabolism and consequently their growth. Using CO₂ atmospheres in combination with HHP treatments, pressure could be lowered without compromising the reduction of microbial counts.     

Fate of Staphylococcus aureus in cheese treated by ultrahigh pressure homogenization and high hydrostatic pressure

We evaluated the influence of ultrahigh pressure homogenization (UHPH) treatment applied to milk containing Staphylococcus aureus CECT 976 before cheese making, and the benefit of applying a further high hydrostatic pressure (HHP) treatment to cheese. The evolution of Staph. aureus counts during 30 d of storage at 8°C and the formation of staphylococcal enterotoxins were also assessed. Milk containing approximately 7.3 log₁₀ cfu/mL of Staph. aureus was pressurized using a 2-valve UHPH machine, applying 330 and 30 MPa at the primary and the secondary homogenizing valves, respectively. Milk inlet temperatures (T_in) of 6 and 20°C were assayed. Milk was used to elaborate soft-curd cheeses (UHPH cheese), some of which were additionally submitted to 10-min HHP treatments of 400 MPa at 20°C (UHPH+HHP cheese). Counts of Staph. aureus were measured on d 1 (24 h after manufacture or immediately after HHP treatment) and after 2, 15, and 30 d of ripening at 8°C. Counts of control cheeses not pressure-treated were approximately 8.5 log₁₀ cfu/g showing no significant decreases during storage. In cheeses made from UHPH treated milk at T_in of 6°C, counts of Staph. aureus were 5.0 ± 0.3 log₁₀ cfu/g at d 1; they decreased significantly to 2.8 ± 0.2 log₁₀ cfu/g on d 15, and were below the detection limit (1 log₁₀ cfu/g) after 30 d of storage. The use of an additional HHP treatment had a synergistic effect, increasing reductions up to 7.0 ± 0.3 log₁₀ cfu/g from d 1. However, for both UHPH and UHPH+HHP cheeses in the 6°C T_in samples, viable Staph. aureus cells were still recovered. For samples of the 20°C T_in group, complete inactivation of Staph. aureus was reached after 15 d of storage for both UHPH and UHPH+HHP cheese. Staphylococcal enterotoxins were found in controls but not in UHPH or UHPH+HHP treated samples. This study shows a new approach for significantly improving cheese safety by means of using UHPH or its combination with HHP.     

Response of two Salmonella enterica strains inoculated in model cheese treated with high hydrostatic pressure

The aim of this work was to determine the response to high hydrostatic pressure and the ability for survival, recovery, and growth of 2 strains of Salmonella enterica (Salmonella enteritidis and Salmonella typhimurium) inoculated in a washed-curd model cheese produced with and without starter culture. Inoculated samples were treated at 300 and 400 MPa for 10 min at room temperature and analyzed after treatment and after 1, 7, and 15 d of storage at 12° C to study the behavior of the Salmonella population. Cheese samples produced with starter culture and treated at 300 and 400 MPa showed maximum lethality; no significant differences in the baroresistant behavior of both strains were detected. Nevertheless, when starter culture was not present, the maximum lethality was only observed in cheese samples treated at 400 MPa, in the case of S. enteritidis. Ability to repair and grow was not observed in model cheese produced with starter culture and cell counts of treated samples decreased after 15 d of storage at 12° C. In cheese produced without starter culture, Salmonella cells showed the ability to repair and grow during the storage period, reaching counts over 3 log₁₀ (cfu/mL) in both applied treatments and serotypes. These results suggest that high hydrostatic pressure treatments are effective to reduce Salmonella population in this type of cheese, but the presence of the starter culture affects the ability of this microorganism to repair and grow during the storage period.     

Physicochemical and functional properties of cowpea protein isolates treated with temperature or high hydrostatic pressure

The effect of thermal (TT, 70 and 90 °C) and high hydrostatic pressure (HHPTs, 200, 400 and 600 MPa) treatments on physicochemical and functional properties of cowpea protein isolates (CPIs) extracted at pH 8.0 (A8) and pH 10.0 (A10) was analyzed. The pH of protein extraction affected some physicochemical properties (surface hydrophobicity (Ho) and denaturation temperature), without affecting the functional properties. Treatments led to the formation of soluble protein aggregates stabilized by disulfide bonds, especially with TT at 90 °C. TT and HHPTs shifted the wavelengths of maximum emission to red and to blue, respectively. All treatments induced unfolding and denaturation. HHPTs was more efficient than TT to enhance gelation and water holding capacities. Interestingly, treated and untreated CPIs exhibited high values of solubility (72–97%). TT and HHPT induced greater changes in physicochemical and functional properties of A8 than in those of A10. Remarkably, functional properties were improved from the less energetic treatments (70 °C, 200 MPa).Industrial relevanceThe comparison between treatments (one traditional and one corresponding to an emerging technology) gives information about the possibility of obtaining modified proteins for different functional purposes. The modified cowpea protein isolates may be used in beverages because of high solubility, in desserts because of gel formation capacity and/or as additives in other foodstuff because of improved water holding capacity. This knowledge would increase the added value of a local production currently marketed without processing.     

Modeling survival of high hydrostatic pressure treated stationary- and exponential-phase Listeria innocua cells

High Hydrostatic Pressure (HHP) inactivation (325–400 MPa; 0–20 min; maximum temperature 30 °C) of cells of Listeria innocua CECT 910 was studied in two different growth phases (exponential and stationary), and the corresponding survival curves were obtained for each case. The curves were fitted to two nonlinear models, the modified Gompertz equation and the Baranyi model. The kinetic constants calculated for both models, µ_max and k_max, indicated that cells in exponential growth phase were more sensitive to pressure than those in stationary phase. Both mathematical models were suitable for describing L. innocua HHP survival curves, rendering kinetic constants that increased with increasing pressure. When considering the experimental models validation, both Gompertz and Baranyi predicted in a similar way, however Baranyi had slightly lower A_f (Accuracy factor) and B_f (Bias factor) values, which indicated better prediction values. In summary, both mathematical models were perfectly valid for describing L. innocua inactivation kinetics under HHP treatment.Industrial relevanceThe mathematical models for inactivation and growth of microorganisms are the foundation of predictive microbiology and are used in risk assessments procedures as part of the food safety management system. Besides, these models together with those applied to inactivation of enzymes and destruction of quality factors are essential to optimize processes and thus to lay the foundations for industrial processing. It is therefore necessary to identify generally applicable kinetic models that will produce primary and secondary kinetic parameters and are statistically reliable as a key tool to predict the behaviour of microorganisms, enzymes and quality factors after processing.     

Effect of phosvitin on lipid and protein oxidation in ground beef treated with high hydrostatic pressure

In this study, we aimed to examine the effect of phosvitin on lipid and protein oxidation of raw and cooked ground beef treated with high hydrostatic pressure (HHP). Ground beef patty with 0, 500, or 1000 mg phosvitin/kg meat was treated with HHP at 0.1, 300, or 600 MPa. Half of the patties were used in a raw meat analysis, and the other half were used in a cooked meat analysis. Phosvitin and HHP treatment at 300 MPa synergistically reduced microbial growth, and HHP treatment at 600 MPa reduced microbial counts to undetectable levels (< 1 log CFU/g) throughout the length of the study in all samples. Phosvitin delayed lipid and protein oxidation in HHP-treated cooked and raw ground beef, respectively. However, phosvitin had no effect on the color changes of raw ground beef attributable to HHP. The results indicated that phosvitin could enhance the stability of lipids and proteins but not color changes of raw ground beef caused by HHP.     

Survival and growth of Yersinia enterocolitica strains inoculated in skimmed milk treated with high hydrostatic pressure

Four human pathogenic strains of Yersinia enterocolitica (serotypes O:1, O:3, O:8, and O:9) were inoculated (7-8 log CFU/ml) in UHT skimmed milk and treated at 300, 400, and 500 MPa for 10 min at 20 degrees C, and then kept at 8 degrees C to assess their evolution for 15 days. Treatments at 400 and 500 MPa caused the highest lethality, generally reaching counts below detection level (1 CFU/ml) in the culture media. At 300 MPa, the most baroresistant serotypes were O:3 and O:8. After 15 days of storage at 8 degrees C, Y. enterocolitica showed growth over 8 log (CFU/ml) in all treatments. Kinetic study of microbial inactivation in skimmed milk was performed with serotype O:8 at 300 MPa, showing a tailing after 35 min of pressure treatment.     

Effect of skim milk treated with high hydrostatic pressure on permeate flux and fouling during ultrafiltration

Ultrafiltration (UF) is largely used in the dairy industry to generate milk and whey protein concentrate for standardization of milk or production of dairy ingredients. Recently, it was demonstrated that high hydrostatic pressure (HHP) extended the shelf life of milk and improved rennet coagulation and cheese yield. Pressurization also modified casein micelle size distribution and promoted aggregation of whey proteins. These changes are likely to affect UF performance. Consequently, this study determined the effect of skim milk pressurization (300 and 600 MPa, 5 min) on UF performance in terms of permeate flux decline and fouling. The effect of HHP on milk proteins was first studied and UF was performed in total recycle mode at different transmembrane pressures to determine optimal UF operational parameters and to evaluate the effect of pressurization on critical and limiting fluxes. Ultrafiltration was also performed in concentration mode at a transmembrane pressure of 345 kPa for 130 or 140 min to evaluate the decline of permeate flux and to determine fouling resistances. It was observed that average casein micelle size decreased by 32 and 38%, whereas β-lactoglobulin denaturation reached 30 and 70% at 300 and 600 MPa, respectively. These results were directly related to UF performance because initial permeate fluxes in total recycle mode decreased by 25% at 300 and 600 MPa compared with nonpressurized milk, critical flux, and limiting flux, which were lower during UF of milk treated with HHP. During UF in concentration mode, initial permeate fluxes were 30% lower at 300 and 600 MPa compared with the control, but the total flux decline was higher for nonpressurized milk (62%) compared with pressure-treated milk (30%). Fouling resistances were similar, whatever the treatment, except at 600 MPa where irreversible fouling was higher. Characterization of the fouling layer showed that caseins and β-lactoglobulin were mainly involved in membrane fouling after UF of pressure-treated milk. Our results demonstrate that HHP treatment of skim milk drastically decreased UF performance.     

Food processing by high hydrostatic pressure

The use of high hydrostatic pressures (HHP) for food processing is finding increased application within the food industry. One of the advantages of this technology is that because it does not use heat, sensory, and nutritional attributes of the product remain virtually unaffected, thus yielding products with better quality than those processed traditional methods. HHP have the ability to inactivate microorganisms as well as enzymes responsible for shortening the life of a product. In addition to lengthening the shelf-life of food products, HHP can modify functional properties of components such as proteins, which in turn can lead to the development of new products. Equipment for large-scale production of HHP processed products are commercially available nowadays. Guacamole, sliced ham, oysters, and fruit juices are some of the products currently available on the market. HHP technology is one of the most promising nonthermal processes.     

超高压与转谷氨酰胺酶协同对碎猪肉凝胶成型的研究

研究了超高压与TGase协同对碎猪肉凝胶成型的影响,采用Box-Behnken实验设计获得的碎猪肉凝胶成型最佳工艺条件为:TGase添加量4.5g/kg,压力水平240MPa,保压时间10min。最佳工艺条件下的碎猪肉凝胶成型制品与鲜猪肉的硬度、粘着性、内聚性、胶凝性差异不显著。超高压与TGase结合的技术,实现了碎猪肉的凝胶成型,为碎猪肉边角料的利用提供了新方法。     

超高压对食品中微生物的影响

超高压是一种新型食品加工技术,已广泛应用于食品的非热加工。在超高压条件下微生物的细胞壁、细胞膜被破坏,引起细胞形态结构的改变;微生物细胞内的结构蛋白、酶等在超高压条件下被钝化,导致微生物正常的代谢功能和增殖能力被破坏;在超高压条件下微生物的蛋白组和基因组也产生了一定的变化,许多与抗逆有关的蛋白质和基因表达上调。     

Optimization of textural properties of reduced-fat and reduced-salt emulsion-type sausages treated with high pressure using a response surface methodology

This study optimized the high pressure conditions for development of a reduced fat sausage. A three-factor-three-level Box–Behnken design was adopted to study the simultaneous effects of one compositional variable (15, 20 and 25% fat content) and two processing variables (150, 200 and 250 MPa high pressure, along with 5, 6 and 7 min high pressure treatment time) on firmness of emulsion-type sausages. Analysis of variance (ANOVA) was performed to evaluate the potential interactive and quadratic effects between these variables. The results revealed that, the optimum processing conditions for an optimum gel setting were 22.19% fat content, 197.30 MPa high pressure and 5.92 min pressure treatment time. The adequacy of the model equation for predicting the optimum response values was effectively verified. In conclusion, the emulsion-type meat sausages using a novel high pressure based processing method were preferred for their improved textural properties and reduced fat content.Industrial relevanceFor health reasons, there is a need to reduce fat content of processed meat products. This study developed a novel processing method using high pressure to produce emulsion-type meat sausages with reduced-fat, with improved functional qualities, including objective appearance, textural properties and sensory evaluation. Importantly, this was achieved with a model, mainly based on prediction of the firmness of the pressure treated sausages with reduced fat contents.     

Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages

The single and combined effects of enterocin AS-48 and high hydrostatic pressure (HHP) on Listeria monocytogenes, Salmonellaenterica, and Staphylococcus aureus was investigated in fuet (a low acid fermented sausage) during ripening and storage at 7 °C or at room temperature. AS-48 (148 AU g⁻¹) caused a drastic 5.5 log cfu g⁻¹ decrease in L. monocytogenes (P < 0.001) and a significant (P < 0.01) inhibition (1.79 logs) for Salmonella at the end of ripening (10 d). After pressurization (400 MPa) and storage Listeria counts remained below 5 cfu g⁻¹ in all fuets containing AS-48 (pressurized or not). HHP alone had no anti-Listeria effect. HHP treatment significantly reduced Salmonella counts, with lowest levels in pressurized fuets with AS-48. S. aureus showed similar growth for all treatments and storage conditions. These results indicate that AS-48 can be applied alone to control L. monocytogenes and combined with HHP treatment to control Salmonella in fuets.