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.

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.

Effect of UHPH on indigenous microbiota of apple juice: a preliminary study of microbial shelf-life.

The effect of ultra high pressure homogenisation (UHPH) at 100, 200 and 300 MPa on apple juice at two different inlet temperatures (4 and 20 degrees C) was studied. Raw and conventional heat treated (PA) apple juice was compared with UHPH treated juice after treatment and during 60 days of storage at 4 degrees C. The microbial quality was studied by enumerating aerobic mesophilic counts (AM), psychrotrophs (PS), moulds and yeasts (MY), lactobacilli (LB), enterobacteriaceae (EB), and faecal coliforms (FC). PA samples were below detection level (< or = -2 log cfu/mL) for all groups, the same as UHPH treated juices at 200 MPa and above but for around 1 log cfu/mL for AM. Those AM counts did not change during 60 days storage at 4 degrees C and proved to be spores. Juices treated at 100 MPa shown little reductions in microbial counts, and surviving microorganisms significantly increased their numbers during storage. This research work showed that non-thermal methods such a UHPH technology may give new opportunities to develop "fresh like" apple juice with a shelf-life equivalent to PA in terms of microbiological characteristics without affecting the product quality.

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

The effect of including citrus fruits (CF) in the rations of dairy ewes on the milk characteristics and biochemical changes of cheeses during ripening was evaluated. For this purpose, 48 lactating ewes (Guirra breed) were divided into 4 homogeneous groups and fed with isoenergetic and isoprotein rations containing CF at 0, 10, 20, and 30% on a dry matter basis in substitution of dry barley and pelleted beet pulp. During the experimental period, 3 batches of bulk milk were collected from each group and semi-hard cheeses were manufactured. Cheeses were sampled at 15, 30, and 60 d of ripening. Milk coagulation parameters and cheese yield were not negatively affected by the inclusion of CF in the ration. Physicochemical composition of cheeses at 60 d showed statistical differences for lower total solids and fat content of 30% CF cheeses. Proteolysis of cheeses measured by water-soluble nitrogen and total free amino acids content was not influenced by the ration. Differences between rations with respect to free fatty acids were significant for medium- and long-chain free fatty acids, and therefore for total content, but differences did not show a trend related to the increase of CF in the diet. The inclusion of CF in the ration of lactating ewes up to levels of 30% did not negatively affect the properties of milk and the biochemical and sensory characteristics of 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.

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 degrees 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 degrees C. In cheese produced without starter culture, Salmonella cells showed the ability to repair and grow during the storage period, reaching counts over 3 log(10) (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.

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 degrees C and the formation of staphylococcal enterotoxins were also assessed. Milk containing approximately 7.3 log(10) 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 degrees 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 degrees 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 degrees C. Counts of control cheeses not pressure-treated were approximately 8.5 log(10) cfu/g showing no significant decreases during storage. In cheeses made from UHPH treated milk at T(in) of 6 degrees C, counts of Staph. aureus were 5.0 +/- 0.3 log(10) cfu/g at d 1; they decreased significantly to 2.8 +/- 0.2 log(10) cfu/g on d 15, and were below the detection limit (1 log(10) 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 log(10) cfu/g from d 1. However, for both UHPH and UHPH+HHP cheeses in the 6 degrees C T(in) samples, viable Staph. aureus cells were still recovered. For samples of the 20 degrees 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.

Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria.

AIMS: To investigate the combined effect of high-pressure treatments (HPT) and milk inoculation with bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Staphylococcus aureus during ripening of raw milk cheese. METHODS AND RESULTS: Cheeses were manufactured from raw milk artificially contaminated with S. aureus at ca 5 log CFU ml(-1), a commercial starter culture and one of seven strains of BP-LAB, added as adjuncts at 0.1%. HPT of cheeses were performed on days 2 or 50 at 300 MPa (10 degrees C, 10 min) or 500 MPa (10 degrees C, 5 min). On day 3, S. aureus counts were 6.46 log CFU g(-1) in control cheese. Milk inoculation with different BP-LAB lowered S. aureus counts on day 3 when compared with control cheese by up to 0.46 log CFU g(-1), HPT at 300 MPa on day 2 by 0.45 log CFU g(-1) and HPT at 500 MPa on day 2 by 2.43 log CFU g(-1). Combinations of BP-LAB with HPT at 300 and 500 MPa on day 2 lowered S. aureus counts on day 3 by up to 1.02 and 4.00 log CFU g(-1) respectively. CONCLUSIONS: The combined effect of milk inoculation with some of the BP-LAB tested and HPT of cheese on S. aureus inactivation was synergistic. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of HPT at lower pressures with BP-LAB inoculation is a feasible system to improve cheese safety in case of deleterious effects on cheese quality caused by HPT at higher pressures.

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.

High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents.

High hydrostatic pressure inactivation of Escherichia coli, Pseudomonas fluorescens, Listeria innocua, Staphylococcus aureus, and Lactobacillus helveticus were studied. These microorganisms were inoculated at a concentration between 10(7) and 10(8) cfu/ml in Ringer solution and in ovine milk adjusted to 0, 6, and 50% fat content to evaluate the baroprotective effect of fat content on inactivation of microorganisms. Treatments of pressurization consisted of combinations of pressure (100 to 500 MPa) and temperature (4, 25, and 50 degrees C) for 15 min. Gram-negative microorganisms were more sensitive than were Gram-positive ones (more destruction P. fluorescens > E. coli > or = List. innocua > Lb. helveticus > S. aureus). Pressurizations at low temperature (4 degrees C) produced greater inactivation on P. fluorescens, List. innocua, and Lb. helveticus than at room temperature (25 degrees C), whereas for E. coli and S. aureus the results were opposite. Ovine milk per se (0% fat) showed a baroprotective effect on all microorganisms, but percentage of fat (6 and 50%) did not show a progressive baroprotective effect in all pressurization conditions or for all microorganisms.

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.

Mechanically recovered poultry meat sausages manufactured with high hydrostatic pressure.

The effect of high pressure processing at high temperature on texture and color of frankfurter-type sausages made with different contents of mechanically recovered poultry meat (MRPM) was evaluated and compared with that of a standard cooking process. Five types of sausages containing 100, 75, 50, 25, and 0% MRPM and 0, 25, 50, 75, and 100% of minced pork meat (MPM), respectively, were manufactured. They were pressurized at 500 MPa for 30 min at 50, 60, 70, and 75 C or cooked at 75 C for 30 min. Pressure-treated sausages were less springy and firm, but more cohesive. Moreover, color of pressurized sausages was lighter and more yellow than that of conventionally cooked sausages. Addition of MPM increased cohesiveness, hardness, and force at 80% compression. Minced pork meat also caused the appearance of sausages to be lighter, less red, and less yellow. Cooked sausages made with MRPM can have an attractive appearance and texture via high pressure processing.

Sensitivity of Staphylococcus aureus and Lactobacillus helveticus in ovine milk subjected to high hydrostatic pressure.

Ovine milk, standardized to 6% fat, was inoculated with Staphylococcus aureus CECT 534 and Lactobacillus helveticus CECT 414 at a concentration of 10(7) cfu/ml and treated by high hydrostatic pressure. Treatments consisted of combinations of pressure (200, 300, 400, 450, and 500 MPa), temperature (2, 10, 25, and 50 degrees C), and time (5, 10, and 15 min). Staphylococcus aureus was highly resistant to pressure; only pressurizations at 50 degrees C of 500 MPa for 15 min achieved reductions of > or = 7.3 log units. For L. helveticus, the number of surviving cells was reduced considerably at pressures of 400 MPa or more (up to 4.5 log units at 50 degrees C for 15 min), and pressure was more effective at low (2 and 10 degrees C) and moderately high (50 degrees C) temperatures than at room temperature (25 degrees C). Both species showed first-order kinetics of destruction in the range 0 to 60 min. The D values for S. aureus were 20 min (2 degrees C at 450 MPa) and 16.7 min (25 degrees C at 450 MPa), and D values for L. helveticus were 7.1 min (2 degrees C at 450 MPa) and 9.1 min (25 degrees C at 450 MPa). Lactobacillus helveticus showed higher rates of survival of pressure than those reported in previous studies for other Lactobacillus spp.

Combined effect of nisin and high hydrostatic pressure on destruction of Listeria innocua and Escherichia coli in liquid whole egg.

High hydrostatic pressure inactivation of Escherichia coli and Listeria innocua inoculated in liquid whole egg was improved significantly (P < 0.05) with nisin addition at concentrations of 1.25 and 5 mg/1. A reduction of almost 5 log10 units in E. coli counts and more than 6 log10 units for L. innocua was obtained at 450 MPa and 5 mg/l of nisin. For this treatment, the two microorganisms were not detectable after 1 month of storage at 4 degrees C. The amount of nisin added did not affect E. coli inactivation at 300 MPa. For L. innocua, 5 mg/l of nisin was more effective than 1.25 mg/l. Nisin showed no effect when samples were stored at 20 degrees C after pressurization, except for samples with L. innocua containing 5 mg/l of nisin and treated with 450 MPa.

Inactivation of Listeria innocua inoculated in liquid whole egg by high hydrostatic pressure.

The resistance of Listeria innocua, as a model microorganism for Listeria monocytogenes, to high hydrostatic pressure in liquid whole egg was studied at several pressures (300, 350, 400, and 450 MPa),temperatures (- 15, 2, and 20 degrees C), and times (5, 10, and 15 min). Listeria innocus was added to liquid whole egg at approximately 10(6) CFU/ml. Listeria innocua was not totally inactivated in any of the treatments. In general, reduction was better at 2 degrees than at room temperature, but the greatest inactivation was obtained at 450 MPa at 20 degrees C for 15 min (over 5 log of reduction), The results indicate that microbial inactivation was increased with prolonged exposure to pressure. D values for Listeria innocua were obtained at 400 MPa for two temperatures (2 and 20 degrees C), and different times (0 to 20 min). The microbial inactivation followed apparent first-order kinetics, exhibiting a decimal reduction time of 7.35 min at 2 degrees C and 8.23 min at 20 degrees C.