Research| Volume 104, ISSUE 3, P2843-2854, March 2021
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# Freezing kinetics and microstructure of ice cream from high-pressure-jet processing of ice cream mix

Open ArchivePublished:January 15, 2021

## ABSTRACT

The effect of high-pressure-jet (HPJ) processing (0–500 MPa) on low-fat (6% fat) ice cream was studied by evaluating physiochemical properties before freezing, during dynamic freezing, and after hardening. An HPJ treatment ≥400 MPa decreased the density, increased the apparent size of colloidal particles, and altered rheological behavior (increased non-Newtonian behavior and consistency coefficients) of low-fat ice cream mix before freezing. During dynamic freezing, the particle size and consistency coefficient decreased but remained higher in 400 MPa–treated samples vs. non-HPJ-treated controls at the conclusion of freezing. The resulting ice creams (400 and 500 MPa–treated) had similar hardness values (3,372 ± 25 and 3,825 ± 14 g) and increased melting rates (2.91 ± 0.13 and 2.61 ± 0.31 g/min) compared with a control sample containing polysorbate 80 (3,887 ± 2 and 1.62 ± 0.25 g/min). Visualization of ice cream samples using transmission electron microscopy provided evidence of casein micelle and fat droplet disruption by HPJ treatment ≥400 MPa. In the 400 MPa–treated samples, a unique microstructure consisting of dispersed protein congregated around coalesced fat globules likely contributed to the altered physiochemical properties of this ice cream. High-pressure-jet processing can alter the microstructure, rheological properties, and hardness of a low-fat ice cream, and further modification of the formulation and processing parameters may allow the development of products with enhanced properties.

## INTRODUCTION

Ice cream mix must contain a minimum of 10% milkfat and 20% total milk solids and be pasteurized and dynamically frozen (i.e., frozen while stirring) to be considered “ice cream” in the United States (21CFR §135.110;
• FDA
Code of Federal Regulations in Title 21.
). Additional functional ingredients (e.g., stabilizers and emulsifiers) are often added to enhance textural properties and stability. Emulsifiers (e.g., polysorbate 80 and mono- and diglycerides) primarily function to enhance the extent of fat destabilization during dynamic freezing, which in turn improves ice cream texture and meltdown properties (
• Goff H.D.
• Hartel R.W.
Mix ingredients.
;
• Hartel R.
• Rankin S.
The effects of fat structures and ice cream mix viscosity on physical and sensory properties of ice cream.
). Stabilizers are a group of ingredients, predominately polysaccharides (e.g., carrageenan), added to ice cream mix formulations to increase viscosity and stability against temperature fluctuations, enhance texture, and slow ice/lactose crystal growth in the frozen product (
• Flores A.A.
• Goff H.D.
Recrystallization in ice cream after constant and cycling temperature storage conditions as affected by stabilizers.
;
• Bahramparvar M.
• Mazaheri Tehrani M.
Application and functions of stabilizers in ice cream.
;
• Goff H.D.
• Hartel R.W.
Mix ingredients.
). Although stabilizers and emulsifiers are helpful in the development and maintenance of ice cream's characteristic properties, many consumers are cautious about the use of unfamiliar ingredients and are pushing for clean labels. As a result, companies are seeking to simplify product formulation without compromising product integrity.
The use of various high-pressure technologies on ice cream mix has shown potential to improve the quality of the final frozen product.
• Huppertz T.
• Smiddy M.A.
• Goff H.D.
• Kelly A.L.
Effects of high pressure treatment of mix on ice cream manufacture.
found that a high hydrostatic pressure treatment (>300 MPa) of ice cream mix significantly increased mix viscosity with increasing pressure treatment, and the resulting high-pressure-treated ice cream had a greater resistance to meltdown than an untreated control.
• Lim S.Y.
• Swanson B.G.
• Ross C.F.
• Clark S.
High hydrostatic pressure modification of whey protein concentrate for improved body and texture of lowfat ice cream.
used high hydrostatic pressure to treat whey protein concentrate in an ice cream mix and obtained a mix with enhanced overrun and foam stability that froze to produce a significantly harder ice cream.
High-pressure-jet (HPJ) technology, a more recent advancement in high-pressure technology, has been shown to enhance viscosity, foam stability, and emulsifying properties in skim milk, skim milk powder, and whole milk (
• Hettiarachchi C.A.
• Corzo-Martínez M.
• Mohan M.S.
• Harte F.M.
Enhanced foaming and emulsifying properties of high-pressure-jet-processed skim milk.
;
• Tran M.
• Roberts R.
• Felix T.L.
• Harte F.M.
Effect of high-pressure-jet processing on the viscosity and foaming properties of pasteurized whole milk.
;
• Hettiarachchi C.A.
• Voronin G.L.
• Harte F.M.
Spray drying of high pressure jet-processed condensed skim milk.
). We previously showed processing of low-fat ice cream mix using HPJ at pressure ≥400 MPa created fat–protein aggregates that increased apparent viscosity and non-Newtonian flow behavior (
• Voronin G.L.
• Roberts R.
• Felix T.L.
• Coupland J.N.
• Harte F.M.
Effect of high pressure jet processing on the physiochemical properties of low fat ice cream mix.
). These aggregates also entrapped air and appeared to sterically hinder ice crystal growth during static freezing. These observations led us to speculate that the viscous mix produced by HPJ might reduce the need for emulsifiers and stabilizers in ice cream manufacturing. However, the mix in that work was not dynamically frozen to create commercially viable low-fat ice cream.
In the present work, we hypothesized that the modified physiochemical properties observed in HPJ-treated (≥400 MPa) low-fat ice cream mix are retained upon freezing and lead to a decreased melting rate and increased hardness and apparent viscosity in the frozen product.

## MATERIALS AND METHODS

### Ice Cream Mix Processing

Pasteurized skim milk, skim milk powder, 40% fat cream, and sugar were purchased from the Penn State Berkey Creamery (University Park, PA) and used to formulate a 13% milk solids-not-fat, 6% fat, and 13% sugar low-fat ice cream mix containing 32% total solids. A control containing an emulsifier (polysorbate 80 at 0.075% wt/wt) was also prepared. Skim milk and cream were heated to 43°C before adding skim milk powder and sugar and mixing until a homogeneous mix was obtained. All samples were pasteurized (71°C for 30 min) under continuous stirring at a moderate speed that did not introduce air, then homogenized in a 2-stage homogenizer (Gaulin, Lake Mills, WI) with the first stage set at 13.8 MPa and the second stage set at 3.4 MPa. Following homogenization, samples were immediately cooled to <4°C and aged for at least 24 h before HPJ processing. Non-HPJ-treated samples with (C-P80) and without (C-0) emulsifiers were considered as controls.

### High-Pressure-Jet Processing

The mixes (4°C) were processed using a Hyperjet 94i-S pump system (Flow Internationals Corp., Kent, WA) set at the appropriate pressure: 100, 200, 300, 400, or 500 MPa. The pressure was achieved by an intensifier pump and maintained by an attenuator before the liquid sample exited the system through a diamond nozzle (10-µm inner diameter). Immediately after this nozzle, the samples were collected in a custom-made tube-in-tube heat exchanger with a counter-current flow of cold water (∼2°C), which cooled the samples to an outlet temperature of <30°C. The samples were then transferred to a refrigerator and held ∼24 h before dynamic freezing.

### Total Solids and Fat

The total solids and fat content of each replicate mix were tested. The total solids and fat content were determined with a Smart-Trac-II (CEM Corp., Matthews, NC).

### Freezing

The ice cream mixes (3 L) were frozen in a Taylor batch freezer (Taylor C117 Batch Freezer, Taylor Freezers, Rockton, IL). The mixes were agitated in the freezer until reaching a temperature of −5°C. When this temperature was reached, the condenser was shut off to continue agitation without further temperature reduction. During this isothermal whipping period, samples were extracted every 3 min for 12 min (starting at time 0) and tested for overrun. Mix samples and dynamically frozen samples at 0, 6, and 12 min were collected and stored (4°C) for particle size determination and confocal scanning laser microscopy (CSLM). After 12 min, the samples were collected in pint containers and stored for 1 wk at −25°C before rheological testing, hardness, and drip-through rate determination and transmission electron microscopy (TEM).

### Measurement of Properties During Dynamic Freezing

#### Overrun

Samples were extracted at the time points described above and immediately added to a tared transparent plastic container (90 mL) and weighed.
The overrun was calculated using Equation 1:
$Overrun=Wt.ofmix-Wt.oficecreamWt.oficecream×100,$
[1]

where Wt = weight, with the non-HPJ-treated ice cream mix (C-0) functioning as the initial mix weight for all overrun calculations. Two overrun measurements were completed for each replicate sample.

#### Particle Size

The particle size was determined by static light scattering using a Malvern MasterSizer 3000 (Malvern Instruments Ltd., Malvern, UK). One drop of each sample was added to the sampling unit with water under continuous agitation (refractive index of 1.33) to bring the obscuration into the optimum range determined by the manufacturer. The particle refractive index and absorbance were set at 1.47 (milkfat) and 0.01, respectively. This procedure was modified from
• Warren M.M.
• Hartel R.W.
Effects of emulsifier, overrun and dasher speed on ice cream microstructure and melting properties.
.

#### Rheological Characterization

Each ice cream sample (8 mL) was melted and placed in Discovery HR-3 (TA Instruments, New Castle, DE) rheometer fitted with a double wall concentric cylinder geometry (40.77-mm inside diameter, 43.88-mm outside diameter, 44.82-mm cup diameter). A preconditioning step involving the sample being held for 30 s at 4°C was used to ensure that the sample was equilibrated to 4°C before measurement. A flow curve was created by plotting shear stress (Pa) over a shear rate range of 1 to 100 s−1. Consistency coefficient and flow index values were determined by fitting the resulting flow curves with a Power Law model (Equation 2) using the Trios software (ver. 4.1.1.33073, TA Instruments):
$σ=kγ˙n,$
[2]

where σ is the shear stress (Pa), k is the consistency coefficient (Pa.sn), $γ˙$ is the shear rate (s−1), and n is the flow behavior index (
• Steffe J.F.
Rheological Methods in Food Process Engineering.
).

#### Confocal Scanning Laser Microscopy

Fluorescein isothiocyanate (10 µL, 0.1% wt/vol in acetone) and Nile Red (10 µL, 0.01% wt/vol in ethanol) were mixed into an aliquot (2 mL) of each sample before freezing and at the conclusion of isothermal whipping. The samples were then observed using an Olympus Fluoview 1000 Confocal Microscope (Olympus Corp., Center Valley, PA) at excitation wavelengths 488 and 633 nm. At these wavelengths, the blue and red probes fluoresced, and the fluorescing images were overlaid to highlight the location of the protein and fat, respectively. This method was modified from
• He W.
• Zhao W.
• Yang R.
Effects of wheat gluten modified by deamidation-heating with three different acids on the microstructure of model oil-in-water emulsion and rheological–physical property of ice cream.
.

### Measurement of Physical Properties after Hardening

#### Hardness

Samples were transferred into pint containers at the conclusion of isothermal whipping and placed into a hardening freezer (−25°C). After hardening for 1 wk, samples were removed from this freezer, tempered for 24 h at −15°C, and tested for hardness using a TA.XT2 (Texture Technologies, Hamilton, MA). The pint containers were sliced (3 cm from the bottom) to reveal a smooth surface for testing and quickly transferred from the freezer and penetrated 3 times in 3 separate locations (20 mm, 2 mm/s) with a cylindrical geometry (TA-55, diameter = 5 mm, length = 35 mm; Texture Technologies). The trigger force was set at 5 N. The hardness was determined by the peak force, which was averaged across the 3 measurements for each sample. This procedure was modified from
• Hartel R.
• Rankin S.
The effects of fat structures and ice cream mix viscosity on physical and sensory properties of ice cream.
.

#### Melting Rate

A disk of ice cream (approximate diameter = 2 cm, height = 1.9 cm, weight = 50 g) was removed from the center portion of each sample and placed onto a wire mesh (3 holes/cm) with a beaker beneath. The sample was then moved into a 22.4°C room, a timer was started, and the weight of dripped portion was recorded for a 60-min period. This procedure was completed in duplicate for each replicate sample. The proportion of melted ice cream (melted weight/initial weight × 100) was plotted against time and the slope of the linear period (20–40 min) was used to calculate melting rate.

### Microstructure Analysis

The TEM procedure was modified from
• Goff H.D.
• Verespej E.
• Smith A.K.
A study of fat and air structures in ice cream.
. A sample (1 mm3) was extracted at −25°C from the inner section of hardened ice cream samples using a razor blade. The samples were immediately immersed in liquid nitrogen. The samples were then transferred using precooled tweezers from liquid nitrogen into vials containing a fixative cocktail of 2.0% (wt/vol) uranyl acetate, 3.0% (vol/vol) glutaraldehyde, and 1.0% (wt/vol) osmium tetroxide in absolute methanol, which was stored in liquid nitrogen before use. These vials then went through a gradual temperature increase to allow for complete freeze substitution using the following Leica EM AFS (Leica Microsystems, Buffalo Grove, IL) program: hold at −80°C for 4 d, increase to −40°C (2°C/h), hold at −40°C for 18 h, and increase to −20°C (2°C/h). It was noted that the ice cream cubes remained floating on the top of the fixative cocktail during the initial temperature stages, so a small piece of paper towel saturated with fixative cocktail was placed over the vials to ensure that all of the sample blocks were immersed in the fixation solution. Furthermore, the samples were gently agitated twice daily to ensure even fixation. At −20°C, the fixation solution was discarded and the samples were washed with precooled methanol 3 times. The gradual infiltration with low-temperature embedding resin Lowicryl HM20 (Marvac Ltd., Halifax, NS, Canada) was completed at −20°C as follows: 1 wash with HM20: 100% methanol 1:2 for 30 min; 1 wash with HM20: 100% methanol 1:1 for 30 min; 1 wash with HM20: 100% methanol 3:1 for 30 min; and 3 changes of 100% HM20 with each wash occurring for 30 min. The samples were then polymerized with a UV light (360 nm) at −20°C for 72 h. Further polymerization was completed at 0°C for 24 h. Resin blocks were trimmed, and thin sections were cut into 80-nm thickness using a diamond knife (Diatome, Hartfield, PA) fitted on a Leica UC6 Ultramicrotome (Leica). The sections were then mounted onto 300-mesh formvar-carbon-coated copper grids (EMS, Hartfield, PA). Poststaining was completed by submersing samples into a drop of 2% aqueous uranyl acetate solution for 8 min followed by an immediate wash in ultrapure water. Then, samples were placed onto a drop of lead citrate solution (0.04%) for 5 min to complete the poststaining. Samples were viewed using a FEI Tecnai 12 BioTwin electron microscope (120 kV; FEI Company, Hillsboro, OR) and representative images were taken with a 4k Eagle CCD camera after viewing multiple fields per sample.

## RESULTS AND DISCUSSION

### Properties of Ice Cream Mixes

The average total solids and fat content for all ice cream mixes was 32.6 ± 0.6% and 6.1 ± 0.2%, respectively. No significant differences in ice cream mix density were observed except for the 400 MPa–treated (0.71 ± 0.08 g/mL) and 500 MPa–treated (0.75 ± 0.02 g/mL) ice cream mix samples, which were significantly (P < 0.05) less dense than the C-0 sample (1.07 ± 0.03 g/mL; Table 1). We have previously reported similar low densities in low-fat ice cream mix processed by HPJ at 400 MPa due to the incorporation of stable air bubbles sucked into the fluid stream as it exits the nozzle (
• Voronin G.L.
• Roberts R.
• Felix T.L.
• Coupland J.N.
• Harte F.M.
Effect of high pressure jet processing on the physiochemical properties of low fat ice cream mix.
). Samples treated at lower HPJ pressures (100–300 MPa) had an average density similar to the controls (i.e., C-0, C-P80).
Table 1Physical properties (±SD) of ice cream mix at 4°C
Sample
C-0 (control) = ice cream mix; C-P80 = low-fat ice cream mix with polysorbate 80; 100–500 MPa = low-fat ice cream mix processed by high-pressure jet at 100, 200, 300, 400, or 500 MPa.
Density (g/mL)Consistency coefficient (Pa·s
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
)
Flow behavior indexD[4,3]
D[4,3] = volume-moment mean diameter.
(μm)
C-01.07 ± 0.03
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
13.9 ± 2.0
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.96 ± 0.03
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.50 ± 0.06
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
C-P801.08 ± 0.00
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
14.9 ± 0.7
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.95 ± 0.00
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.51 ± 0.17
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
100 MPa1.07 ± 0.01
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
13.8 ± 1.6
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
1.00 ± 0.02
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.53 ± 0.08
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
200 MPa1.07 ± 0.02
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
13.3 ± 3.4
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.99 ± 0.04
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.45 ± 0.02
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
300 MPa0.98 ± 0.07
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
23.7 ± 3.2
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.96 ± 0.03
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
3.68 ± 1.94
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
400 MPa0.71 ± 0.08
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
284.4 ± 170.0
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.78 ± 0.04
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
13.06 ± 2.91
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
500 MPa0.75 ± 0.02
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
353.6 ± 122.6
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
0.74 ± 0.05
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
12.91 ± 0.77
Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
a,b Values with different superscripts within a column indicate significant differences at P < 0.05 (Tukey's test).
1 C-0 (control) = ice cream mix; C-P80 = low-fat ice cream mix with polysorbate 80; 100–500 MPa = low-fat ice cream mix processed by high-pressure jet at 100, 200, 300, 400, or 500 MPa.
2 D[4,3] = volume-moment mean diameter.
The flow behavior indexes and consistency coefficients of the ice cream mixes treated at 400 and 500 MPa were significantly different from the controls and from the samples treated with lower HPJ pressures (P < 0.05, Table 1 and Figure 1), with higher consistency coefficients and smaller flow behavior indices (i.e., more viscous and more shear-thinning). These results are consistent with previously reported data in HPJ-treated low-fat ice cream mix (
• Voronin G.L.
• Roberts R.
• Felix T.L.
• Coupland J.N.
• Harte F.M.
Effect of high pressure jet processing on the physiochemical properties of low fat ice cream mix.
).
The apparent volume-moment mean diameter (D[4,3]) values were significantly larger for the 400 MPa–treated (13.06 ± 2.91 µm) and 500 MPa–treated (12.91 ± 0.77 µm) ice cream mixes compared with the controls (C-0 = 0.50 ± 0.06 µm, C-P80 = 0.51 ± 0.17 µm) (Table 1). However, reported apparent particle sizes (especially at HPJ pressures > 300 MPa) should be treated with caution because the protein–lipid complexes have poorly defined refractive indices and shapes. Therefore, it was necessary to support the particle size determinations from light scattering with direct particle observation using CSLM (Figure 2). As expected, both control mixes before freezing (C-0 and C-P80) showed a well-dispersed mixture of protein and fat (Figure 2a and b), whereas ice cream mix processed at 400 MPa and 500 MPa showed aggregation of particles in which fat and protein were present together as a complex along with small, stable air bubbles (Figure 2c and d). These results are similar to previously reported data on the 400-MPa HPJ treatment of low-fat ice cream mix (
• Voronin G.L.
• Roberts R.
• Felix T.L.
• Coupland J.N.
• Harte F.M.
Effect of high pressure jet processing on the physiochemical properties of low fat ice cream mix.
). The presence of the large protein–lipid complexes in the samples treated at 400 MPa and 500 MPa was presumably responsible for the change in rheological properties of these samples (Figure 1), whereas the presence of air was responsible for their lower densities (Table 1).

### Behavior of Ice Cream Mixes During Dynamic Freezing

#### Control Samples (C-0 and C-P80)

The overrun of the C-0 and C-P80 samples increased during the isothermal whipping period at rates of 5.89 and 3.66% overrun per minute, respectively, to statistically similar final values after 12 min (118.8 ± 8.2% and 94.1 ± 10.1%, respectively, Figure 3). In agreement with
• Chang Y.
• Hartel R.W.
Development of air cells in a batch ice cream freezer.
, our results indicated that the emulsifier had little effect on the development of overrun.
After dynamic freezing, the melted C-0 sample had a consistency coefficient of 12.9 ± 2.0 mPa·sn and flow behavior index of 0.97 ± 0.03 (near-Newtonian behavior), whereas the melted C-P80 sample had a consistency coefficient of 9.8 ± 3.4 mPa·sn and a flow behavior index of 1.01 ± 0.06 (near-Newtonian behavior) (Figure 4). No significant differences were detected in the rheological properties of these 2 controls due to the presence of the emulsifier.
The particle sizes (determined by light scattering) for all samples before freezing and during the progression of isothermal whipping are presented in Supplemental Table S1 (https://doi.org/10.3168/jds.2020-19011) and shown in Figure 5 (D[4,3]). The D[4,3] values of the control samples (C-0 and C-P80) increased throughout the isothermal whipping period (Supplemental Table S1). At the end of the isothermal whipping period, the D[4,3] of the C-0 and C-P80 samples were 0.90 ± 0.19 µm and 2.21 ± 0.16 µm, respectively.
Confocal scanning laser microscopy micrographs of melted ice cream after dynamic freezing are shown in Figure 6. At the conclusion of dynamic freezing, the C-0 sample was a mix of protein and fat with some larger destabilized fat globules present (Figure 6a), whereas the C-P80 sample had much larger destabilized fat globules (Figure 6b). In a comparison of the CSLM micrographs of the samples before dynamic freezing (Figure 2) and after dynamic freezing (Figure 6), the greater fat destabilization in the C-P80 sample as a result of freezing is apparent. This outcome was expected because P80 has been shown to destabilize fat globules and allow for partial coalescence (
• Goff H.D.
• Hartel R.W.
Mix ingredients.
;
• Warren M.M.
• Hartel R.W.
Effects of emulsifier, overrun and dasher speed on ice cream microstructure and melting properties.
). The ice cream particle sizes quantified with light scattering (Supplemental Table S1 and Figure 5) agree with what was seen with CSLM.

#### Low-Pressure Regimen (≤300 MPa)

Samples of low-fat ice cream mix processed with low pressure HPJ (≤300 MPa) had physical properties (i.e., rheological properties, density, and particle size) similar to the C-0 samples (see above), so we expected that the ice creams made from these samples would also be similar. Indeed, no significant variations were present in the final overrun values (after isothermal whipping) for the various low-pressure-regimen ice cream samples (Figure 3). The rheological properties (Figure 4) and the particle size parameters (Figure 5) of the low-pressure-regimen samples after isothermal whipping also had no significant differences in comparison with the C-0 samples.

#### High-Pressure Regimen (≥400 MPa)

In contrast, samples of low-fat ice cream mix processed with higher HPJ pressures (400 and 500 MPa) were different from the control and low-pressure-regimen samples (see above), so we expected that the ice creams formed from them would also be different.
The ice cream mixes made with an HPJ treatment ≥400 MPa started with a greater initial overrun (Table 1), so the potential existed for these samples to show exceptionally high overrun after dynamic freezing and isothermal whipping. However, during dynamic freezing, the overrun increased more slowly than in the other samples and reached a statistically similar end-point at the end of isothermal whipping (Figure 3).
The only samples that had significantly (P < 0.05) different consistency coefficients from the melted C-0 sample (12.9 ± 2.0 mPa·sn) were the melted 400 MPa–treated and 500 MPa–treated ice cream samples with consistency coefficients of 21.5 ± 3.7 mPa·sn and 26.2 ± 5.6 mPa·sn, respectively. No significant differences were found in the flow behavior indices of any of the samples, with the 400 MPa–treated and 500 MPa–treated melted ice creams having flow behavior indices of 0.96 ± 0.02 and 0.96 ± 0.03, respectively. In a comparison of the rheological properties of the ice cream mixes before freezing (Figure 1) with those of the melted samples after dynamic freezing (Figure 4), the samples treated at 400 and 500 MPa clearly had far greater alterations in rheological properties during dynamic freezing than the other samples. Specifically, the dynamic freezing process decreased the consistency coefficients for the samples treated at 400 and 500 MPa from 284.4 ± 170.0 mPa·sn and 353.6 ± 122.6 mPa·sn to 21.5 ± 3.7 mPa·sn and 26.2 ± 5.6 mPa·sn, respectively. Furthermore, the non-Newtonian behavior seen in the 400 MPa–treated and 500 MPa–treated ice cream mixes was completely altered, with both samples behaving as near-Newtonian fluids after dynamic freezing (and melting). Despite these major shifts, the 400 MPa–treated and 500 MPa–treated melted ice creams still exhibited significantly (P < 0.05) larger consistency coefficients at the end of freezing than the C-0 sample.
At the conclusion of isothermal whipping, the D[4,3] value of the 400 MPa–treated sample (3.62 ± 1.53 µm; P < 0.05) was significantly larger than the C-0 sample (0.90 ± 0.19 µm, Figure 5). Additionally, unlike all other samples, the particle size of the ice cream mixes treated at 400 and 500 MPa decreased during the initial freezing process and by the time these samples reached −5°C, none of the particle size parameters remained significantly larger than the C-0 sample (Supplemental Table S1; https://doi.org/10.3168/jds.2020-19011). However, growth occurred in the particle sizes of the 400 MPa–treated and 500 MPa–treated samples from 6 to 12 min (D[4,3] increased from 2.81 ± 0.88 µm to 3.62 ± 1.53 µm and from D[4,3] = 1.60 ± 0.64 µm to 2.89 ± 1.17 µm, respectively).
Confocal scanning laser microscopy images of the samples treated at 400 and 500 MPa after dynamic freezing (Figure 6c and d) showed small regions of fat surrounded by protein-rich layers with diameters consistent with light-scattering measurements (Supplemental Table S1). These structures appeared to be the fat–protein complexes seen in the 400 MPa–treated and 500 MPa–treated low-fat ice cream mixes (Figure 2), but they were modified by the physical stresses of freezing. These structures, not present in the control samples or the low-pressure-regimen samples, were presumably responsible for differences in rheological properties (Figure 4) and particle sizes (Figure 5).

### Ice Cream Characterization After Hardening

The effect of HPJ treatment on fat droplets has not previously been visualized using TEM. Transmission electron microscopy images of the control and HPJ-processed hardened ice cream samples are shown in Figure 7, Figure 8, respectively. In the C-0 sample, casein micelles appeared as almost perfectly circular dark structures, whereas small fat globules were lighter in color but maintained well-defined edges as described by
• Goff H.D.
• Verespej E.
• Smith A.K.
A study of fat and air structures in ice cream.
. Distinguishing air cells from coalesced fat droplets was more difficult, but air cells were identified as large structures with rougher edges. In the C-0 sample, some casein micelles appeared to be adsorbed to the surface of fat globules, as they were likely acting as protein emulsifiers. The TEM of the C-0 sample was considered as a baseline for evaluating and comparing the other sample micrographs.
The C-P80 sample had more evidence of fat destabilization and aggregation than the C-0 sample. This destabilized fat network tended to congregate around air bubbles in the ice cream.
• Goff H.D.
• Verespej E.
• Smith A.K.
A study of fat and air structures in ice cream.
observed similar features in ice cream with polysorbate 80. The destabilized fat network likely contributed to the increased hardness and decreased melting rate of the C-P80 sample (Figure 9).
The ice creams made from mix processed at 300 MPa were expected to be similar to the C-0 ice cream sample because many of the properties (e.g., particle size, apparent viscosity) studied in the current work did not significantly differ from the C-0 sample. Indeed, the microstructure seen in the TEM images of the 300 MPa–treated sample had no major differences except for the presence of some larger casein micelles (>200 nm). Similarly sized micelles were visualized by
• Hettiarachchi C.A.
• Corzo-Martínez M.
• Mohan M.S.
• Harte F.M.
Enhanced foaming and emulsifying properties of high-pressure-jet-processed skim milk.
in skim milk processed at an HPJ pressure of 300 MPa.
In contrast, the microstructure of the 400 MPa–treated and 500 MPa–treated ice creams had notably different features than the C-0 sample (Figure 8, items 4 and 5). Fewer intact casein micelles were present in these samples, and dispersed protein was scattered throughout the serum of these ice creams. Smaller, less regular fat structures also seemed to be present in these samples. Again, some ambiguity was present regarding the identification of fat globules versus air cells, but CSLM of the melted ice creams (Figure 6) provided fluorescence-based identification of large coalesced fat globules. In agreement with CSLM, TEM revealed a very thick coating of protein around fat globules in the 400 MPa–treated ice cream. The protein-coated fat globules seen in the 400 MPa–treated sample and CSLM (Figure 6) were not observed in the TEM micrograph of the 500 MPa–treated sample. The disruption of casein micelles and fat droplets likely contributed to the altered rheological properties (Figure 4) and increased hardness (Figure 9).
Processing skim milk using HPJ ≥400 MPa has previously been shown to disrupt native casein micelle structure (
• Hettiarachchi C.A.
• Corzo-Martínez M.
• Mohan M.S.
• Harte F.M.
Enhanced foaming and emulsifying properties of high-pressure-jet-processed skim milk.
).
• Hettiarachchi C.A.
• Corzo-Martínez M.
• Mohan M.S.
• Harte F.M.
Enhanced foaming and emulsifying properties of high-pressure-jet-processed skim milk.
proposed that the disruption of micelles allowed for the release of surface-active casein protein monomers, which contributed to enhanced foamability and emulsification ability in HPJ-treated skim milk. The TEM images of 400 MPa–treated and 500 MPa–treated ice creams also showed the disruption of casein micelles and, in the 400 MPa–treated ice cream, this dispersed protein appeared to congregate around fat globules, which could aid in their stabilization (i.e., emulsification ability).

### Hardness and Melting Rate

Given the ice cream made from mix processed at 400 or 500 MPa HPJ had different rheological properties, particle sizes, and microstructures after freezing than both the control sample (C-0) and the low-pressure-regimen samples (≤300 MPa), we anticipated that the finished ice creams would also have different physical properties.
Significant differences in hardness were noted between the C-P80 ice creams (3,887 ± 1.68 g) and the 200 MPa–treated (2,254 ± 603 g) and 300 MPa–treated (1,589 ± 212 g) ice creams (P < 0.05, Figure 9). The 400 MPa–treated ice creams were the only samples that had significantly (P < 0.05) higher melting rates than the C-0 ice creams (2.91 ± 0.13 and 2.17 ± 0.13 g/min, respectively); however, the 300 MPa–treated (2.83 ± 0.31 g/min), 400 MPa–treated, and 500 MPa–treated (2.61 ± 0.31 g/min) ice creams melted significantly faster than the C-P80 ice creams (1.62 ± 0.25 g/min).
By plotting ice cream hardness as a function of melting rate for the finished ice cream (Figure 9), 4 distinct sample groupings became apparent: C-0, 100 MPa–treated, and 200 MPa–treated ice creams (intermediate hardness and meltdown); C-P80 (harder and more resistant to meltdown); 300 MPa–treated ice cream (softer and less resistant to meltdown); and 400 MPa–treated and 500 MPa–treated ice creams (harder and less resistant to meltdown).
Our results for the C-P80 sample were consistent with data reported by
• Hartel R.
• Rankin S.
The effects of fat structures and ice cream mix viscosity on physical and sensory properties of ice cream.
that showed that an ice cream with added polysorbate 80 (80:20 blend of mono- and diglycerides and polysorbate 80) was both harder and more resistant to meltdown than an ice cream with no emulsifier due to the formation of a destabilized fat network.
As mentioned above, the physical properties of ice creams that were HPJ-treated at pressures ≤300 MPa did not differ from the C-0 sample (Figure 4, Figure 5), so the freezing process and development of structural attributes in the 100 MPa–treated and 200 MPa–treated ice creams were not expected to vary from those of the C-0 sample. The 300 MPa–treated ice creams had significantly lower hardness values and greater melting rates than the C-P80 ice creams. This reduction in hardness and increase in melting rates for the ice creams made from mix processed at 300 MPa were unexpected because none of the physical properties varied significantly from the C-0 samples (Figure 4, Figure 5). Therefore, other structures that were not quantified in the present work (e.g., ice crystals, air cells) likely contributed to these differences.
We hypothesized that the development of large fat–protein complexes in the HPJ would alter ice cream hardness and meltdown properties, similarly to C-P80 ice cream samples in which a matrix of destabilized fat formed. Although the 400 MPa–treated and 500 MPa–treated ice creams had similar hardness values to the C-P80 samples, their melting rates were significantly greater. With this, the large particles observed in the 400 MPa–treated and 500 MPa–treated ice creams clearly did not behave as a typical polysorbate 80–based destabilized fat network. The dispersed protein seen in the TEM images of 400 MPa–treated and 500 MPa–treated ice creams likely contributed to the increased hardness values, but the large coalesced fat globules seen in the TEM images likely failed to support the ice cream structure during melting, increasing the melting rate compared with the C-P80 sample.

## CONCLUSIONS

This study demonstrates the use of HPJ technology to alter the physiochemical and microstructural characteristics in a low-fat ice cream. The HPJ treatment of the mix resulted in the formation of fat–protein complexes that entrapped air and contributed to altered rheological properties (increased non-Newtonian behavior and consistency coefficients). Although we originally hypothesized that the modified characteristics observed in HPJ-treated (≥400 MPa) low-fat ice cream mix would be retained upon dynamic freezing, the large fat–protein complexes progressively broke down during the agitation process. With further modifications of formulation and processing conditions, it may be possible to use the changed properties and microstructural attributes in the ≥400-MPa HPJ-treated and frozen ice cream samples to produce products with greater resistance to shrinkage, improved extrudability, and increased ice crystal stability.

## ACKNOWLEDGMENTS

This research was partially funded by Dairy Management Inc. through the project entitled “High pressure jet spray drying to create novel dairy powders” and by USDA National Institute of Food and Agriculture (Washington, DC) Federal Appropriations under Project PEN04565 and Accession number 1002916. The authors have not stated any conflicts of interest.

## Supplementary Material

• Supplementary appendix

## REFERENCES

• Hartel R.
• Rankin S.
The effects of fat structures and ice cream mix viscosity on physical and sensory properties of ice cream.
J. Food Sci. 2017; 82 (28631811): 1851-1860
• Bahramparvar M.
• Mazaheri Tehrani M.
Application and functions of stabilizers in ice cream.
Food Rev. Int. 2011; 27: 389-407
• Chang Y.
• Hartel R.W.
Development of air cells in a batch ice cream freezer.
J. Food Eng. 2002; 55: 71-78
• FDA
Code of Federal Regulations in Title 21.
• Flores A.A.
• Goff H.D.
Recrystallization in ice cream after constant and cycling temperature storage conditions as affected by stabilizers.
J. Dairy Sci. 1999; 82: 1408-1415
• Goff H.D.
• Hartel R.W.
Mix ingredients.
in: Ice Cream. Springer, US, Boston, MA2013: 45-87
• Goff H.D.
• Verespej E.
• Smith A.K.
A study of fat and air structures in ice cream.
Int. Dairy J. 1999; 9: 817-829
• He W.
• Zhao W.
• Yang R.
Effects of wheat gluten modified by deamidation-heating with three different acids on the microstructure of model oil-in-water emulsion and rheological–physical property of ice cream.
Food Hydrocoll. 2019; 87: 679-690
• Hettiarachchi C.A.
• Corzo-Martínez M.
• Mohan M.S.
• Harte F.M.
Enhanced foaming and emulsifying properties of high-pressure-jet-processed skim milk.
Int. Dairy J. 2018; 87: 60-66
• Hettiarachchi C.A.
• Voronin G.L.
• Harte F.M.
Spray drying of high pressure jet-processed condensed skim milk.
J. Food Eng. 2019; 261: 1-8
• Huppertz T.
• Smiddy M.A.
• Goff H.D.
• Kelly A.L.
Effects of high pressure treatment of mix on ice cream manufacture.
Int. Dairy J. 2011; 21: 718-726
• Lim S.Y.
• Swanson B.G.
• Ross C.F.
• Clark S.
High hydrostatic pressure modification of whey protein concentrate for improved body and texture of lowfat ice cream.
J. Dairy Sci. 2008; 91 (18349223): 1308-1316
• Steffe J.F.
Rheological Methods in Food Process Engineering.
2nd ed. Freeman Press, East Lansing, MI1996
• Tran M.
• Roberts R.
• Felix T.L.
• Harte F.M.
Effect of high-pressure-jet processing on the viscosity and foaming properties of pasteurized whole milk.
J. Dairy Sci. 2018; 101 (29477512): 3887-3899
• Voronin G.L.
• Roberts R.
• Felix T.L.
• Coupland J.N.
• Harte F.M.
Effect of high pressure jet processing on the physiochemical properties of low fat ice cream mix.
J. Dairy Sci. 2020; 103 (32307154): 6003-6014
• Warren M.M.
• Hartel R.W.
Effects of emulsifier, overrun and dasher speed on ice cream microstructure and melting properties.
J. Food Sci. 2018; 83 (29388677): 639-647