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International Journal of

Molecular Sciences Article

Effect of Calcium on Absorption Properties and Thermal Stability of Milk during Microwave Heating Yejun Wu 1,2,3 , Daming Fan 1,2,3,4,5, * ID , Feng Hang 1,5 , Bowen Yan 2,3 , Jianxin Zhao 2,3,4,5, *, Hao Zhang 2,3,4,5 and Wei Chen 1,2,3,4,5 1

2 3 4 5

*

State Key Laboratory of Dairy Biotechnology, Technology Center, Bright Dairy & Food Co., Ltd., Shanghai 200436, China; [email protected] (Y.W.); [email protected] (F.H.); [email protected] (W.C.) State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; [email protected] (B.Y.); [email protected] (H.Z.) School of Food Science and Technology, Jiangnan University, Wuxi 214122, China National Engineering Research Center for Functional Food, Jiangnan University, Wuxi 214122, China Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Wuxi 214122, China Correspondence: [email protected] (D.F.); [email protected] (J.Z.); Tel.: +86-0510-8532-6696 (D.F.); +86-0510-8588-4620 (J.Z.)  

Received: 7 May 2018; Accepted: 8 June 2018; Published: 13 June 2018

Abstract: During heating, there are a lot of physical and chemical changes in milk components, which are mainly reflected in the changes of proteins. Calcium ions in milk react with proteins to precipitate or form gels, and the thermal stability of milk is affected by the type and content of calcium. In this study, different calcium-fortified milk systems were treated by rapid conventional heating (RCV) and microwave heating (MV) to evaluate the effects of forms and concentration of calcium in liquid milk on microwave absorption properties and thermal stability of milk. It was found that the concentration of calcium ions on microwave energy absorption is not a significant influence, while the forms affected the systems dramatically. The thermal stability of milk during MV is remarkably affected by the forms of calcium ions. When adding ionized calcium, the calcium-fortified milk systems had poor thermal stability and severe agglomeration of protein, while the addition of milk calcium had little effect and was almost free from protein coagulation. It could be speculated that the metal ions in the microwave field could create a strong vibration that could trigger protein agglomeration through the combination of the surrounding casein phosphorylates. Keywords: calcium; milk; microwave heating; absorption properties; thermal stability

1. Introduction Milk is one of the oldest natural beverages, which has a high nutritional value and is the best source of nutrition for new life. Milk proteins are mainly composed of casein and whey proteins, including all the essential amino acids. 100 g of common milk contains about 100 mg of calcium. A significative amount of calcium is present as a colloidal form of casein calcium phosphate, which is more likely to be absorbed by the body than the calcium found in vegetables and fruits, thereby calcium-fortified foods are favored by most governments and health organizations. Heating milk has always been a key process in the dairy industry. During heating, there are a lot of physical and chemical changes in milk components [1]. When heating to greater than 60 ◦ C, it causes some significant changes such as whey protein denaturation, changes in casein micelles, reactions between denatured whey proteins and casein micelles, reactions between lactose and proteins, changes in milk globulin membrane proteins, conversion of soluble forms of calcium, magnesium,

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Int. J. Mol.and Sci. 2018, 19, elements x 2 of 11 phosphorus, other to colloidal states, the reduction of pH, and loss of vitamins and bioactive substances. The thermal stability of milk is affected by the type of calcium salt and the milk proteins, changes in milk globulin membrane proteins, conversion of soluble forms of calcium, system [2–6]. magnesium, phosphorus, and other elements to colloidal states, the reduction of pH, and loss of Microwave heating is a typical fast heating method that works by alternating cycle changes vitamins and bioactive substances. The thermal stability of milk is affected by the type of calcium salt in the and microwave field and magnetic field. The polarity orientation of the dipole in milk the milk electric system [2–6]. macromolecules varies with the of the external electromagnetic and under the action Microwave heating is a change typical fast heating method that works byfield, alternating cycle changes in of the the frequency modulation (FM) electromagnetic field of 2.45 GHz, the polar molecules microwave electric field and magnetic field. The polarity orientation of the dipole inrotate milk severalmacromolecules billion times per second. Thechange sharp of friction, collision, vibration, extrusion, and other effects varies with the the external electromagnetic field, and under the action of between rotating molecules produces heat, so field partsofof theGHz, material simultaneously gain heat. thethe frequency modulation (FM) electromagnetic 2.45 the polar molecules rotate several billion times per second. The sharp friction, collision, vibration, extrusion, effects between Both the target activation hypothesis and thermal agitation hypothesis focusand onother the induction of the the rotating molecules produces heat, so parts of the material simultaneously gain heat. Both the dielectric enhancement effect of the dielectric sensing component in the liquid phase of the system. target activation andions thermal hypothesis focus on the induction of themolecular dielectric In the microwave field,hypothesis the calcium haveagitation high dielectric response characteristics. The enhancement effect of the dielectric sensing component in the liquid phase of the system. In the vibrations during heating can affect the interaction of the phosphate groups on the surface of caseins microwave field, the calcium ions have high dielectric response characteristics. The molecular with the calcium ions and thus contribute to protein agglomeration [7,8]. vibrations during heating can affect the interaction of the phosphate groups on the surface of caseins In this study, we investigated how different forms and concentrations of calcium affected the with the calcium ions and thus contribute to protein agglomeration [7,8]. absorption properties and thermal stability of milk and analyzed the differences between milk heated In this study, we investigated how different forms and concentrations of calcium affected the by rapid heat conduction and microwave heating to learn how the quality of calcium-fortified milk is absorption properties and thermal stability of milk and analyzed the differences between milk heated affected the heat different heating methods. Theheating resultstocould about the changes byby rapid conduction and microwave learn provide how the information quality of calcium-fortified milk in milkisduring heat processing and may also provide an important theoretical basis for the extensive affected by the different heating methods. The results could provide information about the changes application ofduring microwave ultra-highand temperature (UHT) an sterilization in the future. in milk heat processing may also provide important theoretical basis for the extensive

application of microwave ultra-high temperature (UHT) sterilization in the future.

2. Results and Discussion

2. Results and Discussion

2.1. The Basic Components of Milk 2.1.components The Basic Components Milk The of milk ofare very complex and contain more than 3000 kinds of compounds, the basic being fat, protein,oflactose, salt,and vitamins, and water. Under conditions, The components milk areinorganic very complex contain more than 3000 kindsnormal of compounds, the the components stable. The basic components blank milkUnder systemnormal are shown in Table basic beingare fat,relatively protein, lactose, inorganic salt, vitamins,ofand water. conditions, the1. components are relatively stable. The basic components of blank milk system are shown in Table 1. Table 1. The basic components of blank milk system. SnF, Ts, FPT Table 1. The basic components of blank milk system. SnF, Ts, FPT Sample Fat/% Lactose/% Calcium/% Cru.Protein 1 /% Tru.Protein 2 /% SnF 3 /% Ts 4 /% FPT 5 /◦ C Sample Fat/% Cru.Protein 1/% Tru.Protein 2/% Lactose/% SnF 3/% Ts 4/% FPT 5/°C Calcium/% Blank milk 3.50 ± 0.19 3.07 ± 0.30 2.91 ± 0.21 4.62 ± 0.11 8.54 ± 0.31 12.08 ± 0.30 0.495 ± 0.14 0.10 ± 0.03 Blank milk 3.50 ± 0.19 3.07 ± 0.30 2.91 ± 0.21 4.62 ± 0.11 8.54 ± 0.31 12.08 ± 0.30 0.495 ± 0.14 0.10 ± 0.03 1

2 Tru.Protein means true protein content, 3 SnF means solids non-fat Cru.Protein means crude protein 1 Cru.Protein means crudecontent, protein content, 2 Tru.Protein means true protein content, 3 SnF means 5 FPT means freezing point. content, 4 Ts means total solids content, 4 5

solids non-fat content, Ts means total solids content, FPT means freezing point.

Particle size analysis is a method thatthat cancan quickly analyze thethe thermal If the the Particle size analysis is a method quickly analyze thermalstability stabilityof ofaasystem. system. If particleparticle size distribution of theofsystem is Gaussian, i.e.,i.e., thethe mean, median, in the the size distribution the system is Gaussian, mean, median,and andfrequency frequency are in same position, the system in a relatively stable state; conversely, thereare areinstability instability factors factors in same position, the system is in is a relatively stable state; conversely, there in the the [9].particle The particle size distribution of blank milk samples is shownininFigure Figure11and andTable Table 2. 2. systemsystem [9]. The size distribution of blank milk samples is shown

Figure 1. The particle size distribution of blank milk system.

Figure 1. The particle size distribution of blank milk system.

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Table 2. The particle size distribution of blank milk system. The Percentage of Particles in Different Distribution/%

Sample Int. J. Mol. Sci. 2018, 19, x

Blank milk

0.1–1 µm

1–10 µm

10–100 µm

90.95 ± 0.23

5.55 ± 0.31

3.50 ± 0.56

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Table 2. The particle size distribution of blank milk system.

2.2. Effects of Calcium on the DielectricThe Properties and Microwave Absorption Properties of Milk Percentage of Particles in Different Distribution/% Sample

0.1–1 μm

1–10 μm

2.2.1. Dielectric Properties of the90.95 Calcium-Fortified Milk Blank milk ± 0.23 5.55Systems ± 0.31

10–100 μm 3.50 ± 0.56

The dielectric property is an inherent response characteristic of a bound charge within a molecule 2.2. Effects of Calcium on the Dielectric Properties and Microwave Absorption Properties of Milk to an applied electric field. The dielectric constant ε0 and dielectric loss factor ε00 are commonly used to express parameters of dielectric properties. TheMilk dielectric 2.2.1.the Dielectric Properties of the Calcium-Fortified Systemsconstant ε0 represents the ability of the calcium-fortified milk systems electromagnetic waves because the electrical properties of The dielectric propertytoisstore an inherent response characteristic of aofbound charge within a 00 represents the the system that affect energy absorption and transfer, and the dielectric loss factor ε molecule to an applied electric field. The dielectric constant ε′ and dielectric loss factor ε′′ are abilitycommonly of the systems electrical energy the formproperties. of heat [10]. is an inhomogeneous used to toconsume express the parameters of in dielectric TheMilk dielectric constant ε′ represents the ability of the calcium-fortified milk systems to store electromagnetic waves because of colloidal dispersion system. The protein colloids and fat particles are dispersed in water that contains the electrical properties of the For system that affect energysubstances, absorption and the dielectric salts, lactose, and whey proteins. nonhomogeneous the transfer, differentand phases have unique lossparameters. factor ε′′ represents thecharges ability of theaccumulate systems to consume electricalphase energyboundaries, in the form of heat the dielectric Electric can at the different causing [10]. Milk is an inhomogeneous colloidal dispersion system. The protein colloids and fat particles are two phases to polarize at the interface. Changes in the dielectric parameters of milk are mainly dispersed in water that contains salts, lactose, and whey proteins. For nonhomogeneous substances, causedthe by different the dipoles in the inhomogeneous material, the polarization of electrons and atoms and the phases have unique dielectric parameters. Electric charges can accumulate at the Maxwell-Wagner effect [11,12]. causing the two phases to polarize at the interface. Changes in the different phase boundaries, Todielectric study the effects ofofdifferent forms caused and concentrations on the dielectric properties parameters milk are mainly by the dipoles of in calcium the inhomogeneous material, the polarization of electrons and atoms and the Maxwell-Wagner effect [11,12]. of milk, here, we chose three sources of calcium: calcium chloride, calcium lactate, and milk calcium. Toconcentrations study the effects of different forms 1.25, and concentrations thechanges dielectricof properties The calcium were 1 (blank), 1.5, 1.75, andof2calcium mg/g. on The the dielectric 0 00 of milk, here, we chose three sources of calcium: calcium chloride, calcium lactate, and milk calcium. constant ε and the dielectric loss factor ε at 2.45 GHz in different milk systems are shown in Figure 2. The calcium concentrations were 1 (blank), 1.25, 1.5, 1.75, and 2 mg/g. The changes of the dielectric 0 As shown in Figure 2a, the ε did not change significantly with different forms of calcium and with constant ε′ and the dielectric loss factor ε′′ at 2.45 GHz in different milk systems are shown in Figure the increase in calcium concentration at room temperature. As shown in Figure 2b, the ε00 fluctuated 2. As shown in Figure 2a, the ε′ did not change significantly with different forms of calcium and with slightly with the increase calcium concentration and the As fluctuations of calcium and milk the increase in calciuminconcentration at room temperature. shown in Figure 2b, thechloride ε′′ fluctuated 00 calcium were with contrary to calcium lactate, moreover,and thethe ε fluctuations of the calcium chloride was and maximal slightly the increase in calcium concentration of calcium chloride milk and fluctuant obviously, while the ε00 of the calcium lactatethe and milk were closer and hadand a couple calcium were contrary to calcium lactate, moreover, ε′′ of the calcium calcium chloride was maximal fluctuant obviously, while the ε′′ of the calcium lactate and milk calcium were closer and had a couple of small fluctuations. That is, with the increase in calcium concentration, the ability of the systems to small fluctuations. That is,energy with theinincrease in calciumfield concentration, the ability the systems absorbofand store the microwave the microwave did not change, but of the ability to to convert absorb and store the microwave energy in the microwave field did not change, but the ability to the absorbed microwave energy into heat energy fluctuated slightly. In addition, the means by which convert the absorbed microwave energy into heat energy fluctuated slightly. In addition, the means the different forms of calcium affected the ability of the system to absorb and store microwave energy by which the different forms of calcium affected the ability of the system to absorb and store were not obviousenergy but had annot effect on the the on system to convert the absorbed microwave microwave were obvious butability had anof effect the ability of the system to convert the energyabsorbed into heat energy; more specifically, the ionized form of calcium, calcium chloride, had the microwave energy into heat energy; more specifically, the ionized form of calcium, calcium greatest influence. chloride, had the greatest influence.

FigureFigure 2. (a) The constant ε0 andε′ (b) loss factor ε00 ofε′′the milk systems. 2. (a)dielectric The dielectric constant anddielectric (b) dielectric loss factor of calcium-fortified the calcium-fortified milk systems.

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2.2.2. Microwave Absorption Properties Properties of of the the Calcium-Fortified Calcium-Fortified Milk Milk Systems Systems The a material in a microwave field cannot reflected by itsonly complex permittivity, The response responseofof a material in a microwave field be cannot be only reflected by its complex as it can also be affected by the impedance matching problem and others. The microwave absorption permittivity, as it can also be affected by the impedance matching problem and others. The properties the material are the comprehensive of its dielectric properties, microwaveofabsorption properties of the materialperformance are the comprehensive performance of its magnetic dielectric properties the macroscopic morphology [13]. Reflection loss (RL)[13]. is anReflection importantloss parameter to properties,and its magnetic properties and the macroscopic morphology (RL) is an evaluate the absorption performance of the material, and the RL of the material reflects its absorption, important parameter to evaluate the absorption performance of the material, and the RL of the release loss ability of the microwave [14–16]. We measured the RL of [14–16]. the blank milk systemsthe at materialand reflects its absorption, release and loss ability of the microwave We measured different observe absorption properties. As shown in Figure 3a, the RL ofAs theshown blank RL of thethicknesses blank milk to systems at the different thicknesses to observe the absorption properties. at GHz tendency to increase and then decreased with the thickness increasing and in 2.45 Figure 3a,first the showed RL of thea blank at 2.45 GHz first showed a tendency to increase and then decreased the reachedincreasing a maximum of − 24.627 dB at a thickness of 1 cm.ofTherefore, cana withsystems the thickness andabsorption the systems reached a maximum absorption −24.627 dBit at be concluded that the absorption properties of the material were not linearly related to its thickness thickness of 1 cm. Therefore, it can be concluded that the absorption properties of the material were and there was an optimal thicknessand at the specified of 2.45 GHz. From above, a standard not linearly related to its thickness there was anfrequency optimal thickness at the specified frequency of thickness 1 cmabove, was chosen to conduct the follow-up studies. 2.45 GHz.of From a standard thickness of 1 cm was chosen to conduct the follow-up studies. It can be be seen seen from from Figure Figure3b 3bthat thatthe thecalcium calciumconcentration concentrationincreased increasedfrom from1 1mg/g mg/gto to22mg/g mg/g at 2.45 GHz, and the RL of the systems did not not change change significantly, significantly, while the different forms of calcium production affected the system system in in the the following following ways: ways: (i) in in the the range range of of 11 to to 1.25 1.25mg/g, mg/g, with the increase of calcium calcium concentration, concentration,the theeffect effectofofcalcium calciumchloride, chloride, calcium lactate, and milk calcium calcium lactate, and milk calcium on on systems consistent, andRL the RL decreased slightly; (ii)range in theofrange of21.25 tothe 2 mg/g, thethe systems waswas consistent, and the decreased slightly; (ii) in the 1.25 to mg/g, effect the effect of forms the three forms ofon calcium on thediffered systemsasdiffered as theconcentration calcium concentration of the three of calcium the systems the calcium increased.increased. Calcium Calcium lactatehad almost had no effect thedB). RL (in dB). In contrast, chloride andcalcium milk calcium lactate almost no effect and the and RL (in In contrast, calciumcalcium chloride and milk made made RL fluctuate the effect of calcium chloride greater. RL fluctuate being being the effect of calcium chloride greater.

3. The Thereflection reflectionloss loss (RL) of (a) blank at different thicknesses the calciumFigure 3. (RL) of (a) the the blank milkmilk at different thicknesses and (b)and the (b) calcium-fortified fortified at a thickness milk at a milk thickness of 1 cm. of 1 cm.

2.3. The Thermal Stability of the Calcium-Fortified Calcium-Fortified Milk Milk Systems Systems under under the the Microwave Microwave Field Field 2.3.1. Comparison of the Microwave Heating Heating and and Rapid Rapid Conventional Conventional Heating Heating Curves Curves During MV, material. MV, the the thermal thermal and and non-thermal non-thermal effects were simultaneously applied to the material. In this study, study, multistage multistage MV MV programs programs were were used used to to match match the the RCV RCV curves curves to achieve similar heating rates so that we could study the non-thermal effects effects of the microwave microwave on on the the systems. systems. The root mean square error (RMSE) was calculated calculated according to Equation (1) to evaluate evaluate the the accuracy accuracy between the MV and RCV data [17]. [17]. v u u1 N (1) RMSE = t1 ∑ ( TMV − TRCV )2 (1) RMSE = N n=1 − As shown in Figure 4, the RMSE value was 1.77, representing the difference in the average As shown in Figure 4, the RMSE value was 1.77, representing the difference in the average temperatures of RCV and MV. This result indicated that the MV curve fits well with RCV processing. temperatures of RCV and MV. This result indicated that the MV curve fits well with RCV processing.

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Figure4.4.Comparison Comparisonof ofthe themicrowave microwaveheating heating(MV) and rapid rapid conventional conventional heating heating (RCV) (RCV) curves. curves. Figure (MV) and

2.3.2. The The Thermal Thermal Stability Stability of of the the Milk Milk during during the the Heating Heating Process Process 2.3.2. Under the the same and 1.51.5 mg/g milkmilk calcium-fortified milkmilk systems were Under same MV MVconditions, conditions,the theblank blank and mg/g calcium-fortified systems heated to various temperatures and their particle size changed significantly, as shown in Table 3. were heated to various temperatures and their particle size changed significantly, as shown in Table 3. Withthe theincrease increaseof oftemperature, temperature,the thesize sizeof ofparticles particlesin intwo twomilk milksystems systemsshifted shiftedto tobe be larger largerand and the the With percentage of particles (10–100 μm) increased to 15.72% and 14.51%, respectively, when the systems percentage of particles (10–100 µm) increased to 15.72% and 14.51%, respectively, when the systems wereheated heatedto to95 95 ◦°C by MV. MV.This This could could be be that that the the heating heating temperature temperature was was too too high, high, causing causing intense intense were C by aggregation of of milk milk protein protein particles particles and and denaturation denaturation of of whey whey protein, protein, and and leading leading to to the the whey whey aggregation protein to form protein polymers with itself or with casein. Subsequently, we conducted experiments protein to form protein polymers with itself or with casein. Subsequently, we conducted experiments at 95 95 ◦°C to investigate investigate the the effects effects of of the the different different calcium calcium forms, forms, concentrations, concentrations, and and heating heating methods methods at C to on the thermal stability of milk. on the thermal stability of milk. Table3.3. The Thepercentage percentageof ofdifferent differentparticle particlesizes sizes in in the the different differentmilk milksystems systemsat atvarious various temperatures. temperatures. Table Samples Samples

Temperature 0–1 μm Temperature 0–1 µm 25 °C 90.95 ± 0.23 ◦ ± 0.23 45 °C 25 C 82.2890.95 ± 0.59 Blank milk systems 45 ◦ C 76.3382.28 ± 0.59 65 °C ± 0.88 Blank milk systems ◦ ± 0.88 95 °C 65 ◦ C 71.6976.33 ± 0.45 95 C 71.69 ± 0.45 45 °C 69.84 ± 0.69 milk calcium-fortified ◦ ± 0.69 65 °C 45 C 69.8369.84 ± 0.54 milk systems calcium-fortified systems95 °C 65 ◦ C 70.3569.83 ± 0.54 ± 0.73 95 ◦ C 70.35 ± 0.73

1–10 μm 1–10 µm 5.55 ± 0.31 5.55 ± 0.31 7.28 ± 0.42 7.28± ± 0.42 10.66 0.74 10.66 ± 0.74 12.59 ± 0.51 12.59 ± 0.51 15.62 ± 0.51 15.62 ± 0.51 15.65 ± 0.72 15.65 ± 0.72 15.14 ± 0.93 15.14 ± 0.93

10–100 μm 10–100 µm 3.50 ± 0.56 3.50 ± ±0.56 9.44 0.60 9.44 ± 0.60 13.01 ± 0.62 13.01 ± ±0.62 15.72 0.54 15.72 ± 0.54 14.54 ± 0.72 14.54 ± ±0.72 14.52 0.49 14.52 ± ±0.49 14.51 1.21 14.51 ± 1.21

2.3.3. The Effects of Different Calcium Forms on the Thermal Stability of Milk 2.3.3. The Effects of Different Calcium Forms on the Thermal Stability of Milk Figure 5 shows the particle size distribution for the milk calcium, calcium lactate and calcium Figure 5 shows thesystems particle at size for the milk calcium, calcium and calcium chloride fortified milk 95distribution °C. In the milk calcium-fortified systems,lactate the particles were ◦ chloride fortified milk systems at 95 C. In the milk calcium-fortified systems, the particles were mainly mainly distributed in 0.1 to 1 μm, and 0.82 μm were the most common. However, in the case of distributed in 0.1and to 1calcium µm, andchloride, 0.82 µm the were the most common. in the case of calcium calcium lactate particles were mainlyHowever, distributed in 10 to 340 μm andlactate a few and calcium chloride, the particles were mainly distributed in 10 to 340 µm and a few in 1.0 tosystems 10 µm. in 1.0 to 10 μm. These showed that the effect of milk calcium on the thermal stability of milk These showed effect ofaggregation. milk calciumInoncontrast, the thermal stability of milk littlehad anda was little and that had the no protein calcium chloride andsystems calciumwas lactate had no protein aggregation. In contrast, calcium chloride and calcium lactate had a great influence on great influence on the systems, forming larger particles and substantial protein aggregation. the systems, forming substantial protein aggregation. The addition oflarger milk particles calcium and made the systems’ structure more uniform. It also created a The addition of milk calcium made the systems’ structure more uniform. It also created a buffering buffering effect so that the pH of the systems remained stable and had little effect on the thermal effect so that the pH of the systems remained stable and had little effect on the thermal stability [18]. stability [18]. However, the addition of ionic calcium in the form of either calcium chloride or calcium However, the addition of ionic thebalance form ofofeither calcium chloride calciumthe lactate could lactate could have altered the calcium original in salt the milk systems. We or observed following have altered the original salt balance of the milk systems. We observed the following effects: colloidal effects: colloidal calcium phosphate dissolved; casein lost its electrostatic charge and agglomerated; calcium dissolved; caseintolost its electrostatic and and agglomerated; fat was constantly fat wasphosphate constantly aggregated form larger fat charge particles; calcium precipitated after aggregated to form larger fat particles; and calcium precipitated after polymerization to form larger polymerization to form larger particles. These effects showed that the calcium chloride and calcium lactate fortified milk systems were less thermostable because internal polymerization occurred and

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particles. These effects Int. J. Mol. Sci. 2018, 19, x Int. J. Mol. Sci. 2018, 19, x

showed that the calcium chloride and calcium lactate fortified milk systems 6 of 11 6 of 11 were less thermostable because internal polymerization occurred and the fat granules and casein particles that were originally small gradually to form more large particles.to The large fat the fat fat granules granules and casein particles particles that were werepolymerized originally small small gradually polymerized form more the and casein that originally gradually polymerized to form more granules and casein particles resulted and in increased agglomeration [19]. large particles. The large fat granules casein particles resulted in increased agglomeration [19]. large particles. The large fat granules and casein particles resulted in increased agglomeration [19].

The particle particle size size distribution calcium-fortified milk milk systems systems at at 95 95 ◦°C. °C. Figure 5. 5. The distribution of of different different calcium-fortified calcium-fortified milk systems at C. Figure 95

2.3.4. The The effects effects of of different calcium concentrations onon the thermal stability of milk milk The Effects ofdifferent Differentcalcium Calciumconcentrations Concentrations the Thermal Stability of Milk 2.3.4. on the thermal stability of For the the milk milk calcium-fortified calcium-fortified systems, systems, as as shown shown in in Figure Figure 6, 6, the the particle particle sizes sizes were consistently milk calcium-fortified systems, as shown in Figure 6, the particle sizes were were consistently consistently For distributed in 0.1 to 1 μm, indicating that different calcium concentrations had little effect on the the distributed in in 0.1 0.1 to to 1 μm, µm, indicating that different calcium concentrations concentrations had had little little effect effect on on distributed thermal stability stability of of the the systems systems under under the the microwave microwave field. field. Also, Also, there there was was no no protein protein aggregation. aggregation. thermal aggregation.

Figure 6. 6. The The particle particle size size distribution distribution of of different different concentrations concentrations of of milk milk calcium-fortified calcium-fortified milk milk Figure Figure 6. The particle size distribution of different concentrations of milk calcium-fortified milk systems systems at 95 °C. systems at 95 ◦ C.at 95 °C.

2.3.5. The Effects Effects of Different Different Heating Methods Methods on the the Thermal Thermal Stability Stability of of Milk Milk 2.3.5. 2.3.5. The The Effects of of Different Heating Heating Methods on on the Thermal Stability of Milk For the the 1.5 1.5 mg/g mg/g milk milk calcium-fortified calcium-fortified systems, systems, as as shown shown in in Figure Figure 7, 7, no no significant significant differences differences For For the 1.5 mg/g milk calcium-fortified systems, as shown in Figure 7, no significant differences were seen seen in in the the particle particle size size distribution distribution curves curves by by MV MV or or RCV, RCV, and and the the particle particle sizes sizes were were roughly were roughly were seen in the particle size distribution curves by MV or RCV, and the particle sizes were roughly distributed in 0.1 to 1 μm, indicating that the different heating methods had little effect on the thermal distributed in 0.1 to 11 μm, indicating that the different heating methods had little effect on the thermal distributed in 0.1 to µm, indicating that the different heating methods had little effect on the thermal stability of of the the milk milk systems. systems. stability stability of the milk systems.

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Theparticle particle size distribution of different concentrations milk calcium-fortified milk Figure 7.7.The size distribution of different concentrations of milkofcalcium-fortified milk systems ◦ systems at 95 °C. at 95 C.

3. Materials 3. Materials and and Methods Methods 3.1. Materials

milkand andmilk milk calcium (Bright Dairy & Food Co.,Shanghai, Ltd., Shanghai, Anhydrous Pure milk calcium (Bright Dairy & Food Co., Ltd., China); China); Anhydrous calcium calcium chloride and lactate calcium(Sinopharm lactate (Sinopharm Ltd., Shanghai, chloride and calcium ChemicalChemical Reagent Reagent Co., Ltd.,Co., Shanghai, China). China). 3.2. Preparation of 3.2. Preparation of Calcium-Fortified Calcium-Fortified Milk Milk Systems Systems Referring to tothe thecalcium calcium content in high-grade calcium its preparation content in high-grade calcium milk milk and itsand preparation methodmethod (Patent (Patent CN 103651846 [20], calcium concentrations of 1 (blank), 1.25,1.75, 1.5, and 1.75,2and 2 mg/g number:number: CN 103651846 A) [20],A) calcium concentrations of 1 (blank), 1.25, 1.5, mg/g were were chosen. chosen. A 100-g milk system waswas prepared for each The amounts of calcium 100-gcalcium-fortified calcium-fortified milk system prepared for experiment. each experiment. The amounts of chloride, calcium lactate, milkand calcium toadded the system shown Table 4. calcium chloride, calciumand lactate, milk added calcium to theare system areinshown in Table 4. Table 4. Table 4. Amounts Amounts of of calcium calcium chloride, chloride, calcium calcium lactate lactate and and milk milk calcium calcium added added to to the the system. system.

Calcium Content Calcium Content 1.00 mg/g 1.00 mg/g 1.25 mg/g 1.25 mg/g 1.50 mg/g 1.50 mg/g 1.751.75 mg/g mg/g 2.002.00 mg/g mg/g

Calcium Calcium Chloride Chloride 0 0 0.0694 g ± 0.0002 0.0694 g ± 0.0002 0.1387 g ± 0.0003 0.1387 g ± 0.0003 0.2081 g g± ± 0.0001 0.2081 0.0001 0.2775 g ± 0.0001 0.2775 g ± 0.0001

Calcium Lactate Calcium Lactate 0 0 0.1927 g ± 0.0001 0.1927 g ± 0.0001 0.3854 g ± 0.0000 0.3854 g ± 0.0000 0.5781 ± 0.0002 0.5781 g ±g0.0002 0.7708 g ± 0.0003 0.7708 g ± 0.0003

Calcium MilkMilk Calcium 0 0 0.0975 g ± 0.0002 0.0975 g ± 0.0002 0.1951 g ± 0.0002 0.1951 g ± 0.0002 g ± 0.0000 0.29260.2926 g ± 0.0000 0.3902 g ± 0.0001 0.3902 g ± 0.0001

3.3. Determination of Dielectric Properties 3.3. Determination of Dielectric Properties The ε′ and ε′′ values were measured using a vector network analyzer (E5071C, Agilent, Santa The ε0 and ε00 values were measured using a vector network analyzer (E5071C, Agilent, Clara, CA, USA) with an open-ended coaxial line, connected to a high-temperature probe (85070E, Santa Clara, CA, USA) with an open-ended coaxial line, connected to a high-temperature probe Agilent) [21,22]. The probe was calibrated with air, a short circuit, and water, respectively. Each (85070E, Agilent) [21,22]. The probe was calibrated with air, a short circuit, and water, respectively. sample was measured in triplicate. Each sample was measured in triplicate. 3.4. Determination Reflection Loss Loss (RL) (RL) 3.4. Determination of of Reflection The RL RL versus for liquid liquid materials was improved improved based based on on the the arch by adding The versus frequency frequency for materials was arch method method by adding polytetrafluoroethylene (PTFE) a wall wall material material on on an an aluminum aluminum plate, plate, to to make make aa specialized specialized polytetrafluoroethylene (PTFE) as as a container (Figure (Figure 8a). 8a). The improved arch method included included aa vector vector network network container The testing testing system system of of the the improved arch method analyzer (HP8720B, Hewlett Packard Co., Palo Alto, CA, USA) and standard horn antennas in an an analyzer (HP8720B, Hewlett Packard Co., Palo Alto, CA, USA) and standard horn antennas in anechoic chamber chamberasasa afunction function frequency from 2.32.5toGHz 2.5 GHz (Figure 8b)In[23]. In this system, testing anechoic of of frequency from 2.3 to (Figure 8b) [23]. this testing system, the signal was emitted from the transmitter and reflected by a metal reflecting plate, then the the signal was emitted from the transmitter and reflected by a metal reflecting plate, then the reflected reflected signal was received by the receivier and passed into the vector network analyzer, so that a

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Int. J.frequency Mol. Sci. 2018, established 19, 1747 8 of 11of the certain a power reference. The difference between the received power metal plate and the sample to be measured was converted into a reflection loss.

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signal was received by the receivier and passed into the vector network analyzer, so that a certain frequency established a power areference. The difference between the received of the metalofplate certain frequency established power reference. The difference between thepower received power the and the sample measured converted a reflection metal plate and to thebesample to bewas measured wasinto converted into loss. a reflection loss.

Figure 8. The improved specialized container for liquid samples (a) and a schematic diagram of the archFigure method testing system (b). Figure Theimproved improved specialized containerfor forliquid liquidsamples samples (a) (a) and and aa schematic schematic diagram diagram of the 8.8.The specialized container system (b). (b). arch method testing system

3.5. Rapid Conventional Heating Method 3.5. Rapid Heating Method Method 3.5. Rapid Conventional Conventional Heating In terms of heating conditions, a HAAKE stainless steel oil bath (AC200, Thermo Scientific, Waltham, In terms a HAAKE stainless steelwas oil bath (AC200, Thermo Waltham, MA, USA) was ofused forconditions, RCV while temperature monitored online Scientific, using a Scientific, thermocouple In terms ofheating heating conditions, athe HAAKE stainless steel oil bath (AC200, Thermo MA, USA) was used for RCV while the temperature was monitored online using a thermocouple Waltham, (Shifu MA, USA) was used while the temperature was monitored online using sample a thermometer Instrument Co., for Ltd.,RCV Wuhu, China) [24], as shown in Figure 9. A 50 g of blank thermometer (Shifu Instrument Co., Ltd., Wuhu, China) [24], as shown in Figure 9. A 50 g of blank sample thermocouple thermometer (Shifu Instrument Co., Ltd., Wuhu, China) [24], as shown in Figure 9. was weighed into a quartz beaker and set it at 195 °C, a temperature determined by a previous experiment, was into a quartz andinto set itaat 195 °C, a temperature a previous experiment, 50weighed gstirrer of blank sample wasbeaker weighed quartz itdetermined at 195 ◦ C, aby temperature determined keptAthe open (45 kr/min) during heating to beaker ensureand thatset the sample was heated evenly and reliably. kept the stirrerexperiment, open (45 kr/min) heating ensure that the sample was heated evenly by a previous keptduring the stirrer opento(45 kr/min) during heating to ensure thatand thereliably. sample To achieve a rapid heat transfer, the control time was set to less than 3 min. The milk was heated to the To achieve a rapid heat transfer, the control time was set to less than 3 min. The milk was heated the was heated evenly and reliably. To achieve a rapid heat transfer, the control time was set to lesstothan specified temperature (45, temperatureprobe probe to internal temperature. ◦detect temperature (45,65, 65,95 95°C) °C)using using the thetemperature temperature detect thethe internal temperature. The The 3specified min. The milk was heated to the specified (45, 65,to95 C) using the temperature probe real-time temperature ofofthe recordedtotoplot plota atemperature temperature curve as reference the reference for the real-time thesystem system was was curve aswas the forplot the to detect temperature the internal temperature. The recorded real-time temperature of the system recorded to microwave heating conditions. To themicrowave sampletemperature temperature from rising past thethe specified heating conditions. Toprevent prevent the sample from rising pastprevent the specified limit, limit, amicrowave temperature curve as the reference for the heating conditions. To sample the sample was immediately cooled in an ice bath [25]. the sample was immediately cooled in an bath [25]. temperature from rising past the specified limit, the sample was immediately cooled in an ice bath [25].

Figure 9. The device of rapid conventional heating: a HAAKE stainless steel oil bath. Figure 9. The deviceofofrapid rapid conventional conventional heating: stainless steelsteel oil bath. Figure 9. The device heating:a HAAKE a HAAKE stainless oil bath. 3.6. Microwave Heating Method

3.6. Microwave HeatingMultiSYNTH Method An advanced microwave synthesis platform (MultiSYNTH, Milestone, Sorisole, Italy) was used toMultiSYNTH simulate the RCV heating curves [26], platform as shown (MultiSYNTH, in Figure 10. A 4 Milestone, g of the sample An advanced microwave synthesis Sorisole, was weighed into a quartz tube that was 1.2 cm in diameter. The tube was placed in a polyester sleeve

Italy) was used to simulate the RCV heating curves [26], as shown in Figure 10. A 4 g of the sample was weighed into a quartz tube that was 1.2 cm in diameter. The tube was placed in a polyester sleeve

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3.6. Microwave Heating Method An advanced MultiSYNTH microwave synthesis platform (MultiSYNTH, Milestone, Sorisole, Italy) was used to2018, simulate Int. J. Mol. Sci. 19, x the RCV heating curves [26], as shown in Figure 10. A 4 g of the sample 9 of 11 was weighed into a quartz tube that was 1.2 cm in diameter. The tube was placed in a polyester withoutwithout electromagnetic absorption and then and placed intoplaced the cavity poolsample of the MultiSYNTH sleeve electromagnetic absorption then into sample the cavity pool of the microwave synthesizer (Milestone, Sorisole, Italy). Sorisole, The synthesizer were microwave MultiSYNTH microwave synthesizer (Milestone, Italy). settings The synthesizer settingssinglewere mode processing, a microwave frequency of 2.45 GHz and the vibration frequency at frequency 10%. The microwave single-mode processing, a microwave frequency of 2.45 GHz and the vibration MultiSYNTH detectedsystem the real-time temperature of thetemperature test tube using at 10%. The system MultiSYNTH detected the real-time of the infrared test tubetemperature using the probe. Bytemperature adjusting the vibration frequencythe of vibration the samplefrequency pool, the of microwave synthesizer the infrared probe. By adjusting the sample pool, the allowed microwave sample to receive radiation and to have aradiation uniform and temperature When the synthesizer alloweduniform the sample to receive uniform to have adistribution. uniform temperature temperature reached temperature, thethe sample was immediatelythe cooled in anwas ice bath. Multiple distribution. When the preset temperature reached preset temperature, sample immediately replications were performed during the preliminary stages to determine the power necessary to match cooled in an ice bath. Multiple replications were performed during the preliminary stages to determine bath.necessary The MV procedure canoil be bath. seen in Table the oil power to match the The MV5.procedure can be seen in Table 5.

Figure 10. The device of Microwave heating: a MultiSYNTH microwave synthesizer. synthesizer.

Table 5. Microwave heating procedure procedure for for aa 44 gg sample sample of of the the calcium-fortified calcium-fortified milk milksystems. systems. Heating Stages Power/W Heating Stages Power/W First stage 72 First stage Second stage 39 72 Second stage Third stage 33 39 Third stage 33 Fourth stage 31 Fourth stage 31

Time/s 15 50 45 45

Time/s 15 50 45 45

3.7. Analysis of Milk Composition 3.7. Analysis of Milk Composition A Milko-ScanTM FT1 multifunction dairy analyzer (Foss, Hillerød, Denmark) was used to A Milko-ScanTM FT1 multifunction dairy analyzer (Foss, Hillerød, Denmark) was used to compare the differences between the compositions of the calcium-milk composite system when compare the differences between the compositions of the calcium-milk composite system when treated by RCV or by MV. The components tested included fat content (FAT, %), crude protein treated by RCV or by MV. The components tested included fat content (FAT, %), crude protein content content (Cru.Prot, %), true protein content (Tru.Prot, %), lactose content (Lactose, %), solids non-fat (Cru.Prot, %), true protein content (Tru.Prot, %), lactose content (Lactose, %), solids non-fat content content (SnF, %), total solids content (Ts, %), freezing point (FPT, °C). (SnF, %), total solids content (Ts, %), freezing point (FPT, ◦ C). 3.8. Analysis of Particle Size 3.8. Analysis of Particle Size A BT-9300H laser particle size distribution instrument (Bettersize Instruments Co., Ltd., A BT-9300H laser particle size distribution instrument (Bettersize Instruments Co., Ltd., Dandong, Dandong, China) with a BT-600-type circulating dispersion pump was used. The samples which were China) with a BT-600-type circulating dispersion pump was used. The samples which were placed at placed at 20 ± 1 °C for 30 min before testing consisted of controls (blanks), milk samples treated via 20 ± 1 ◦ C for 30 min before testing consisted of controls (blanks), milk samples treated via RCV and RCV and MW. All samples were not diluted. A moderate number of samples were added to the MW. All samples were not diluted. A moderate number of samples were added to the sample cell with sample cell with water as the dispersion medium, opened the ultrasonic dispersion and collected measurements at a rotational speed of 320 rpm to obtain the particle size distribution. 3.9. Data Analysis All statistical analyses were performed with Origin 2017 and SPSS. The data are presented as mean ± SD for each group. The differences between the mean values of the groups were analyzed

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water as the dispersion medium, opened the ultrasonic dispersion and collected measurements at a rotational speed of 320 rpm to obtain the particle size distribution. 3.9. Data Analysis All statistical analyses were performed with Origin 2017 and SPSS. The data are presented as mean ± SD for each group. The differences between the mean values of the groups were analyzed using a one-way variance analysis with Duncan’s multiple range tests. A p value of less than 0.05 was considered to indicate statistical significance. 4. Conclusions We investigated the effects of different forms and concentrations of calcium on the microwave absorption properties and the thermal stability of milk. The results showed that the effect of different forms and concentrations of calcium on the absorption of microwave energy was not significant, but the presence of ionic calcium had a relatively great influence. Compared with RCV and unheated, the calcium-fortified milk systems treated by MV had no significant loss of nutrients. Furthermore, the milk systems with added ionized calcium had poor thermal stability and severe aggregation of proteins during MV, while the addition of milk calcium had little effect and there was almost no protein aggregation in the systems. In fast-paced modern life, the quality of rejuvenating liquid milk is a focus for consumers. Microwave heating is a typical fast method for reheating, but its safety has always been controversial. This study investigated the microwave heating process of calcium-fortified milk systems, which can provide a theoretical basis for the study of the mechanisms that underlie the dielectric intervention effect of calcium-induced milk proteins in microwave fields and lay a foundation for the development of microwave sterilization technology. Author Contributions: Conceptualization, Y.W. and D.F.; Methodology, Y.W.; Validation, F.H. and B.Y.; Formal Analysis, Y.W.; Investigation, B.Y.; Resources, D.F.; Data Curation, Y.W.; Writing—Original Draft Preparation, Y.W.; Writing—Review and Editing, D.F.; Visualization, B.Y.; Supervision, H.Z.; Project Administration, J.Z.; Funding Acquisition, W.C. Acknowledgments: This study was supported by The Key Projects in the National Science & Technology Pillar Program during the Open Project Program of State Key Laboratory of Dairy Biotechnology, Bright Dairy & Food Co., Ltd. (SKLDB2015-001) and the program of “Collaborative innovation center of food safety and quality control in Jiangsu Province”. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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