High pressure processing effect on different Listeria spp. in a commercial starter-free fresh cheese.

In this study, both microbial inactivation and growth of Listeria spp. inoculated in commercial free-starter fresh cheese was evaluated after high-pressure processing (HPP). HPP conditions (300, 400, 500 and 600 MPa at 6 °C for 5 min) and inoculum level (3-4 or 6-7 log CFU/g of cheese), as well as differences among strains inoculated (Listeria innocua, L. monocytogenes CECT 4031 and L. monocytogenes Scott A) were investigated. Inactivation and generation of sublethal injury were determined after HPP using ALOA (Agar Listeria according to Ottaviani and Agosti) and TAL (Thin Agar Layer) plating methods, respectively. Listeria inactivation increased with the pressure applied, presenting some statistical differences between the employed strains, inoculum level and sublethal injury. The highest lethality values were obtained at 600 MPa for the three strains tested, although the 500 MPa treatment presented high lethality for L. innocua and L. monocytogenes CECT 4031. After treatment, L. innocua and L. monocytogenes CECT 4031 counts in fresh cheese increased gradually during cold storage. By contrast, counts in cheeses inoculated with L. monocytogenes Scott A did not change significantly (p ≥ 0.05), being this strain the most pressure resistant and with the slowest growth rate. The manuscript present information supporting that, strains with high-level resistance should be employed during inactivation studies, instead of surrogate microorganisms. Application of HPP treatments of 500 MPa and especially 600 MPa on fresh cheeses would be effective to eliminate the most resistant microorganism to a level that should not present a public health risk under normal conditions of distribution and storage.

Microbiological stabilization of tiger nuts' milk beverage using ultra-high pressure homogenization. A preliminary study on microbial shelf-life extension.

Tiger nuts' milk beverages are highly perishable products. For this reason, the interest of food industry for their commercialization makes necessary the application of preservation treatments to prolong their shelf-life. In the current study, the effect of ultra-high pressure homogenization (UHPH) on the microbiological and sensory qualities of tiger nuts' milk beverage was evaluated. Characteristics of UHPH-treated products (at 200 and 300 MPa, with inlet temperature of 40 °C) were compared with those of raw (RP) and conventionally homogenized-pasteurized (H-P) beverages, after treatment and during cold storage at 4 °C. Microbiological quality of beverages was studied by enumerating total counts, psychrotrophic bacteria, lactobacilli, enterobacteria, molds and yeasts, and mesophilic spores. Evolution of color and sensory characteristics of beverages were also determined. Microbiological shelf-life of the tiger nuts' milk beverages was extended from 3 to 25, 30 and 57 days by applying H-P and UHPH treatments at 200 and 300 MPa, respectively. Color of beverages was the only attribute that differentiated UHPH samples from the others, with greater luminosity and whiteness. Hence, UHPH treatments showed to be an alternative to the conventional H-P for obtaining tiger nuts' milk beverages with an improved microbiological shelf-life and good sensorial characteristics.

Ultra-High Pressure Homogenization improves oxidative stability and interfacial properties of soy protein isolate-stabilized emulsions.

Ultra-High Pressure Homogenization (100-300MPa) has great potential for technological, microbiological and nutritional aspects of fluid processing. Its effect on the oxidative stability and interfacial properties of oil-in-water emulsions prepared with 4% (w/v) of soy protein isolate and soybean oil (10 and 20%, v/v) were studied and compared to emulsions treated by conventional homogenization (15MPa). Emulsions were characterized by particle size, emulsifying activity index, surface protein concentration at the interface and by transmission electron microscopy. Primary and secondary lipid oxidation products were evaluated in emulsions upon storage. Emulsions with 20% oil treated at 100 and 200MPa exhibited the most oxidative stability due to higher amount of oil and protein surface load at the interface. This manuscript addresses the improvement in oxidative stability in emulsions treated by UHPH when compared to conventional emulsions.

Compositional and biochemical changes during cold storage of starter-free fresh cheeses made from ultra-high-pressure homogenised milk.

The aim of the present study was to evaluate the effects of using ultra-high pressure homogenisation (UHPH) on the composition and biochemistry of starter-free fresh cheeses and to monitor their evolution during cold storage as an alternative to conventional treatments applied in the production of fresh cheese such as conventional pasteurisation and homogenisation-pasteurisation. Although both homogenisation treatments increased cheese moisture content, cheeses from UHPH-treated milk showed lower moisture loss during storage than those from conventionally homogenised-pasteurised milk. Lipolysis and proteolysis levels in cheeses from UHPH-treated milk were lower than those from conventionally treated milk samples. Although, oxidation was found to be the major drawback, in general terms, high quality starter-free fresh cheeses were obtained from UHPH-treated milk.

Ultra-High Pressure Homogenization enhances physicochemical properties of soy protein isolate-stabilized emulsions.

The effect of Ultra-High Pressure Homogenization (UHPH, 100-300MPa) on the physicochemical properties of oil-in-water emulsions prepared with 4.0% (w/v) of soy protein isolate (SPI) and soybean oil (10 and 20%, v/v) was studied and compared to emulsions treated by conventional homogenization (CH, 15MPa). CH emulsions were prepared with non-heated and heated (95°C for 15min) SPI dispersions. Emulsions were characterized by particle size determination with laser diffraction, rheological properties using a rotational rheometer by applying measurements of flow curve and by transmission electron microscopy. The variation on particle size and creaming was assessed by Turbiscan® analysis, and visual observation of the emulsions was also carried out. UHPH emulsions showed much smaller d3.2 values and greater physical stability than CH emulsions. The thermal treatment of SPI prior CH process did not improve physical stability properties. In addition, emulsions containing 20% of oil exhibited greater physical stability compared to emulsions containing 10% of oil. Particularly, UHPH emulsions treated at 100 and 200MPa with 20% of oil were the most stable due to low particle size values (d3.2 and Span), greater viscosity and partial protein denaturation. These results address the physical stability improvement of protein isolate-stabilized emulsions by using the emerging UHPH technology.

Inactivation of Bacillus spores inoculated in milk by Ultra High Pressure Homogenization.

Ultra High-Pressure Homogenization treatments at 300 MPa with inlet temperatures (Ti) of 55, 65, 75 and 85 °C were applied to commercial Ultra High Temperature treated whole milk inoculated with Bacillus cereus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus coagulans, Geobacillus stearothermophilus and Bacillus subtilis spores in order to evaluate the inactivation level achieved. Ultra High-Pressure Homogenization conditions at 300 MPa with Ti = 75 and 85 °C were capable of a spore inactivation of ∼5 log CFU/mL. Furthermore, under these processing conditions, commercial sterility (evaluated as the complete inactivation of the inoculated spores) was obtained in milk, with the exception of G. stearothermophilus and B. subtilis treated at 300 MPa with Ti = 75 °C. The results showed that G. stearothermophilus and B. subtilis have higher resistance to the Ultra High-Pressure Homogenization treatments applied than the other microorganisms inoculated and that a treatment performed at 300 MPa with Ti = 85 °C was necessary to completely inactivate these microorganisms at the spore level inoculated (∼1 × 10(6) CFU/mL). Besides, a change in the resistance of B. licheniformis, B. sporothermodurans, G. stearothermophilus and B. subtilis spores was observed as the inactivation obtained increased remarkably in treatments performed with Ti between 65 and 75 °C. This study provides important evidence of the suitability of UHPH technology for the inactivation of spores in high numbers, leading to the possibility of obtaining commercially sterile milk.

Effect of moderate inlet temperatures in ultra-high-pressure homogenization treatments on physicochemical and sensory characteristics of milk.

The effect of ultra-high-pressure homogenization (UHPH) on raw whole milk (3.5% fat) was evaluated to obtain processing conditions for the sterilization of milk. Ultra-high-pressure homogenization treatments of 200 and 300 MPa at inlet temperatures (Ti) of 55, 65, 75, and 85 °C were compared with a UHT treatment (138 °C for 4s) in terms of microbial inactivation, particle size and microstructure, viscosity, color, buffering capacity, ethanol stability, propensity to proteolysis, and sensory evaluation. The UHPH-treated milks presented a high level of microbial reduction, under the detection limit, for treatments at 300 MPa with Ti of 55, 65, 75, and 85 °C, and at 200 MPa with Ti = 85 °C, and few survivors in milks treated at 200 MPa with Ti of 55, 65, and 75 °C. Furthermore, UHPH treatments performed at 300 MPa with Ti = 75 and 85 °C produced sterile milk after sample incubation (30 and 45 °C), obtaining similar or better characteristics than UHT milk in color, particle size, viscosity, buffer capacity, ethanol stability, propensity to protein hydrolysis, and lower scores in sensory evaluation for cooked flavor.

Influence of fat replacement by inulin on rheological properties, kinetics of rennet milk coagulation, and syneresis of milk gels.

The objective of this study was to evaluate the effect of inulin as a fat replacer on the rheological properties, coagulation kinetics, and syneresis of milk gels. A randomized factorial design, replicated 3 times, with 3 inulin concentrations (0, 3, and 6%), 2 levels of fat (<0.2 and 1.5%), and 3 coagulation temperatures (27, 32, and 37°C) was used. The coagulation process was monitored using near-infrared spectrometry, small amplitude oscillatory rheometry, and visual coagulation indexes. The syneresis was evaluated by volumetric methods. Inulin addition increased the rates of aggregation and curd firming reactions in the casein gels. The observed effect, which was more evident on the aggregation reaction, depended on the concentration of inulin and the coagulation temperature. Addition of 6% inulin reduced the clotting time by approximately 26% and the time at which the gel reached a storage modulus equal to 30 Pa by approximately 36%. The optical parameter R'max, defined as the maximum value of change in light backscatter profile/change in time (where R' = dR/dt), was used to calculate an approximation of the temperature coefficients (Q10) for milk coagulation. Increasing fat concentration induced a consistent increase in all the optical, rheological, and visual parameters studied, although the observed trend was not statistically significant. The addition of inulin at a level of 6% produced a reduction in syneresis and increased the curd yield by approximately 30%. It was concluded that the addition of inulin affects the kinetics of milk coagulation and the cutting time and, therefore, the use of inline sensors such as near-infrared spectrometry may be necessary for optimal process control.

Effect of fat content and homogenization under conventional or ultra-high-pressure conditions on interactions between proteins in rennet curds.

The objective of this study was to investigate the influence of conventional and ultra-high-pressure homogenization on interactions between proteins within drained rennet curds. The effect of fat content of milk (0.0, 1.8, or 3.6%) and homogenization treatment on dissociation of proteins by different chemical agents was thus studied. Increasing the fat content of raw milk increased levels of unbound whey proteins and calcium-bonded caseins in curds; in contrast, hydrophobic interactions and hydrogen bonds were inhibited. Both homogenization treatments triggered the incorporation of unbound whey proteins in the curd, and of caseins through ionic bonds involving calcium salts. Conventional homogenization-pasteurization enhanced interactions between caseins through hydrogen bonds and hydrophobic interactions. In contrast, ultra-high-pressure homogenization impaired hydrogen bonding, led to the incorporation of both whey proteins and caseins through hydrophobic interactions and increased the amount of unbound caseins. Thus, both homogenization treatments provoked changes in the protein interactions within rennet curds; however, the nature of the changes depended on the homogenization conditions.

Effect of the inclusion of artichoke silage in the ration of lactating ewes on the properties of milk and cheese characteristics during ripening.

The effect of including artichoke silage in the rations of dairy ewes on milk characteristics and biochemical changes of ripened cheeses was evaluated. Four groups of lactating ewes were fed rations containing 0, 10, 20, or 30% artichoke silage on a dry matter basis. Bulk milk samples were collected 3 times during the feeding period, and semi-hard cheeses were manufactured and sampled during ripening. Milk composition and cheese yield were not affected by diet. Inclusion of 20 and 30% artichoke silage reduced the firmness of the curds at a level only detected by the Gelograph (Gelograph-NT, Gel-Instrumente, Thalwil, Switzerland) probe. Inclusion of artichoke silage in ewes' diet decreased fat and total free fatty acids content of these cheeses and increased total free amino acids content. Despite the effect of diet on cheese ripening characteristics, the overall sensory scores for cheeses corresponding to artichoke silage diets were statistically higher than those for the control cheeses.

Proteolysis of yogurts made from ultra-high-pressure homogenized milk during cold storage.

Proteolysis was investigated in yogurts made from milk that was ultra-high-pressure homogenized at 200 or 300 MPa and at 30 or 40 degrees C and compared with those produced from heat-treated milk containing 3% skim milk powder. To evaluate changes in the protein fraction, samples were analyzed at d 1, 7, 14, 21, and 28 of storage for residual caseins, peptides, and total free amino acids. Results showed that yogurts from heat-treated milk and 300 MPa-treated milk presented similar levels of residual caseins, as well as similar profiles of soluble peptides and total free amino acids. On the contrary, greater amounts of hydrophobic peptides were detected in yogurts made from 200 MPa-treated milk at both 30 and 40 degrees C, especially at the end of storage. In all treatments studied, caseins were hydrolyzed and hydrophobic peptides were increased during storage, as reflected by the increase in soluble nitrogen at the end of the storage.

Effects of high pressure on proteolytic enzymes in cheese: relationship with the proteolysis of ewe milk cheese.

Ewe milk cheeses were submitted to 200, 300, 400, and 500 MPa (2P to 5P) at 2 stages of ripening (after 1 and 15 d of manufacturing; P1 and P15). The high-pressure-treated cheeses showed a more important hydrolysis of beta-casein than control and 2P1 cheeses. Degradation of alpha(s1)-casein was more important in 3P1, 4P1, and P15 cheeses than control and 2P1 cheeses. The 5P1 cheeses exhibited the lowest degradation of alpha(s)-caseins, probably as a consequence of the inactivation of residual chymosin. Treatment at 300 MPa applied on the first day of ripening increased the peptidolytic activity, accelerating the secondary proteolysis of cheeses. The 3P1 cheeses had extensive peptide degradation and the highest content of free amino acids. Treatments at 500 MPa, however, decelerated the proteolysis of cheeses due to a reduction of microbial population and inactivation of enzymes.

Effects of ultra-high pressure homogenization on microbial and physicochemical shelf life of milk.

The effect of ultra-high pressure homogenization (UHPH) on microbial and physicochemical shelf life of milk during storage at 4 degrees C was studied and compared with a conventional heat preservation technology used in industry. Milk was standardized at 3.5% fat and was processed using a Stansted high-pressure homogenizer. High-pressure treatments applied were 100, 200, and 300 MPa (single stage) with a milk inlet temperature of 40 degrees C, and 200 and 300 MPa (single stage) with a milk inlet temperature of 30 degrees C. The UHPH-treated milks were compared with high-pasteurized milk (PA; 90 degrees C for 15 s). The microbiological quality was studied by enumerating total counts, psychrotropic bacteria, lactococci, lactobacilli, enterococci, coliforms, spores, and Pseudomonas. Physicochemical parameters assessed in milks were viscosity, color, pH, acidity, rate of creaming, particle size, and residual peroxidase and phosphatase activities. Immediately after treatment, UHPH was as efficient (99.99%) in reducing psychrotrophic, lactococci, and total bacteria as was the PA treatment, reaching reductions of 3.5 log cfu/mL. Coliforms, lactobacilli, and enterococci were eliminated. Microbial results of treated milks during storage at 4 degrees C showed that UHPH treatment produced milk with a microbial shelf life between 14 and 18 d, similar to that achieved for PA milk. The UHPH treatments reduced the L* value of treated milks and induced a reduction in viscosity values of milks treated at 200 MPa compared with PA milks; however, these differences would not be appreciated by consumers. In spite of the fat aggregates detected in milks treated at 300 MPa, no creaming was observed in any UHPH-treated milk. Hence, alternative methods such as UHPH may give new opportunities to develop fluid milk with an equivalent shelf life to that of PA milk in terms of microbial and physicochemical characteristics.

Effect of heat and high-pressure treatments on microbiological quality and immunoglobulin G stability of caprine colostrum.

Caprine colostrums (6 batches) were subjected to heat (56 degrees C for 60 min and 63 degrees C for 30 min) and high-pressure (400 and 500 MPa for 10 min at 20 degrees C) treatments at laboratory scale, and analyses of the main microbial groups and the extent of IgG denaturation (determined by immunodiffusion) were performed. Overall mean microbial values in raw colostrums were: total count, 5.55 log cfu/mL; Enterobacteriaceae, 2.64 log cfu/mL; lactococci, 5.41 log cfu/mL; lactobacilli, 2.34 log cfu/mL; and enterococci, 4.06 log cfu/mL. Neither Salmonella spp. nor Listeria monocytogenes were detected, whereas coagulase-positive staphylococci were found in various colostrum samples with an overall mean of 1.02 log cfu/mL. Heat and high-pressure treatments significantly reduced total count (1.47 log), lactococci (1.45 log), enterococci (2.47 log), and Enterobacteriaceae, whereas lactobacilli and coagulase-positive staphylococci counts were reduced to undetectable levels, but differences between technological treatments were not statistically significant. High-pressure treatments were as efficient in reducing the bacterial population as were heat pasteurization treatments: 95.50 and 96.93% for pressure treatments of 400 and 500 MPa, and 91.61 and 97.59% for heat treatments of 56 degrees C for 60 min and 63 degrees C for 30 min, respectively. All treatments assayed produced a reduction in colostrum IgG concentration (27.53, 23.58, 23.33, 22.09, and 17.06 mg/mL for raw, heat-treated at 56 degrees C for 60 min or 63 degrees C for 30 min, and pressure-treated at 400 and 500 MPa, respectively), but differences were only observed between raw colostrums and those pressure-treated at 500 MPa. This laboratory-scale study indicated that 20- to 30-mL volumes of goat colostrum could be heated and pressure-treated (400 MPa) to produce hygienic colostrum without affecting IgG concentration.

Effects of ultra-high pressure homogenization on the cheese-making properties of milk.

The effects of single- or 2-stage ultra-high pressure homogenization (UHPH; 100 to 330 MPa) at an inlet temperature of 30 degrees C on the cheese-making properties of bovine milk were investigated. Effects were compared with those from raw, heat-pasteurized (72 degrees C for 15 s), and conventional homogenized-pasteurized (15 + 3 MPa, 72 degrees C for 15 s) treatments. Rennet coagulation time, rate of curd firming, curd firmness, wet yield, and moisture content of curds were assessed. Results of particle size and distribution of milk, whey composition, and gel microstructure observed by confocal laser scanning microscopy were analyzed to understand the effect of UHPH. Single-stage UHPH at 200 and 300 MPa enhanced rennet coagulation properties. However, these properties were negatively affected by the use of the UHPH secondary stage. Increasing the pressure led to higher yields and moisture content of curds. The improvement in the cheese-making properties of milk by UHPH could be explained by changes to the protein-fat structures due to the combined effect of heat and homogenization.

Effects of high pressure treatment on volatile profile during ripening of ewe milk cheese.

The effect of high-pressure treatment on the volatile profile of ewe milk cheeses was investigated. Cheeses were submitted to 200, 300, 400 and 500 MPa at 2 stages of ripening (after 1 and 15 d of manufacturing) and volatile compounds were assayed at 15 and 60 d of ripening. High-pressure treatment altered the balance of volatile profile of cheeses, limiting the formation of acids, alcohols, ketones, aldehydes, and sulfur compounds and enhancing the formation of 2,3-butanedione. In general, cheeses pressurized at 15 d of ripening were more similar to untreated cheeses than those treated at 1 d. Cheeses treated at 300 MPa after 1 d of manufacturing were characterized by higher levels of free amino acids, ethanol, ethyl esters, and branched-chain aldehydes, whereas cheeses treated at 500 MPa after 1 d of manufacturing had lower microbial populations, showed the highest abundance of 2,3-butanedione, pyruvaldehyde, and methyl ketones, and the lowest abundance of alcohols.

Proteolysis in Manchego-type cheese salted by brine vacuum impregnation.

A new salting procedure based on the brine vacuum impregnation of porous products was tested on Manchego-type cheese and compared with conventional brine immersion. Its effect on cheese proteolysis throughout a 90-d ripening period was determined. Three cheese regions were evaluated (the rind, the middle, and the internal regions). The parameters analyzed were total N, water-soluble N, soluble N in trichloroacetic acid and soluble N in phosphotungstic acid by using the Kjeldahl method, casein profile by urea-PAGE, and peptide profile of the water soluble nitrogen extract by reverse-phase HPLC. Free amino acid formation was monitored with a spectrophotometric method by using a Cd-ninhydrin reagent. Globally, proteolysis was significantly affected by ripening stage (increasing throughout all the maturation period studied) and cheese region (rind showed a proteolysis pattern different from the middle and internal regions). The salting procedure only affected cheese proteolysis in the rind, whereas conventional brine-salted cheeses showed lower proteolysis than vacuum-impregnated cheeses.

Analysis of major ovine milk proteins by reversed-phase high-performance liquid chromatography and flow injection analysis with electrospray ionization mass spectrometry.

Ovine milk proteins were analyzed both by coupling HPLC and electrospray ionization mass spectrometry (ESI-MS) and by flow injection analysis and ESI-MS detection after separation and collection of fractions from gel permeation chromatography. These methods resolved the four ovine caseins and whey proteins and made it possible to study the complexity of these proteins associated with genetic polymorphism, post-translational changes (phosphorylation and glycosylation) and the presence of multiple forms of proteins. The experimental molecular masses of ewe milk proteins were: 19,373 for kappa-casein 3P; 25,616 for alpha(s2)-casein 10P; 23,411 for alpha(s1)-casein C-8P; 23,750 for beta-casein 5P; 18,170 and 18,148 for beta-lactoglobulins A and B; 14,152 for alpha-lactalbumin A and 66,322 for serum albumin.

Analysis of major caprine milk proteins by reverse-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry.

Major proteins from caprine milk were separated by preparative gel permeation and cation-exchange fast protein liquid chromatography and were characterized by flow injection analysis by electrospray ionization mass spectrometry. In addition, proteins from whole skim milk and whole casein were analyzed by coupling reverse-phase HPLC and electrospray ionization mass spectrometry by two different chromatographic methods. These methods successfully resolved the major caprine milk proteins and main casein variants. The experimental molecular masses of major milk proteins and variants were: 19,302 for kappa-CN 2P; 25,599 for alphas2-CN A-11P; 25,514 for alphas2-CN B-10P; 23,370 for alphas1-CN A-8P; 23,345 for alphas1-CN B-8P; 23,264 for alphas1-CN E-8P; 18,817 for alphas1-CN F-3P; 23,835 for beta-CN 6P; 18,181 for beta-LG; 14,180 for alpha-LA and 66,318 for serum albumin.

Proteolytic specificity of chymosin on caprine alpha s1-caseins A and F.

From hydrolysis experiments carried out on alpha s1-caseins A and F at pH 5.2 in the presence of 30 g NaCl/l, i.e. the conditions encountered in many young goats' cheeses, it was found that minima of 19 and 9 bonds were sensitive to chymosin in variants A and F respectively. Variant A was hydrolysed faster than variant F and the proteolytic pattern (reversed-phase HPLC and polyacrylamide agarose gel electrophoresis) differed between the variants. Hydrolysates from both variants had a number of cleavage sites in common (Leu20-Leu21, Phe23-Ala24 and Phe32-Arg33 in both variants, Leu101-Lys102 and Leu64-Lys65, Leu120-His121 and Leu83-His84, Leu142-Ala143 and Leu105-Ala106, Leu149-Phe150 and Leu112-Phe113, Leu156-Asp157 and Leu119-Asp120, Trp164-Tyr165 and Trp127-Tyr128 in variants A and F respectively), while other bonds were split only in variant A (Leu16-Asn17, Glu18-Asn19, Phe28-Pro29, Ile44-Gly45, Tyr80-Ile81, Gln82-Lys83, Tyr91-Leu92, Tyr94-Leu95, Leu109-Glu110 and Phe179-Ser180). Major cleavage sites appeared to be at Phe23-Val24, Leu142-Ala143 and Trp164-Tyr165 for variant A, and Phe23-Val24 and Leu64-Lys65 for variant F. Cleavage site Phe23-Val24 could be the origin of the first breakdown product from goat alpha s1-caseins A and F visible in polyacrylamide agarose gel electrophoresis.