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Effects of pressurized thermal processing on native proteins of raw skim milk and its concentrate

Open ArchivePublished:January 14, 2021DOI:https://doi.org/10.3168/jds.2020-19542

      ABSTRACT

      Heating, pressurization, and shearing can modify native milk proteins. The effects of pressurized heating (0.5 vs. 10 MPa at 75 or 95°C) with shearing (1,000 s−1) on proteins of raw bovine skim milk (SM, ∼9% total solids) and concentrated raw skim milk (CSM, ∼22% total solids) was investigated. The effects of evaporative concentration at 55°C and pressurized shearing (10 MPa, 1,000 s−1) at 20°C were also examined. Evaporative concentration of SM resulted in destabilization of casein micelles and dissociation of αS1- and β-casein, rendering CSM prone to further reactions. Treatment at 10 MPa and 1,000 s−1 at 20°C caused substantial dissociation of αS1- and β-casein in SM and CSM, with some dissociated caseins forming shear-induced soluble aggregates in CSM. The pressure applied at 10 MPa induced compression of the micelles and their dissociation in SM and CSM at 75 or 95°C, resulting in reduction of the micelle size. However, 10 MPa did not alter the mineral balance or whey proteins denaturation largely, except by reduction of some β-sheets and α-helices, due to heat-induced conformational changes at 75 and 95°C.

      Key words

      INTRODUCTION

      Heat treatments are commonly applied to raw bovine milk in the dairy industry to achieve microbial safety and to extend shelf life (e.g., pasteurization and sterilization) or as a preparatory step to enhance the functional properties of some dairy products (e.g., cheese and yogurt). Heating of milk, mostly above 70°C, can result in physicochemical changes to proteins, predominantly denaturation and aggregation, depending on temperature and time combination. Unlike caseins, whey proteins are relatively heat-labile. Being the most abundant whey protein, β-LG usually leads heat-induced protein denaturation and subsequent aggregation with other whey proteins, such as α-LA, BSA, immunoglobulins, and lactoferrin (LF), as well as the caseins, mainly κ-CN, via thiol, disulfide, electrostatic, and hydrophobic interactions (
      • Patel H.A.
      • Singh H.
      • Anema S.G.
      • Creamer L.K.
      Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk.
      ;
      • Wijayanti H.B.
      • Bansal N.
      • Deeth H.C.
      Stability of whey proteins during thermal processing: A review.
      ;
      • Bogahawaththa D.
      • Chandrapala J.
      • Vasiljevic T.
      Thermal denaturation of bovine β-lactoglobulin in different protein mixtures in relation to antigenicity.
      ).
      Concentration of milk by removal of water is an essential step of producing milk powders and other milk concentrates (e.g., evaporated milk and condensed milk), which leads to dissociation of the casein micelles into the serum phase, modification of mineral distribution, conformational changes of whey proteins, and association of β-LG with the casein micelles (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ). These modifications can lead to further destabilization of the casein micelles and conformational changes of β-LG during heating, resulting in compromised heat stability of proteins, depending on the concentration level and combination of temperature and time (
      • Huppertz T.
      Heat stability of milk.
      ;
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      Structural changes of milk proteins during heating of concentrated skim milk determined using FTIR.
      ). The milk can also be subjected to many mechanical forces, including shear, at various steps during commercial thermal processing (e.g., mixing, stirring, pumping, flowing through pipes, and spraying), which can induce whey protein denaturation, influence protein aggregation, and modify the casein micelles (
      • Mediwaththe A.
      • Bogahawaththa D.
      • Grewal M.K.
      • Chandrapala J.
      • Vasiljevic T.
      Structural changes of native milk proteins subjected to controlled shearing and heating.
      ;
      • Bogahawaththa D.
      • Vasiljevic T.
      Shearing accelerates denaturation of β-lactoglobulin and α-lactalbumin in skim milk during heating.
      ).
      Application of high hydrostatic pressure (HHP), especially at higher levels (100–1,000 MPa), has been recognized as a promising nonthermal method for pasteurization and sterilization of dairy products and for modification of the functional properties of milk proteins. However, HHP treatments can result in denaturation of whey proteins and their interaction with other proteins to a varying degree, depending on protein type and the combination of pressure and holding time. α-LA is the most pressure-stable (>400 MPa at 40°C for 30 min), and β-LG appears to be the most pressure-sensitive (>100 MPa at 4°C for 30 min;
      • Huppertz T.
      • Fox P.F.
      • Kelly A.L.
      High pressure treatment of bovine milk: Effects on casein micelles and whey proteins.
      ). β-LG tends to form aggregates mainly with κ-CN at ≥600 MPa (
      • Huppertz T.
      • Fox P.F.
      • Kelly A.L.
      High pressure treatment of bovine milk: Effects on casein micelles and whey proteins.
      ;
      • Patel H.A.
      • Singh H.
      • Anema S.G.
      • Creamer L.K.
      Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk.
      ;
      • Bogahawaththa D.
      • Buckow R.
      • Chandrapala J.
      • Vasiljevic T.
      Comparison between thermal pasteurization and high pressure processing of bovine skim milk in relation to denaturation and immunogenicity of native milk proteins.
      ). Furthermore, HHP treatments at >200 MPa can modify the casein micelle size. For instance, the micelle size increased (∼25%) reversibly at 250 MPa but decreased (∼50%) irreversibly at ≥300 MPa in raw skim milk (SM;
      • Huppertz T.
      • Fox P.F.
      • Kelly A.L.
      High pressure treatment of bovine milk: Effects on casein micelles and whey proteins.
      ). Pressurization (HHP) of raw SM at the level of 100 to 600 MPa caused the increase of αS1- and β-CN in the serum phase due to weakening of hydrophobic interactions and solubilization of colloidal calcium phosphate (CCP;
      • Huppertz T.
      • Fox P.F.
      • Kelly A.L.
      Dissociation of caseins in high pressure-treated bovine milk.
      ).
      The application of pressure (>100 MPa) combined with moderate temperature (40–70°C) can modify the physicochemical properties of milk proteins differently than a pressure treatment at ≤30°C. For instance, pressurization at 200 to 800 MPa with 70°C resulted in significantly greater denaturation of β-LG and α-LA and larger particle size in reconstituted SM compared with to the corresponding pressure treatments performed at 20°C (
      • Anema S.G.
      Heat and/or high-pressure treatment of skim milk: Changes to the casein micelle size, whey proteins and the acid gelation properties of the milk.
      ). When SM was pressurized at 100 to 300 MPa at different temperature levels (25–60°C), the increase in temperature resulted in gradual increase in denaturation of β-LG at the respective pressure levels (
      • López-Fandiño R.
      • Olano A.
      Effects of high pressures combined with moderate temperatures on the rennet coagulation properties of milk.
      ). When raising the temperature from 10 to 40°C at 200 MPa, the particle size of SM increased due to protein aggregation, whereas at ≥400 MPa the degree of disintegration of the casein micelle increased (
      • Anema S.G.
      • Lowe E.K.
      • Stockmann R.
      Particle size changes and casein solubilisation in high-pressure-treated skim milk.
      ).
      According to the existing literature (
      • Anema S.G.
      Heat and/or high-pressure treatment of skim milk: Changes to the casein micelle size, whey proteins and the acid gelation properties of the milk.
      ;
      • Huppertz T.
      • Vasiljevic T.
      • Zisu B.
      • Deeth H.C.
      Novel processing technologies: Effects on whey protein structure and functionality.
      ;
      • Nunes L.
      • Tavares G.M.
      Thermal treatments and emerging technologies: Impacts on the structure and techno-functional properties of milk proteins.
      ;
      • Wijayanti H.B.
      • Brodkorb A.
      • Hogan S.A.
      • Murphy E.G.
      Thermal denaturation, aggregation, and methods of prevention.
      ), it appears that no scientific studies have investigated the effects of low pressure (≤10 MPa) combined with heating (>70°C) and shearing on native and concentrated milk proteins. However, such combinations are frequently applied in milk processing, especially heating the milk at >100°C and milk homogenization (55–80°C and 10–25 MPa;
      • Bylund G.
      Dairy Processing Handbook.
      ). For instance, UHT treatment (138–145°C for 3–5 s) is performed under pressurized conditions (∼0.4 MPa;
      • Deeth H.C.
      • Datta N.
      Heat treatment of milk: Ultra-high temperature treatment (UHT): Heating systems.
      ). Hence, the current study aimed to examine the effects of pressurized (10 vs. 0.5 MPa) heat treatments (75 or 95°C) with a constant shearing (1,000 s−1) on physicochemical and structural changes to native proteins of raw SM and its concentrate. Findings of this work will help in the development or optimization of thermal processing parameters to achieve desired product characteristics.

      MATERIALS AND METHODS

      Sample Preparation

      Murray Goulburn Cooperative Co. Ltd. (Laverton North, VIC, Australia) kindly provided raw bovine milk. Upon delivery, the milk was skimmed by centrifugation, and sodium azide at 0.01% (wt/wt) was added to control potential microbial activities (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ). The standard oven drying method (105°C) was used to determine TS content of the resultant SM, which was 9 ± 0.3% (wt/wt). The SM was then divided into 2 portions, one of which was concentrated by evaporation at 55°C for about 100 min using an R-100 rotary evaporator (John Morris Scientific, Deepdene, VIC, Australia) as described previously (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ) to obtain skim milk with 22 ± 0.2% (wt/wt) TS, which was termed concentrated skim milk (CSM).

      Sample Treatment

      Both SM and CSM samples were heated to 2 temperature levels (75 or 95°C) at a ∼5°C/min heating and cooling rate under 2 low-pressure conditions (0.5 or 10 MPa) using a cup-and-bob geometry (CC 25/PR-SN; Anton Paar, Ostfildern, Germany) placed in a pressure cell (CC25/PR-150; Anton Paar) mounted on a Physica MCR 301 rheometer (Anton Paar), as explained previously (
      • Mediwaththe A.
      • Bogahawaththa D.
      • Grewal M.K.
      • Chandrapala J.
      • Vasiljevic T.
      Structural changes of native milk proteins subjected to controlled shearing and heating.
      ). The required pressure levels were generated and maintained using a compressed air system linked to the pressure cell and monitored by an associated software (Rheoplus, Anton Paar). A constant shear at 1,000 s−1 was applied with all the treatments, and the cooling process was terminated at room temperature (∼20°C). Another aliquot of SM and CSM was pressurized to 10 MPa at 20°C with constant shearing, using the same system, to examine the combined effects of pressure and shearing. Total treatment times (heating and cooling or holding) at 95, 75, and 20°C were ∼30, 22, and 20 min, respectively. Untreated samples of SM and CSM at ∼20°C were considered the controls. After the treatments, an aliquot of all the treated and the control samples was ultra-centrifuged at 100,000 × g for 1 h at 20°C using a Beckman Ultra L-70 (Beckman Coulter Pty Ltd., Lane Cove West, NSW, Australia) to separate the supernatant (serum phase) from the pellet, keeping the other aliquot as the intact milk (termed “bulk milk”) before analysis.

      Particle Size and Zeta Potential Measurement

      Average particle size and zeta potential of all the treated and control milk samples (bulk milk) was measured at 20°C using a Nano-ZS Zetasizer (Malvern Instruments, Malvern, UK) after diluting them in a simulated milk ultrafiltrate as reported previously (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ). Refractive indices of the casein micelle in milk and the simulated milk ultrafiltrate were set at 1.57 and 1.34, respectively.

      Mineral Content Determination

      Calcium, magnesium, and phosphorus content in all the treated and control milk samples and their serum phases were determined using an ICP Multitype inductively coupled plasma atomic emission spectrometer (Shimadzu Corporation, Kyoto, Japan) as described previously (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ). All the samples were ashed and then dissolved in 1 M nitric acid before determining the mineral content.

      Polyacrylamide Gel Electrophoresis

      The SDS-PAGE analysis was performed under nonreducing and reducing (using β-mercaptoethanol) conditions for all the treated and control milk samples and their serum phase following the method explained previously (
      • Bogahawaththa D.
      • Chandrapala J.
      • Vasiljevic T.
      Thermal denaturation of bovine immunoglobulin G and its association with other whey proteins.
      ). Gels containing 30% acrylamide and 10% SDS were used for electrophoresis, and protein bands were stained with Coomassie Brilliant Blue (Sigma-Aldrich Pty Ltd., Castle Hill, NSW, Australia). The gel images were captured using the ChemiDoc Imaging System (Bio-Rad Laboratories, Gladesville, NSW, Australia).

      Fourier-Transform Infrared Spectroscopy

      All the milk samples (treated and control) were scanned using a Frontier Fourier-transform infrared spectrometer (PerkinElmer, Waltham, MA). Every spectrum was an average of 16 scans at 4 cm−1 resolution in absorbance mode with background (water) subtraction. Changes of secondary structure of the proteins were analyzed using second derivative form of all the spectra within broad amide I region (1700–1,600 cm−1) by Spectragryph software (v.1.2.7;
      • Bogahawaththa D.
      • Chandrapala J.
      • Vasiljevic T.
      Thermal denaturation of bovine immunoglobulin G and its association with other whey proteins.
      ).

      Statistical Analysis

      The entire experiment was replicated with raw milk obtained on 2 different occasions (n ≥ 6). Data were analyzed by ANOVA and Tukey test, considering the level of significance at P ≤ 0.05, using SAS statistical software (v. 9.2; SAS Institute Inc., Cary, NC).

      RESULTS AND DISCUSSION

      Effect of Evaporative Concentration on Milk Proteins

      To investigate the effects of the evaporative concentration process on milk proteins, a comparison was made between the control (untreated) sample of SM (∼9% wt/wt of TS) and the control (without further processing) sample of CSM (∼22% wt/wt of TS). Particle size of the control CSM significantly increased (Table 1) compared with the control SM. This can be mainly related to dense packing of the casein micelles during the evaporative concentration and subsequently enhanced protein interactions, especially association of β-LG with the casein micelles (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ). The temperature (55°C) applied during the evaporative concentration was higher than that (30°C) required to dissociate β-LG dimers into monomers and thereby expose hydrophobic sites, leading to increased protein interactions (
      • Wijayanti H.B.
      • Bansal N.
      • Deeth H.C.
      Stability of whey proteins during thermal processing: A review.
      ;
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ). Furthermore, a substantial release of αS1- and β-CN into the serum phase, as observed in the nonreducing SDS-PAGE results (Figure 1, B1 and D1), indicated a considerable dissociation of the casein micelles during the concentration step. This can be attributed to solubilization of CCP as well as to weakening of electrostatic interactions and hydrogen bonding, leading to destabilization of the casein micelles (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ).
      • Anema S.G.
      • Klostermeyer H.
      Heat-induced, pH-dependent dissociation of casein micelles on heating reconstituted skim milk at temperatures below 100°C.
      reported that ∼7% of β-CN and ∼5% of αS-CN were in the serum phase of reconstituted SM (10% wt/wt of TS) after heating at 50 to 60°C for 15 min, and this temperature level had the greatest effect on dissociation of the caseins into the serum phase within the 20 to 100°C range. Hence, the 55°C applied during the concentration phase also contributed to the dissociation of αS1- and β-CN in the current study.
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      reported similar results to the current study: for instance, increase in size of the casein micelle, increase in the soluble caseins in the serum phase, and related destabilization of the casein micelles when raw SM (9% wt/wt of TS) was evaporatively concentrated into 17 or 25% wt/wt of TS using the equivalent conditions.
      Table 1The average particle size and zeta potential of skim milk and concentrated skim milk samples subjected to different pressurized thermal treatments
      SampleTemperature (°C)Pressure (MPa)Particle size (nm)Zeta potential (mV)
      Skim milk (~9% wt/wt TS)Control (~20)NA
      NA = not applicable.
      178.9 ± 1.7
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −18.3 ± 1.0
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      2010179.7 ± 2.6
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −17.7 ± 0.3
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      750.5177.3 ± 1.9
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −17.9 ± 2.1
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      7510173.3 ± 2.2
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −18.8 ± 0.8
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      950.5191.2 ± 4.1
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −18.4 ± 0.5
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      9510184.6 ± 1.4
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −16.4 ± 0.7
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      Concentrated skim milk (~22% wt/wt TS)Control (~20)NA186.6 ± 2.3
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −17.8 ± 1.9
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      2010187.9 ± 5.8
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −17.0 ± 0.7
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      750.5188.1 ± 1.5
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −16.9 ± 0.5
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      7510179.2 ± 1.6
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −17.3 ± 1.1
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      950.5195.9 ± 1.2
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −17.0 ± 0.5
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      9510190.1 ± 1.5
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      −15.9 ± 1.6
      Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      a–e Mean values without a common superscript letter in the same column indicate significant difference (P ≤ 0.05).
      1 NA = not applicable.
      Figure thumbnail gr1
      Figure 1Sodium dodecyl sulfate-PAGE images of skim milk (SM) and concentrated skim milk (CSM) samples (bulk milk) subjected to different pressurized thermal treatments and their supernatants (serum phase) obtained via ultracentrifugation after treatments. A1 and C1 are the SM and CSM bulk samples, and B1 and D1 are their serum phases, respectively, under nonreducing (NR) conditions. A2, B2, C2, and D2 are the corresponding reducing (R) images. Lanes (L) are as follows: L1 = molecular weight marker, L2 = control, L3 = 10 MPa at 20°C, L4 = 0.5 MPa at 75°C, L5 = 10 MPa at 75°C, L6 = 0.5 MPa at 95°C, and L7 = 10 MPa at 95°C. Protein bands are β-LG, α-LA, IgG, lactoferrin (LF), BSA, αS1-CN, αS2-CN, β-CN, and κ-CN.
      Some structural changes of the protein secondary structures were also observed via Fourier-transform infrared spectrometry (Figure 2) upon concentration. For instance, a slight increase in antiparallel β-sheets (∼1638–1,632 cm−1), aggregated β-sheets (∼1690–1,685 cm−1), and α-helices (∼1653–1,651 cm−1) (
      • Bogahawaththa D.
      • Chandrapala J.
      • Vasiljevic T.
      Thermal denaturation of bovine β-lactoglobulin in different protein mixtures in relation to antigenicity.
      ;
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ) was observed in the control CSM compared with the control SM (Figure 2A). These changes can be related to increased protein concentration and, thereby, close molecular packing (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ). We detected no significant changes of the final mineral balance when the soluble minerals of SM (Table 2: Ca 12.1, Mg 3.6, and P 13.6 mM) were compared with concentration-normalized soluble minerals of CSM (Ca 13.5, Mg 3.4, and P 14.5 mM). A slight increase in Ca and P in the serum phase of the control CSM (concentration normalized), compared with the control SM, indicated solubilization of CCP to some extent, which was, however, not prominent, due to movement of Ca and P into the micellar phase during the concentration phase (
      • Nieuwenhuijse J.
      • Timmermans W.
      • Walstra P.
      Calcium and phosphate partitions during the manufacture of sterilized concentrated milk and their relations to the heat stability.
      ). The zeta potential did not change (P > 0.05) after the concentration step (Table 1), as reported previously (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ).
      Figure thumbnail gr2
      Figure 2Second derivative of the Fourier-transform infrared spectra obtained from skim milk (SM) and concentrated skim milk (CSM) samples subjected to the different pressurized thermal treatments and their untreated controls (Control). (A) Control and samples of SM and CSM treated at 20°C and 10 MPa; (B) SM samples treated at 0.5 or 10 MPa and at 75 or 95°C; (C) CSM samples treated at 0.5 or 10 MPa and at 75 or 95°C.
      Table 2The total and soluble mineral concentration of skim milk and concentrated skim milk samples subjected to different pressurized thermal treatments
      SampleTemperature (°C)Pressure (MPa)Mineral concentration (mM)
      CaMgP
      Skim milk (~9% wt/wt total solids)Control (~20)NA
      NA = not applicable.
      Total minerals35.2 ± 2.25.2 ± 0.230.5 ± 1.4
      Control (~20)NASoluble minerals12.1 ± 0.2
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      3.6 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      13.6 ± 0.3
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      201010.6 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      3.0 ± 0.0
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      11.8 ± 0.0
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      750.510.0 ± 0.3
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      3.1 ± 0.0
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      11.7 ± 0.3
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      751010.1 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      2.9 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      11.6 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      950.510.0 ± 0.5
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      3.4 ± 0.2
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      12.0 ± 0.5
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      95109.2 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      2.8 ± 0.0
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      10.9 ± 0.2
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      Concentrated skim milk (~22% wt/wt total solids)Control (~20)NATotal minerals81.8 ± 1.612.3 ± 0.272.6 ± 1.8
      Control (~20)NASoluble minerals33.1 ± 1.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      8.3 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      35.4 ± 0.8
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      201029.5 ± 0.8
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      8.1 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      32.4 ± 0.7
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      750.530.5 ± 0.9
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      8.4 ± 0.3
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      33.3 ± 0.4
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      751030.3 ± 0.6
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      8.4 ± 0.0
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      35.2 ± 0.4
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      950.522.9 ± 1.2
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      7.9 ± 0.2
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      28.8 ± 0.7
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      951021.8 ± 0.7
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      7.5 ± 0.1
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      29.1 ± 0.3
      The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      a–f The mean values of the same soluble mineral without a common superscript letter indicate significant difference (P ≤ 0.05).
      1 NA = not applicable.

      Influence of Pressurized Shearing at 20°C on Milk Proteins

      Any changes of the proteins that can be observed between the control (∼20°C) and samples of SM or CSM treated at 10 MPa at 20°C with 1,000 s−1 can be related to the combined effect of pressure (10 MPa) and shearing (1,000 s−1). We detected no significant change (only a slight increase) of the particle size of SM or CSM following the 10-MPa, 20°C treatment (Table 1). From the SDS-PAGE nonreducing image (Figure 1, B1), slightly intense αS1- and β-CN bands appeared in the serum phase of the SM treated at 10 MPa and 20°C compared with those of the control, whereas the whey protein bands did not change. This indicated that 10 MPa and 20°C under shear treatment contributed to destabilization of the casein micelles slightly, which resulted in dissociation of the caseins into the serum phase without altering the micelle size considerably. Because the low pressure applied (10 MPa) at 20°C is unlikely to change the micellar structure (
      • Huppertz T.
      • Fox P.F.
      • Kelly A.L.
      Dissociation of caseins in high pressure-treated bovine milk.
      ;
      • Anema S.G.
      • Lowe E.K.
      • Stockmann R.
      Particle size changes and casein solubilisation in high-pressure-treated skim milk.
      ), destabilization of the casein micelles can be ascribed to the applied shear or the combination of shear and pressure. Reversible destabilization of the casein micelle, leading to increase in its size, was reported when raw SM was sheared at 1,000 s−1 and 20°C in an equivalent experimental setting, due to fluid grads created by shearing in the flow direction (
      • Mediwaththe A.
      • Bogahawaththa D.
      • Grewal M.K.
      • Chandrapala J.
      • Vasiljevic T.
      Structural changes of native milk proteins subjected to controlled shearing and heating.
      ).
      In contrast, very faint αS1- and β-CN bands were observed from the serum phase of CSM treated at 10 MPa and 20°C under nonreducing SDS-PAGE (Figure 1, D1) compared with those of the control. The intensity of these bands, however, was mostly similar under reducing SDS-PAGE conditions (Figure 1, D2). This suggested that the dissociated caseins were involved in formation of soluble aggregates potentially induced by shearing. Apart from the caseins, it appeared that some whey proteins, especially minor whey proteins (IgG, LF, and BSA), were also involved in this aggregation process, because they displayed relatively more intense bands, as indicated by the reducing SDS-PAGE of the CSM treated at 10 MPa and 20°C, compared with that of the control (Figure 1, D2). However, the participation of β-LG and α-LA in the protein aggregation was not obvious under these treatment conditions. The denser molecular packing, particularly of larger molecules such as minor whey proteins (IgG, LF, and BSA), and deterioration of protein stability take place during the concentration step (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      FTIR analysis of physiochemical changes in raw skim milk upon concentration.
      ). In addition to this, shear-induced structural modifications (
      • Mediwaththe A.
      • Bogahawaththa D.
      • Grewal M.K.
      • Chandrapala J.
      • Vasiljevic T.
      Structural changes of native milk proteins subjected to controlled shearing and heating.
      ,
      • Mediwaththe A.
      • Chandrapala J.
      • Vasiljevic T.
      Shear-induced behaviour of native milk proteins heated at temperatures above 80°C.
      ;
      • Bogahawaththa D.
      • Vasiljevic T.
      Shearing accelerates denaturation of β-lactoglobulin and α-lactalbumin in skim milk during heating.
      ) can create an environment to induce inter- and intraprotein interactions, leading to their aggregation. Pressurization and shearing at 20°C did not change the secondary structure of the proteins in SM or CSM substantially, probably due to least physicochemical changes in major whey proteins (β-LG and α-LA), as discussed above. However, some reduction of the antiparallel β-sheets and α-helices was observed that can be related to the dissociation of β-LG dimers into monomeric form (
      • Lefèvre T.
      • Subirade M.
      Structural and interaction properties of β-Lactoglobulin as studied by FTIR spectroscopy.
      ) and partial unfolding of the native conformation of β-LG induced by shearing (
      • Mediwaththe A.
      • Bogahawaththa D.
      • Grewal M.K.
      • Chandrapala J.
      • Vasiljevic T.
      Structural changes of native milk proteins subjected to controlled shearing and heating.
      ). The soluble Ca, Mg, and P in SM and CSM largely decreased following the pressurized shear treatment, compared with those of the control, possibly due to shifting of the mineral balance induced by shear. Similar to the concentration step, the zeta potential of SM or CSM did not change significantly (P > 0.05) after this treatment.

      Effects of Pressurized Thermal Processing at 75 and 95°C on Milk Proteins

      Examination of the effects of low pressure (10 MPa) on milk proteins at 75 or 95°C is the main focus of this section, and effect of temperature was considered when relevant. Thus, 10 MPa was compared with 0.5 MPa (0.5 MPa was considered the base pressure level) at each temperature level separately (75 or 95°C), where constant shearing (1,000 s−1) was also applied in all treatments. The average particle size of SM was slightly reduced after treatment at 10 MPa and 75°C compared with 0.5 MPa at 75°C. On the other hand, the particle size significantly decreased in CSM subjected to 10 MPa at 75°C compared with that treated at 0.5 MPa and 75°C (Table 1). A mostly similar trend was observed in both SM and CSM at 95°C, as the particle size of the samples treated at 10 MPa was reduced more than those subjected to 0.5 MPa pressure. Pressurization can substantially reduce casein micelle size due to compression, as observed in reconstituted SM after high-pressure and low-temperature treatments (100–200 MPa, 20°C, 20 min;
      • Anema S.G.
      • Lowe E.K.
      • Stockmann R.
      Particle size changes and casein solubilisation in high-pressure-treated skim milk.
      ). Furthermore, increase in treatment temperature from 10 to 40°C at 100 MPa exhibited a decreasing trend of micelle size (
      • Anema S.G.
      • Lowe E.K.
      • Stockmann R.
      Particle size changes and casein solubilisation in high-pressure-treated skim milk.
      ). The micelle size of SM significantly increased when a range of high-pressure treatments (200–800 MPa) were applied at 70°C for 30 min, compared with their counterparts at 20°C, using a high-pressure processor equipped with a water jacket (
      • Anema S.G.
      Heat and/or high-pressure treatment of skim milk: Changes to the casein micelle size, whey proteins and the acid gelation properties of the milk.
      ), potentially due to association of the denatured β-LG with the casein micelles (
      • Huppertz T.
      • Fox P.F.
      • Kelly A.L.
      High pressure treatment of bovine milk: Effects on casein micelles and whey proteins.
      ) or to the formation of large protein aggregates due to various interactions between caseins and whey proteins (
      • Bogahawaththa D.
      • Buckow R.
      • Chandrapala J.
      • Vasiljevic T.
      Comparison between thermal pasteurization and high pressure processing of bovine skim milk in relation to denaturation and immunogenicity of native milk proteins.
      ). Hence, the observations in the current study suggest that the low-pressure, high-temperature treatments (10 MPa, ≥5°C) had effects on casein micelles similar to those of high-pressure, low-temperature treatment (100 MPa, 20°C, ∼20 min) applied using HHP systems (
      • Anema S.G.
      • Lowe E.K.
      • Stockmann R.
      Particle size changes and casein solubilisation in high-pressure-treated skim milk.
      ;
      • Anema S.G.
      Heat and/or high-pressure treatment of skim milk: Changes to the casein micelle size, whey proteins and the acid gelation properties of the milk.
      ).
      When comparing the protein bands between SM treated with 0.5 MPa at 75°C, versus 10 MPa at 75°C, and their serum phases, no substantial changes were observed for whey proteins in the nonreducing SDS-PAGE images (Figure 1, A1, B1). However, we observed relatively intense αS1- and β-CN bands from the serum phase of SM treated with 10 MPa at 75°C, compared with those treated with 0.5 MPa at 75°C (Figure 1, B1). A similar trend of results was observed between SM treated with 0.5 MPa at 95°C and 10 MPa at 95°C and their serum phases under nonreducing SDS-PAGE conditions. These intense casein bands observed at 10 MPa, compared with 0.5 MPa, at 75 or 95°C, can be related to further pressure-induced destabilization of the casein micelles and dissociation of the caseins due to solubilization of CCP and interruption of hydrogen bonds (
      • Patel H.A.
      • Singh H.
      • Anema S.G.
      • Creamer L.K.
      Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk.
      ), which were also affected by heating (
      • Anema S.G.
      Effect of milk concentration on heat-induced, pH-dependent dissociation of casein from micelles in reconstituted skim milk at temperatures between 20 and 120°C.
      ). This observation appears to accord with an effect of the high-pressure, low-temperature treatments (≥100 MPa, 20°C, ∼20 min) on the casein micelles (
      • López-Fandiño R.
      • De la Fuente M.A.
      • Ramos M.
      • Olano A.
      Distribution of minerals and proteins between the soluble and colloidal phases of pressurized milks from different species.
      ;
      • Huppertz T.
      • Fox P.F.
      • Kelly A.L.
      Dissociation of caseins in high pressure-treated bovine milk.
      ;
      • Anema S.G.
      • Lowe E.K.
      • Stockmann R.
      Particle size changes and casein solubilisation in high-pressure-treated skim milk.
      ).
      Furthermore, no apparent changes in the αS1- and β-CN bands were noticed between 75 and 95°C at each pressure level (0.5 or 10 MPa) in SM, indicating heat stability of the micellar structure (
      • Anema S.G.
      Effect of milk concentration on heat-induced, pH-dependent dissociation of casein from micelles in reconstituted skim milk at temperatures between 20 and 120°C.
      ). However, the whey proteins in SM increasingly denatured at 95°C, displaying substantially faint bands of β-LG and α-LA as well as disappearance of BSA and IgG bands compared with those treated at 75°C (
      • Bogahawaththa D.
      • Vasiljevic T.
      Shearing accelerates denaturation of β-lactoglobulin and α-lactalbumin in skim milk during heating.
      ), without obvious pressure dependence, confirming their high heat-lability (
      • Wijayanti H.B.
      • Bansal N.
      • Deeth H.C.
      Stability of whey proteins during thermal processing: A review.
      ,
      • Wijayanti H.B.
      • Brodkorb A.
      • Hogan S.A.
      • Murphy E.G.
      Thermal denaturation, aggregation, and methods of prevention.
      ). It also appeared that these whey proteins formed aggregates with the involvement of κ-CN at 95°C, as seen on the stacking gel of the nonreducing SDS-PAGE (Figure 1, B1), which disappeared in the reducing gels (Figure 1, B2) due to reduction of the covalent bonds by β-mercaptoethanol. This confirmed that the protein aggregates were primarily formed by thiol (disulfide) bonds, as reported previously (
      • Bogahawaththa D.
      • Vasiljevic T.
      Shearing accelerates denaturation of β-lactoglobulin and α-lactalbumin in skim milk during heating.
      ). The formation of protein aggregates was also indicated by the substantially increased particle size in the SM samples treated at 95°C compared with those treated at 75°C, and appeared to be pressure-dependent. The whey protein bands of CSM and its serum phase did not display substantial changes between 0.5 and 10 MPa treatments at 75 or 95°C, as indicated by the nonreducing SDS-PAGE images (Figure 1, C1 and D1).
      The αS1- and β-CN bands of the serum phase of CSM treated with 10 MPa were more intense than those of the samples treated with 0.5 MPa at 75 or 95°C (Figure 1, D1). Similar results were observed in the corresponding SM samples (with relatively faint bands due to the low protein concentration) and can be discussed in the same way in relation to the pressure-induced destabilization of the casein micelles. This also agrees with the substantially smaller average particle size of CSM treated at 10 MPa compared with that treated at 0.5 MPa at 75 or 95°C. However, the αS1- and β-CN bands of the serum phase of CSM treated at 75°C were prominent, whereas those treated at 95°C were relatively faint depending on the applied pressure. These casein bands in the serum phase of SM appeared to change depending only on the pressure (0.5 MPa vs. 10 MPa) but not the temperature levels (75 and 95°C).
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      Structural changes of milk proteins during heating of concentrated skim milk determined using FTIR.
      reported no changes in the αs- and β-CN bands of the serum phase when raw SM (9% wt/wt TS) was heated from 75 to 110°C, whereas intensity of these casein bands gradually reduced in the serum phase of the CSM (17 or 25% wt/wt TS) during the same heating ramp. This gradual reduction of αs- and β-CN bands in the serum phase of the CSM was ascribed to their reassociation with the casein micelles, which largely dissociated (reversibly) at 75°C.
      • Anema S.G.
      Effect of milk concentration on heat-induced, pH-dependent dissociation of casein from micelles in reconstituted skim milk at temperatures between 20 and 120°C.
      reported that when reconstituted CSM (17.5 or 25% wt/wt TS) was heated from 20 to 120°C, the maximum dissociation of the casein micelles was observed at 60 to 80°C, which resulted in the highest content of soluble αs- and β-CN in the serum phase. However, κ-CN progressively dissociated from the casein micelles with increase in temperature from 75 to 110°C in raw SM (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      Structural changes of milk proteins during heating of concentrated skim milk determined using FTIR.
      ) and from 20 to 120°C in reconstituted SM (
      • Anema S.G.
      Effect of milk concentration on heat-induced, pH-dependent dissociation of casein from micelles in reconstituted skim milk at temperatures between 20 and 120°C.
      ) regardless of their TS (unconcentrated or concentrated), and formed soluble and insoluble aggregates with the involvement of the whey proteins, predominantly β-LG and α-LA (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      Structural changes of milk proteins during heating of concentrated skim milk determined using FTIR.
      ). A similar behavior was demonstrated by κ-CN in the current study. The κ-CN band in the serum phase of SM or CSM treated at 95°C was more intense, due to disintegration of thiol (disulfide)-linked κ-CN and whey-protein soluble aggregates, than those treated at 75°C, depending on the pressure applied (Figure 1, B2, D2).
      The Fourier-transform infrared spectrometry results of SM and CSM demonstrated substantial reduction of β-sheet and α-helix structural elements in the samples treated at 10 MPa compared with the 0.5 MPa treatment at 75 or 95°C (Figure 2, B, C). This could be ascribed mainly to loss of native confirmation of the whey proteins, as observed previously, after the high-pressure, low-temperature treatments (≥100 MPa, 20–30°C, ∼20 min;
      • Maresca P.
      • Ferrari G.
      • Leite Júnior, B.R.C.
      • Zanphorlin L.M.
      • Ribeiro L.R.
      • Murakami M.T.
      • Cristianini M.
      Effect of dynamic high pressure on functional and structural properties of bovine serum albumin.
      ;
      • Bogahawaththa D.
      • Buckow R.
      • Chandrapala J.
      • Vasiljevic T.
      Comparison between thermal pasteurization and high pressure processing of bovine skim milk in relation to denaturation and immunogenicity of native milk proteins.
      ). Treatment of CSM with 10 MPa at 95°C resulted in a relatively greater loss of α-helices and appearance of a few new peaks in the β-sheet region (∼1638–1,610 cm−1;
      • Bogahawaththa D.
      • Chandrapala J.
      • Vasiljevic T.
      Thermal denaturation of bovine β-lactoglobulin in different protein mixtures in relation to antigenicity.
      ), displaying some molecular rearrangements. We found no major changes (P > 0.05) in zeta potential between SM or CSM treated with 0.5 MPa and 10 MPa at 75 or 95°C. However, SM and CSM had slightly less negative surface potential after treatment at 10 MPa and 95°C compared with those of the control, indicating protein aggregation mainly induced by elevated temperature and shearing (
      • Mediwaththe A.
      • Chandrapala J.
      • Vasiljevic T.
      Shear-induced behaviour of native milk proteins heated at temperatures above 80°C.
      ), as observed from the nonreducing SDS-PAGE.
      The pressure applied (10 MPa) did not modify the mineral balance of SM or CSM significantly at 75 or 95°C (except Mg in SM at 95°C and P in CSM at 75°C), compared with that at 0.5 MPa, although the casein micelle was substantially altered in the current study in a similar way as if it were subjected to a high-pressure, low-temperature treatment (≥100 MPa, 20°C, ∼20 min). High-pressure treatments at ≥100 MPa and 20°C for 15 to 30 min resulted in an increase in soluble minerals (Ca, P, and Mg) in the serum phase of SM (
      • López-Fandiño R.
      • De la Fuente M.A.
      • Ramos M.
      • Olano A.
      Distribution of minerals and proteins between the soluble and colloidal phases of pressurized milks from different species.
      ) and reconstituted milk protein concentrates (
      • Cadesky L.
      • Walkling-Ribeiro M.
      • Kriner K.T.
      • Karwe M.V.
      • Moraru C.I.
      Structural changes induced by high-pressure processing in micellar casein and milk protein concentrates.
      ), due to disintegration of the casein micelles and movement of Ca, P, and Mg into the serum phase. In contrast, heat treatment can result in movement of the mineral balance into the micellar phase mainly by association of Ca and P with the casein micelles (
      • Gaucheron F.
      The minerals of milk.
      ). We observed a substantial reduction of Ca, P, and Mg in the serum phase of SM and CSM with increase in treatment temperature from 20 to 95°C, regardless of the pressure applied; this effect was greater in CSM than in SM, as observed previously (
      • Markoska T.
      • Huppertz T.
      • Grewal M.K.
      • Vasiljevic T.
      Structural changes of milk proteins during heating of concentrated skim milk determined using FTIR.
      ). Hence, the effect of low pressure (10 MPa) on the mineral balance in SM and CSM appeared to be mostly counterbalanced by the effect of temperature (75 or 95°C) in the current study.
      The heat and pressure treatments govern denaturation of the whey proteins differently in the milk. The heat-induced denaturation of whey proteins occurs following increased hydrophobic interactions and reduction of soluble Ca and P in the serum phase. On the contrary, the pressure treatments induce denaturation of the whey proteins through diminished hydrophobic interactions and increased solubility of Ca and P (
      • Anema S.G.
      Heat and/or high-pressure treatment of skim milk: Changes to the casein micelle size, whey proteins and the acid gelation properties of the milk.
      ). Because the temperature levels were more severe than the pressure applied in the current study, in terms of denaturation of whey proteins (
      • Huppertz T.
      • Fox P.F.
      • Kelly A.L.
      High pressure treatment of bovine milk: Effects on casein micelles and whey proteins.
      ;
      • Patel H.A.
      • Singh H.
      • Anema S.G.
      • Creamer L.K.
      Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk.
      ), the influence of low pressure (10 MPa) on denaturation of whey proteins appeared to be offset by the effect of the high temperature (75 or 95°C). Hence, in the current study, we observed that the increase in temperature from 20 to 95°C accelerated the denaturation of β-LG, α-LA, IgG, LF, and BSA, whereas we detected no significant effect of 10 MPa on denaturation of these whey proteins at 75 or 95°C, as previously discussed.

      CONCLUSIONS

      All the variables tested had individual or combined effects on native milk proteins. Evaporative concentration resulted in the destabilization of the casein micelles and dissociation of αS1- and β-casein into the serum phase. Generally, CSM appeared to be more prone to further modifications than SM. Pressurized shearing at 20°C contributed to formation of soluble aggregates in CSM, driven mainly by dissociated caseins and minor whey proteins. Temperatures of 75 and 95°C influenced the caseins, whey proteins, and mineral balance, whereas low pressure (10 MPa) appeared to influence the micellar structure regardless of temperature. Treatment of 10 MPa at 75 or 95°C can result in dissociation of casein micelles and reduction of their size in both SM and CSM, mostly in a similar way to that found in a high-pressure, low-temperature treatment (≥100 MPa, 20°C, ∼20 min). However, the applied pressure did not greatly modify the mineral balance or accelerate denaturation of whey proteins, due to a countereffect exerted by heating. The pressurization contributed to substantial loss of the secondary structure during shearing at 20°C and heating at 75 or 95°C. These results need to be considered for optimum processing of dairy systems.

      ACKNOWLEDGMENTS

      The authors have not stated any conflicts of interest.

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