High-pressure homogenisation of raw bovine milk. Effects on fat globule size distribution and microbial inactivation

Whole raw milk was processed using a 15 L h⁻¹ homogeniser with a high-pressure (HP) valve immediately followed by a cooling heat exchanger. The influence of homogenisation pressure (100–300 MPa) and milk inlet temperature T_in (4°C, 14°C or 24°C) on milk temperature T₂ at the HP valve outlet, on fat globule size distribution and on the reduction of the endogenous flora were investigated. The T_in values of 4–24°C led to milk temperatures of 14–33°C before the HP valve, mainly because of compression heating. High T_in and/or homogenisation pressure decreased the fat globule size. At 200 MPa, the d_4.3 diameter of fat globules decreased from 3.8±0.2 (control milk) to 0.80±0.08 μm, 0.65±0.10 or 0.37±0.07 μm at T_in=4, 14°C or 24°C, respectively. A second homogenisation pass at 200 MPa (T_in=4°C, 14°C or 24°C) further decreased d_4.3 diameters to about 0.2 μm and narrowed the size distribution. At all T_in tested, an homogenisation pressure of 300 MPa induced clusters of fat globules, easily dissociated with SDS, and probably formed by sharing protein constituents adsorbed at the fat globule surface. The total endogenous flora of raw milk was reduced by more than 1 log cycle, provided homogenisation pressure was ⩾200 MPa at T_in=24°C (T₂∼60°C), 250 MPa at T_in=14°C (T₂∼62°C), or 300 MPa at T_in=4°C (T₂∼65°C). At all T_in tested, a second pass through the HP valve (200 MPa) doubled the inactivation ratio of the total flora. Microbial patterns of raw milk were also affected; Gram-negative bacteria were less resistant than Gram-positive bacteria.     

Effect of high pressure carbon dioxide on alkaline phosphatase activity and quality characteristics of raw bovine milk

Novel food processing techniques would always be pursuits of researchers and food industry to avoid unfavorable thermal effects, especially in dairy and milk processing. In this study, effects of high pressure carbon dioxide (HPCD) on the activity of alkaline phosphatase (ALP) and main quality indices of raw bovine milk at 20 MPa using a batch system were investigated. A complete inactivation of ALP activity as exposure to HPCD treatment at 50 °C and 20 MPa for 50 min was observed. The protein and lactose content of HPCD-treated bovine milk hold steady, while pH value and total solids content decreased, turbidity and average particle size increased significantly (p < 0.05). Although a significant decrease of viscosity (p < 0.05) was observed, the Newtonian flow behavior of raw bovine milk did not alter. More obvious change of quality characteristics of raw bovine milk were observed as subjected to HPCD treatment at higher temperature or treated for longer period. Therefore, a compromise between controlling endogenous enzymes (and/or spoilage and pathogenic microorganisms) and retention of original/fresh like quality of foods should be introduced due to the nature of HPCD processing. It's suggested to keep raw bovine milk with low ALP activity and great quality treated with a batch HPCD apparatus at 20 MPa and 50 °C for 20 min.     

Kinetics of thermal inactivation of alkaline phosphatase in bovine and caprine milk and buffer

The thermal inactivation of alkaline phosphatase (ALP) in raw bovine and caprine milk was investigated in the temperature range 54 to 69 °C. To assess the stabilizing effect of milk compounds on ALP, inactivation experiments were also carried out in 0.1 m potassium phosphate buffer, pH 6.6. Each set of inactivation experiments was fitted simultaneously using kinetic models that were based on either one-step or two-step mechanisms. The parameters of the Arrhenius equation showed that the stabilization effect of milk compounds on ALP had an entropic character. They also indicated a different structure of bovine and caprine milk ALPs, which was reflected by a higher stability of the bovine milk enzyme.     

Alkaline phosphatase and microbial inactivation by pulsed electric field in bovine milk

The effects of pulsed electric field (PEF) treatments at field intensities of 25–37 kV cm^− 1 and final PEF treatment temperatures of 15 °C and 60 °C on the inactivation of alkaline phosphatase (ALP), Total Plate Count (TPC), Pseudomonas and Enterobacteriaceae counts were determined in raw skim milk. At 15 °C, PEF treatments of 28 to 37 kV cm^− 1 resulted in 24–42% inactivation in ALP activity and < 1 log reduction in TPC and Pseudomonas count, while the Enterobacteriaceae count was reduced by at least 2.1 log units to below the detection limit of 1 CFU mL^− 1. PEF treatments of 25 to 35 kV cm^− 1 at 60 °C resulted in 29–67% inactivation in ALP activity and up to 2.4 log reduction in TPC, while the Pseudomonas and Enterobacteriaceae counts were reduced by at least 5.9 and 2.1 logs, respectively, to below the detection limit of 1 CFU mL^− 1. Kinetic studies suggested that the effect of field intensity on ALP inactivation at the final PEF treatment temperature of 60 °C was more than twice that at 15 °C. A combined effect was observed between the field intensity and temperature in the inactivation of both ALP enzyme and the natural microbial flora in raw skim milk.Industrial relevanceMilk has been pasteurised to ensure its safety and extend its shelf life. However, the need for retaining heat-sensitive nutrient and sensory properties of milk has resulted in interest in the application of alternative technologies. The results of the current study suggest that PEF as a non-thermal process can be employed for the treatment of raw milk in mild temperature to achieve adequate safety and shelf life while preserving the heat-sensitive enzymes, nutrients and bioactive compounds.     

Dephosphorylation of bovine casein by milk alkaline phosphatase.

The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.     

Enzymic differentiation between curds of heated and raw milk. I. milk alkaline phosphatase

Alkaline phosphatase, which is present in curds made from raw milk as well as from boiled milk, does not serve to differentiate them. It was found that incubation time or acidity (lactic acid equivalent of curd acidity incubated aseptically with heated milk) does not revive heat-denatured phosphatase. The reappearance was found to be due to phosphatase of microbial origin; it was not a case of reactivation of the denatured enzyme. Low intensity, but increasing slowly, of the activity of regenerated phosphatase in curd (permeability of the microbial cells to the testing reagents is probably limiting in the rate of reaction), presence of the enzyme in the sonicated cells of curd-forming microflora and absence of activity in the supernatant of the growth medium suggest that the formed enzyme is an intracellular one.     

High pressure homogenisation of raw whole bovine milk (a) effects on fat globule size and other properties

Although widely adopted by the chemical and pharmaceutical industries in recent years, little published data is available regarding possible applications of high pressure homogenisation for dairy products. The objective of this work was to compare the effects of conventional (18 MPa, two-stage) and single or two-stage high pressure homogenisation (HPH) at 50-200 MPa on some properties of raw whole bovine milk (approximately 4% fat). Fat globule size decreased as HPH pressure increased and, under certain conditions of temperature and pressure, HPH yielded significantly smaller fat globules than conventional homogenisation. Fat globule size was also affected by milk inlet temperature. The pH of all homogenised milk samples decreased during 24 h refrigerated storage. Total bacterial counts of milk were decreased significantly (P < 0.05) for milk samples HPH-treated at 150 or 200 MPa. Whiteness and rennet coagulation properties of milk were unaffected or enhanced, respectively, as homogenisation pressure was increased. Average casein micelle size decreased slightly when skim milk was homogenised at 200 MPa. Thus, HPH treatment has several, potentially significant, effects on milk properties.     

Effects of combination treatments of nisin and high-intensity ultrasound with high pressure on the microbial inactivation in liquid whole egg

The consecutive combinations of nisin with high hydrostatic pressure (nisin-HHP) and ultrasound with high hydrostatic pressure (US-HHP) were explored to achieve enhanced microbial inactivation in liquid whole egg processing. The HHP processing conditions were fixed to either 250 MPa for 886 s or 300 MPa for 200 s at the treatment temperature of 5 °C, which have been determined as the optimum HHP processing conditions considering egg protein coagulation and microbial inactivation kinetics. Between the two types of combinations, the nisin-HHP combination showed more promising results. The addition of nisin prior to pressure treatments significantly increased the lethal effects of HHP against Listeria seeligeri up to 5 log cycles. Because the individual effects of each nisin and HHP on the Listeria were almost negligible, the Listeria reductions are considered to be due to the synergistic action of nisin and HHP. However, the combination of nisin-HHP on E. coli showed exactly the same degree of inactivation by HHP alone, which supports protection mechanism of gram negative bacteria against the action of nisin. The US-HHP combination caused no reductions of Listeria and only a slightly increased inactivation of E. coli under the experimental conditions.     

Effects of high pressure treatment on indigenous enzymes in bovine milk: Reaction kinetics,inactivation and potential application

High pressure-induced inactivation of the indigenous milk enzymes alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and phosphohexoseisomerase (PHI) was studied in the pressure range 400–800 MPa at temperatures between 5 and 40 °C. With respect to pressure stability the following ranking was observed: ALP>GGT>PHI. PHI was inactivated after pressure treatment at 500 MPa and 20 °C for 10 min. In terms of reaction kinetics, inactivation of GGT followed first-order reaction kinetics in the range of 400–800 MPa whereas a reaction order of 1.5 was found for ALP. Reactivation of pressure-treated ALP was observed at low enzyme activity resulting from severe pressure treatment and 2 h storage at 35 °C. The influence of process temperature on the pressure-induced inactivation of GGT and ALP was limited in the range 5–40 °C.     

Significance of frictional heating for effects of high pressure homogenisation on milk

High pressure homogenisation (HPH) is a novel dairy processing tool, which has many effects on enzymes, microbes, fat globules and proteins in milk. The effects of HPH on milk are due to a combination of shear forces and frictional heating of the milk during processing; the relative importance of these different factors is unclear, and was the focus of this study. The effect of milk inlet temperature (in the range 10-50 degrees C) on residual plasmin, alkaline phosphatase, lactoperoxidase and lipase activities in raw whole bovine milk homogenised at 200 MPa was investigated. HPH caused significant heating of the milk; outlet temperature increased in a linear fashion (0.5887 degrees C/ degrees C, R2=0.9994) with increasing inlet temperature. As milk was held for 20 s at the final temperature before cooling, samples of the same milk were heated isothermally in glass capillary tubes for the same time/temperature combinations. Inactivation profiles of alkaline phosphatase in milk were similar for isothermal heating or HPH, indicating that loss of enzyme activity was due to heating alone. Loss of plasmin and lactoperoxidase activity in HPH milk, however, was greater than that in heated milk. Large differences in residual lipase activities in milks subjected to heating or HPH were observed due to the significant increase in lipase activity in homogenised milk. Denaturation of beta-lactoglobulin was more extensive following HPH than the equivalent heat treatment. Inactivation of plasmin was correlated with increasing fat/serum interfacial area but was not correlated with denaturation of beta-lactoglobulin. Thus, while some effects of HPH on milk are due to thermal effects alone, many are induced by the combination of forces and heating to which the milk is exposed during HPH.     

Effect of heat treatments and homogenisation pressure on the acid gelation properties of recombined whole milk

The homogenisation pressure and the order of heating and homogenisation were varied in the preparation of recombined whole milks. The fat globule sizes decreased from ∼2 to 0.2 μm as the homogenisation pressure increased from 50 to 850 bar for samples heated before (HEHO) or after (HOHE) homogenisation. For acid gels prepared from the milks, small increases in elastic modulus of the set gels were observed with decreasing fat globule size. The yield strain of the acid gels increased linearly with decreasing fat globule size, and HOHE gels had higher yield strains than HEHO gels. The yield stress of the set gels increased with decreasing fat globule size, and the yield stress for HOHE gels were higher than the HEHO gels. Confocal micrographs of the gels revealed an almost continuous protein network structure without pores for gels from milks treated at low homogenisation pressures, and the large fat globules did not actively interact with the strands in the gel network. In contrast, a porous protein network structure with distinct strands was observed for the samples treated at high homogenisation pressures, and the small fat globules were intimately involved in the strands in the network structure. The HOHE gels had a more porous protein network with thicker strands than the HEHO gels. The size of the fat globules and their incorporation in the protein network during acidification is proposed to affect the acid gel structure and properties.     

超高压对双孢蘑菇的杀菌效果和动力学的研究

以细菌总数、大肠菌群、酵母菌和霉菌数为对象,研究了超高压(High hydrostatic pressure,HHP)处理对双孢蘑菇(Agaricus bisporus)的杀菌效果和杀菌动力学。双孢蘑菇在300、400、500、600MPa压力下,室温下分别用HHP处理2.5～25min。结果表明:随压力的升高和时间的延长,杀菌效果增强;霉菌、酵母对压力较为敏感,400MPa处理2.5min可将其全部杀死;300MPa处理2.5min可完全杀灭双孢蘑菇中的大肠菌群。应用Weibull模型对不同处理条件下双孢蘑菇的杀菌效果进行拟合,拟合动力学曲线的决定系数R2均大于0.97,拟合效果较好。提出了双孢蘑菇的HHP杀菌的最优杀菌工艺参数,即600MPa处理5min,该条件即可以有效杀灭双孢蘑菇中的微生物。     

Effect of single and combined UV-C and ultra-high pressure homogenisation treatments on inactivation of Alicyclobacillus acidoterrestris spores in apple juice

Alicyclobacillus acidoterrestris is a spore-forming bacterium that can survive thermal pasteurization and acidic conditions. It produces changes in the odour and flavour of fruit juices leading to economical loses. A. acidoterrestris CECT 7094 spores were inoculated in clarified and cloudy apple juices (Golden delicious var.) in the range of 5–6 log₁₀ spores/mL and submitted to different short-wave ultraviolet light (UV-C) doses (7.2–28.7 J/mL) and ultra-high pressure homogenisation (UHPH) treatments (100–300 MPa), including their combination. A. acidoterrestris could be inactivated in clarified apple juice at a level of 4.8 log₁₀ CFU/mL by a 300 MPa-UHPH treatment when the inlet temperature was 80 °C. UV-C treatments showed to be more efficient achieving a lethality of 5.5 log₁₀ CFU/mL with a dose of 21.5 J/mL at 20 °C. In cloudy apple juice (2357 NTU) UV-C treatments were less efficient with a maximum lethality of 4.07 CFU/mL after a dose of 28.7 J/mL. A previous application of UHPH contributed with UV-C to obtain higher reductions of A. acidoterrestris spores at the doses of 14.3 and 21.5 J/mL compared with UV-C single treatments. On the other hand, this previous treatment also changed the properties of particles in the matrix which apparently reduced the effectiveness of UV-C at 28.7 J/mL.     

Effects of high‐pressure homogenisation on physicochemical characteristics of partially skimmed milk

The changes in partially skimmed milk (0.5% fat) physicochemical properties and proteins after high‐pressure homogenisation (HPH) at 100, 200 and 300 MPa were investigated. Processing parameters and changes in pH, ethanol precipitation stability, lightness, whey protein denaturation, hydrophobicity and viscosity were evaluated. No significant differences were found between milk pH and nonprotein nitrogen content before and after HPH. Ethanol stability, lightness and hydrophobicity increased when pressure was increased from 100 MPa to 300 MPa. Whey protein denaturation, evaluated through noncasein nitrogen, occurred only at 200 to 300 MPa, and viscosity increased just at 300 MPa. Therefore, HPH changed some milk physicochemical characteristics, mainly those related to protein content. These results highlight that HPH processing is a promising technology to improve partially skimmed milk mouth feel being suitable for dairy products manufacturing.     

Kinetics of alkaline phosphatase and lactoperoxidase inactivation,and of beta-lactoglobulin denaturation in milk with different fat content

In the context of identifying intrinsic time temperature integrators (TTIs) for evaluating heat processing of milk, the extent to which milk fat content has an effect on alkaline phosphatase (ALP) and lactoperoxidase (Lpo) inactivation and on beta-lactoglobulin (beta-Ig) denaturation kinetics was studied. Inactivation and denaturation kinetics were analysed in whole, semi-skimmed and skimmed milk. In previous experiments (isothermal and non-isothermal heating conditions), heat inactivation of ALP and Lpo and heat denaturation of beta-Ig were found to follow first order kinetics. This allowed experimental design to be simplified. Data analysis was performed by non-linear regression and results were evaluated by construction of joint confidence regions. The possible effect of milk fat was illustrated by temperature time tolerance (TTT-) diagrams. Although initial ALP activity was lower in skimmed milk compared with semi-skimmed or whole milk, kinetics were comparable and fat content did not seem to substantially affect the ALP test result for pasteurized milk. Unlike ALP, Lpo inactivation and beta-Ig denaturation kinetics differed significantly in milk with different fat content. Differences between Lpo inactivation kinetics were relatively small and acceptable in the context of quantifying the process impact. Denaturation of beta-Ig, on the other hand, seemed to be enhanced at higher milk fat content (> 72 degrees C).     

The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure

Camembert-type cheese was produced from: raw bovine milk; raw milk inoculated with 2 or 4 log CFU/ml Listeria monocytogenes; raw milk inoculated with L. monocytogenes and subsequently pressure-treated at 500 MPa for 10 min at 20 °C; or uninoculated raw milk pressure-treated under these conditions. Cheeses produced from both pressure-treated milk and untreated milk had the typical composition, appearance and aroma of Camembert. Curd and cheese made from inoculated, untreated milk contained large numbers of L. monocytogenes throughout production. An initial inoculum of 1.95 log CFU/ml in milk increased to 4.52 log CFU/g in the curd and remained at a high level during ripening, with 3.85 log CFU/g in the final cheese. Pressure treatment inactivated L. monocytogenes in the raw milk at both inoculum levels and the pathogen was not detected in any of the final cheeses produced from pressure-treated milk. Therefore high pressure may be useful to inactivate L. monocytogenes in raw milk that is to be used for the production of soft, mould-ripened cheese.

Industrial relevance

This paper demonstrates the potential of high pressure (HP) for treatment of raw milk to be used in the manufacture of soft cheeses. HP treatment significantly reduced the level of Listeria monocytogenes in the raw milk and so allowed the production of safer non-thermally processed camembert-like soft cheese.     

压致升温及其对超高压下微生物失活的影响

压力与温度的结合能有效地杀灭芽孢杆菌的芽孢。在绝热情况下,压致升温对芽孢的杀灭有很大影响。本文主要阐述了压力处理过程中介质与食品的压致升温情况,同时由于不同的绝热升温产生的热传递造成高压容器内的温度不均一。因此,在压力处理过程中,控制热效应对芽孢杀灭的效果起重要作用。     

Influence of ultra-high pressure homogenisation on physicochemical and sensorial properties of orange juice in comparison with conventional thermal processing

The effect of different ultra-high pressure homogenisation (UHPH) treatments on physicochemical and sensorial properties of orange juice was studied in comparison with thermal pasteurisation (90 °C, 1 min). UHPH treatments consisted on combinations of two inlet temperatures (10 or 20 °C) and three pressures (100, 200 and 300 MPa). Effect of treatments was assessed on general quality parameters (colour, pH, °Brix, titratable acidity, reducing sugars and non-enzymatic browning index), particle size distribution and cloud stability. None of the UHPH treatments caused significant differences in the °Brix, reducing sugars, pH and non-enzymatic browning index with respect of fresh or pasteurised juice. Only titratable acidity was significantly lower when inlet temperature of UHPH treatments was 20 °C. UHPH treatments significantly reduced the particle size and in consequence the cloudiness and the total colour value (∆E*) increased. The overall consumer acceptability of UHPH and pasteurised juices was similar.     

Changes in the surface protein of the fat globules during ultra-high pressure homogenisation and conventional treatments of milk

Disruption of fat globules upon homogenisation provokes a reduction of their size and a concomitant increase in their specific surface area. In order to overcome this phenomenon, the milk fat globule membrane (MFGM) adsorbs non-native MFGM proteins. The aim of the present study was to examine the effects of UHPH conditions (temperature and pressure) on the milk fat globule and the surface proteins by comparison with conventional treatments applied in the dairy industry. Transmission electron microscopy and SDS-PAGE revealed major differences. In UHPH-treated milk, casein micelles were found to be adsorbed on the MFGM in a lesser extent than in conventional homogenisation–pasteurisation. However, greater adsorption of directly bonded casein molecules, released by UHPH through the partial disruption of casein micelles, was observed especially at high UHPH pressures. Adsorption of whey proteins on the MFGM of conventionally homogenised–pasteurised milk was mainly through intermolecular disulfide bonds with MFGM material, whereas in UHPH-treated milk, disulfide bonding with both indirectly and directly adsorbed caseins was also involved.     

Combined thermal and high pressure inactivation kinetics of tomato lipoxygenase

The combined isothermal (10–60 °C) and isobaric (0.1–650 MPa) inactivation kinetics of lipoxygenase (LOX) extracted from tomatoes and reconstituted in a tomato purée were studied. Thermal inactivation of LOX at atmospheric pressure proceeded in the temperature range of 45–65 °C. LOX inactivation did not follow first order kinetics; the data could be fitted assuming that the two isoforms of LOX with different thermostability were present. Combined thermal and high pressure inactivation occurs at pressures in the range of 100–650 MPa combined with temperatures from 10–60 °C, and followed first-order kinetics. In the high-temperature/low-pressure range, (T≥50 °C and P≤300 MPa) an antagonistic effect is observed, therefore, the Arrhenius and Eyring equation cannot be used over the entire temperature and pressure range. Small temperature dependence is found in the low-temperature/high pressure range. A third degree polynomial model was successfully applied to describe the temperature–pressure dependence of the inactivation rate constants, which can be useful to predict inactivation rate constants of tomato LOX reconstituted in tomato purée in the temperature–pressure range studied.