**3. Result and Discussion**

### *3.1. Determination of Steady Shear Rheological Properties of Ice Cream Mix for the Formulation Optimization*

In this study, the data of the steady shear rheological properties were used for the formulation optimization of ice cream mixtures. The flow curves of the ice cream mix obtained from 17 different trial points are shown in Figure 1. There was a decrease in the slope of the shear rate versus shear stress graphs of the ice cream mixes, indicating that the viscosity of all samples decreased with increasing shear rate. The reduction in viscosity can be explained by the structural breakdown of the intermolecular interaction [35]. The ice cream mixes showed shear-thinning flow characteristics, which is typical flow behavior for an ice cream mix. The shear-thinning behavior is an important factor in choosing the pump size for mixing [35].

**Figure 1.** Steady shear rheological behavior of the ice cream mixes.

Power law model parameters ( *K* and *n* values) and determination coefficient ( *R*2) calculated for 17 different points created with the trial design are shown in Table 1. *R*<sup>2</sup> values of the power law model were higher than 0.99. This shows that the power law model is suitable for determining the flow behavior properties of ice cream mixes. According to the ice cream formulations, *K* and *n* values differed and were found as 4.01–26.05 Pasn and 0.23–0.38, respectively. *n* values lower than 1 indicated that all ice cream mixtures exhibited the non-Newtonian pseudoplastic flow behavior (Table 1). Dairy products generally exhibit shear-thinning (pseudoplastic) behavior with flow behavior indexes of 0 < *n* <1[36]. As seen in Table 1, the sample −10 (0.3% XG, 2.5% fat, 1% CSOB) showed the lowest *K* value (4.01 Pasn), while the sample-11 (0.4% XG, 12.5% fat, 3% CSOB) exhibited the highest *K* value (26.05 Pasn). The *K* value of the samples increased with increasing CSOB, gum, and fat content. These results showed that increasing the *K* value with the increase in CSOB can improve the shear-thinning properties of ice cream mixes. The desired consistency values can be obtained by using CSOB even at low-fat and gum content. Thus, CSOB can be used for improving rheological properties in low-fat ice cream.

### *3.2. The Effect of Model Parameters on K and n Value and Determination Optimum Formulation*

Figure 2 presents the impact of gum, fat, and CSOB in the formulation on *K* value. The increase in fat, gum, and CSOB, as shown in Figure 2, resulted in an increase in the *K* value of the ice cream mixes. The structure of CSOB, which contains polysaccharides with high water-holding ability, can explain this phenomenon. This polysaccharide structure offers excellent water retention and stabilizing capabilities. CSOB can be adsorbed at the interface area and has surface-active qualities, in addition to its stabilizer action. Because of these CSOB characteristics, the *K* value of the ice cream mixes increased. Furthermore, increasing the amount of gum resulted in a considerable rise in the *K* value, particularly in formulations including xanthan gum at a specific level. The increase in *K* value in both increases in fat, CSOB, and gum content can be explained by the synergistic impact of the ice cream mix's components.

**Figure 2.** Response surface plot showing the effect of model parameters on the *K* value of ice cream mixes. (**a**): XG-CSOB, (**b**): XG-Fat, (**c**): CSOB-Fat (A: XG (xanthan gum), B: CSOB (chia seed oil by-product), C: fat (milk fat), *K*: consistency coefficient).

Table 2 showed that the response surface method (RSM) and the quadratic model were used to describe the influence of formulation components on the flow behavior parameters (*K*, *n*) of ice-cream mixtures. As seen in Table 2, the ANOVA analysis of variance was used to statistically evaluate the influence of dependent variables on the *K* value of the ice cream mixes.


**Table 2.** Quadratic model parameter's corresponding *K* value.

XG: xanthan gum, CSOB: chia seed oil by-product, *K*: consistency coefficient.

The *R*<sup>2</sup> and Adjusted *R*<sup>2</sup> values of the model used were determined as 0.9864 and 0.9618, respectively. The differences between Adjusted *R*<sup>2</sup> and predicted *R*<sup>2</sup> was lower than 0.2, and the lack of fit value was found as insignificant. These results indicated that the quadratic model could be successfully used to describe the effect of formulation on the *K* value of the samples. The *p*-value of the model was lower than 0.05, indicating that the model terms significantly affected the *K* value. In this model, A, B, C, AB, C<sup>2</sup> are significant model terms. The linear effect of all independent variables was significant. The interaction and model terms A and B, and the quadratic effect of C was also be found as significant.

Steady shear rheological analyzes showed that the increase in the amount of CSOB in the formulation resulted in a significant increase in the *K* values of the samples. Chia seeds affect fat binding and gel-forming properties due to the functional properties of dietary fiber. Olivos-Lugo et al. [37] reported that chia seeds are high in dietary fiber (34.6%), oil contents (32.2%), and protein (24.6%). Therewith, Akcicek and Karasu [20] suggested that CSOB could be used as a fat replacer in a low-fat salad dressing. Considering the properties of CSOB in this study, it is understood that it has a stabilizer feature due to its polysaccharide content and that the proteins it contains can be adsorbed in the interface area and have emulsifier quality. Proteins show surface-active properties and decrease the interfacial tension, which is predicted to cause an increase in consistency coefficient. The stabilizer feature comes from the branched polysaccharide structure in its content, and these polysaccharides can hold water [12]. Based on these properties, CSOB causes an increase in consistency coefficient (*K*) and can be used in the production of low-fat ice cream. With the increase in the *K* value of the mixtures, the *n* value decreases and shows pseudoplastic behavior, which is the typical flow behavior characteristic of ice cream. Due to the solid particles in CSOB, it significantly increases the *K* value by affecting the viscosity of the ice cream mixture. The fat, emulsifier, stabilizer, and CSOB used in ice cream mixtures significantly increase the *K* consistency coefficient of the mixtures. The main reason for this is the synergistic interaction between the components. The *K* value of the full-fat control sample was used to determine the optimum formulation. The sample with the highest desirability value and minimum fat content was selected as the optimum formulation. The optimum formulation was determined as 2.5% fat, 0.29% XG, 2.51% CSOB.

### *3.3. Rheological Properties of Optimum and Control Ice Cream Mixtures*

The steady shear, frequency sweep, and thixotropic properties of the samples produced based on optimum formulation were compared with the values of the full-fat and low-fat control sample. The flow curves of optimum and control ice cream samples were given in Figure 3. The ice cream mixtures performed a pseudoplastic (shear-thinning) rheological behavior; that is, the shear stress increased with increasing shear rate (Figure 3). Pseudoplastic rheological behavior is the typical behavior to characterize the flow properties of most ice cream mixtures [38,39]. The optimum and high-fat control samples showed similar viscosity curves. The power law model was used to determine the consistency coefficient (*K*) and flow behavior index (*n*) values of the optimal and control ice cream mixtures. Table 3 represents the power law model parameters (*K* and *n* values) as well as the determination coefficient (*R*2). *K* values varied from 3.46 to 5.66 Pasn, *n* values from 0.30 to 0.33, and *R*<sup>2</sup> values higher than 0.99. While the *K* value of the full-fat control samples and the *K* value of the sample containing CSOB were found to be statistically insignificant, the *K* value of both samples was found to be significantly higher than the low-fat control. This result showed that an ice cream mix similar to the consistency properties of full-fat ice cream could be produced with the use of CSOB.

**Figure 3.** Steady shear rheological behavior of ice cream mixes. FF-IC: full-fat ice cream, LF-IC: low-fat ice cream, CBLF-IC: low-fat ice cream with chia seed oil by-product.

The dynamic rheological properties of ice creams produced from optimum and highfat and low-fat control ice cream formulations were investigated. The frequency sweep test can simulate the liquid behavior of ice cream samples during chewing in the mouth [40], which helps to comprehensively evaluate the impact of CSOB addition on ice cream quality. Increasing *G* and *G* values of samples with increasing frequency is evidence of gel-like behavior in ice cream samples [41]. As seen in Figure 4, the value of *G* of all samples was higher than the value of *G*, indicating that the solid character of all ice cream samples is more dominant than the liquid character. As seen in Figure 4, the *G* value of FF-IC (12.5% fat) and CBLF-IC (2.5% fat) samples were almost at the same level, which explained that 10% fat can be compensated with 2.51% CSOB. As can be seen from the graph, the LF-IC instance has the lowest *G* and the lowest *G* . The *G* and *G* values of the LF-IC (2.5% fat, 0.35% XG) were lower than the *G* and *G* values of the CBLF-IC (2.5% fat, 0.29% XG, 2.51% CSOB). As it can also be seen in Table 3, the CBLF-IC has an elastic modulus value similar to FF-IC with full-fat content (12.5%). Aziz et al. [42] investigated the effect of

adding okra gum on the rheological, textural, and melting properties of low-fat ice cream samples. It was stated that *G* values were higher than *G* values for all ice cream samples. Substituting the fat content in ice cream with okra gum increased the viscous modulus (*G*) values of the samples. Previous studies on viscoelastic properties were consistent with the results of our study in terms of *G* and *G* values. Synergetic interactions between CSOB and ice cream ingredients can lead to improved food quality and expanded food applications due to enhanced functional properties. It may also have commercial potential for cost reduction.


**Table 3.** Steady shear, dynamic, and 3-ITT rheological model parameters of FF-IC, LF-IC, and CBLF-IC.

FF-IC: full-fat ice cream, LF-IC: low-fat ice cream, CBLF-IC: low-fat ice cream with chia seed oil by-product. Different lowercase letters in the same line indicate a statistical difference (*p* < 0.05).

> The dynamic rheological parameters (*K*, *K*, *<sup>n</sup>*, and *n* values) were also calculated by using the power law model (Table 3). The *R*<sup>2</sup> values of the model were found in the range of 0.97–0.98. As can be seen in Table 3, the *K* and *K* values of the samples were in the range of 3.09–23.41 and 2.46–8.46, respectively; the values of *n* and *n* were found in the range of 0.268–0.535 and 0.212–0.288, respectively. The *K* values were higher than the *K* values for all samples. Accordingly, all of the ice cream samples showed a viscoelastic solid character. When CSOB was added to low-fat ice cream, the *K* and *K* values increased when compared to the low-fat ice cream sample.

> All ice cream samples in the third interval exhibited thixotropic behavior (Figure 5). This result indicated that all ice cream samples could recover their viscoelastic character after high sudden deformation during food processing, such as homogenization or pumping. For ice cream combinations, this flow behavior is desirable. Akcicek and Karasu [20] investigated the thixotropic behavior of salad dressing samples stabilized by chia seed oil by-products and found that recoverable characteristics in the third interval are similar to our findings. The current investigation found that CSOB enhanced the thixotropic behavior of ice cream samples following rapid deformation.

**Figure 4.** Dynamic rheological properties of the ice cream mixes. (**A**): Storage modulus (*G*) vs. ω, (**B**): loss modulus (*G* ) vs. ω. FF-IC: full-fat ice cream, LF-IC: low-fat ice cream, CBLF-IC: low-fat ice cream with chia seed oil by-product.

Parameters (G0, Ge, *k*, G0/Ge) were obtained with the second-order structural kinetic model. G0, Ge, G0/Ge, *k* × 1000, and *R*<sup>2</sup> values were 8.42–21.12, 14.81–38.73, 1.760–1.834, 11.10–27.00, and 0.995–0.996, respectively. FF-IC showed the highest G0, Ge, G0/Ge, and k × 1000 values, indicating that the full-fat control sample (FF-IC) showed the highest thixotropic behavior. The amount of fat in an O/W emulsion has a significant impact on its rheological characteristics. Therefore, the decrease in the fat content of the ice cream samples causes weak rheological properties, especially the recoverable character, as the fat content of the ice cream samples decreases. Although CBLF-IC has low-fat content (2.5%), FF-IC and CBLF-IC showed similar thixotropic behavior and viscoelastic solid character so the higher recoverable behavior obtained with CSOB addition can be explained by more intermolecular interactions by the formation of small aggregates of hydrocolloid. These rheological properties indicate that CSOB can be utilized to enhance the rheological properties and thixotropy of low-fat ice cream samples.

**Figure 5.** 3-ITT rheological properties of ice cream mixes. FF-IC: full-fat ice cream, LF-IC: low-fat ice cream, CBLF-IC: low-fat ice cream with chia seed oil by-product.

### *3.4. Emulsion Stability and Microstructure Properties of Ice Cream Mixes*

The emulsion stability of the ice cream mixes was determined by the thermal loop test. The thermal loop test could be used as a fast method to measure emulsion stability by subjecting to different numbers of thermal cycles. The temperature fluctuations during processing, production, storage, and transportation stages were simulated in this test [31]. The structural or morphological changes due to the applied thermal stress are determined by the change of modules (*G\**, *G*) from cycle to cycle. Figure 6 shows the change in the *G\** value after applied thermal stress. As can be seen, after 10 applied thermal cycles, a slight increase in the *G\** value of all samples was observed. This insignificant change indicates that all samples are resistant to thermal stress and have high emulsion stability. The percent change (Δ*G\**) in the *G\** values of the samples was calculated as 14.37, 10.10, 8.37 for FF-IC, LF-IC, and CBLF-IC, respectively. This may indicate that the sample containing CSOB may show higher stability than other samples.

The fat particle size (d32), PDI value, and zeta potential values of the samples were found as 0.494–1.305 μm, (−39.13)–(−28.90) mV and 0.493–0.741, respectively (Table 3). As can be seen, the sample containing CSOB exhibited lower particle size and PDI value. These results are consistent with the thermal loop test. A decrease was observed in the zeta potential values of the samples as the water ratio increased. However, all samples have a sufficient zeta potential value. Figure 7 shows the milk fat particle distribution. The high-fat control sample and the sample containing CSOB have homogeneous droplet distribution and low-fat droplet size. These results indicated that the use of CSOB could have a positive effect on the fat droplet size and distribution, and emulsion stability in ice cream.

**Figure 6.** The change in the *G*\* value after applying the thermal loop. (**a**) CBLF-IC: low-fat ice cream with chia seed oil by-product, (**b**) FF-IC: full-fat ice cream, (**c**) LF-IC: low-fat ice cream.

**Figure 7.** Light microscopy pictures of the ice cream mixtures. FF-IC: full-fat ice cream, LF-IC: low-fat ice cream, CBLF-IC: low-fat ice cream with chia seed oil by-product. Samples were characterized at room temperature at 20× magnification.

### *3.5. Quality Parameters of Ice Cream*

### 3.5.1. Thermal Properties of Ice Cream

The thermal properties of the ice cream samples are presented in Table 4. The freezing points (Tf) of FF-IC, LF-IC, and CBLF-IC were obtained as −3.66, −3.35, and −3.99 ◦C, respectively. The freezing points of the ice cream samples were significantly differed (*p* < 0.05). The freezing point temperature of ice cream is closely related to the serum phase concentration and the soluble biopolymer concentration. Generally, the Tf value decreases as the serum phase concentration increases or the molecular weight of the soluble biopolymers decreases [43,44]. Soukoulis, Lebesi and Tzia [33] investigated the effect of different fiber contents on the thermal properties of ice cream. Similar to our study, a significant decrease was observed in the Tf values of ice creams when 2% wheat and oat fiber were added. Researchers explained this result by increasing the serum phase concentration due to the high water holding capacity of wheat and oat fiber. They reported an increase in the Tf value with the addition of apple fiber and inulin, which have higher soluble fiber content. The high water holding capacity of the insoluble fibers in the CSBO content may have caused a decrease in the Tf value. In addition, the use of less XG in the sample containing CSOB may have caused a decrease in the less soluble biopolymer substance in the serum phase, thus significantly reducing the Tf value. The higher Tf value obtained from LF-IC could be due to a decrease in solid concentration by reducing fat contents.

**Table 4.** Thermal properties of the control ice cream mixtures and optimum ice cream mixture contained CSOB.


FF-IC: full-fat ice cream, LF-IC: low-fat ice cream, CBLF-IC: low-fat ice cream with chia seed oil by-product. Different letters in the same column indicate a statistical difference (*p* < 0.05).

The melting resistance of ice cream represents the ability of ice cream to resist melting when exposed to high temperatures. The heating system in the DSC provides the temperature that causes the formation of an endothermic peak. The melting enthalpy ( ΔH) is calculated by integrating with the area of the endothermic peak and is the amount of energy leaving the system. It occurs as a change in the overall internal energy of food [45]. In our study, the melting enthalpy values of the samples were found as 166.0, 199.3, and 146.8 J/g for the FF-IC, LF-IC, and CBLF-IC, respectively. The freezable water amount is a critical parameter affecting ice melting enthalpy in ice cream. Melting enthalpy is less in ice creams with lower freezable water content [46]. The enthalpy value of the sample CBLF-IC (containing CSOB) was found to be lower than that of FF-IC and LF-IC (the full-fat and low-fat control samples, respectively). These results can be explained by the interaction between water and CSOB. The addition of CSOB to ice creams mix could increase the amount of bound water and reduced the amount of freezable water thanks to its water-binding capacity, which may lead to a decrease in enthalpy. In addition, as the fat reduction in ice cream samples was balanced by adding water, it caused high ice formation in ice cream. However, the CSOB produced a balanced effect by chemically binding the free water, preventing excessive ice crystal formation. The lower enthalpy value of ice cream containing CSOB than FF-IC despite higher water content could be explained by the higher water retention capacity and lowering amount of freezable water.

ΔT values were found as 14.16, 15.95, and 17.05 for the samples CBLF-IC, LF-IC, and FF-IC, respectively. The sample containing CSOB had the lowest ΔT value than control samples. The temperature range ( ΔT) could be used as an indicator of the uniformity of the size distribution of ice crystals. Therefore, a narrow melting temperature range indicates a more homogeneous distribution of ice crystals melting in a smaller temperature range [47]. The enrichment of ice creams in terms of polysaccharides and protein with CSOB facilitates the formation of tiny ice crystals, contributing to the improvement of texture perception and stability of ice crystals during cold storage.

### 3.5.2. Overrun Properties of Ice Cream

The overrun values are shown in Table 4. There was a significant difference between the overrun values of the samples. The sample containing CSOB exhibited the highest overrun value, while the low-fat control sample exhibited the lowest overrun value. The increase in the overrun value with the addition of CSOB may be due to the protein and high molecular carbohydrates in the CSOB content. Proteins play an important role in increasing foam stability thanks to their emulsification properties [48]. With the addition of CSOB to the formulation, both the protein content is gained, and the high molecular carbohydrate ratio is increased. Thus, an increase in the overrun value was observed with the addition of CSOB. Akalın et al. [49] reported that dietary fibers obtained from orange, apple, and wheat provided a significant increase in overrun values compared to the control sample. On the other hand, Mansour et al. [50] reported that the addition of datary fiber powder significantly reduced the overrun value of the ice cream samples. Some researchers have suggested that there is a relationship between the overrun value and the rheological properties [6,26]. Samakradhamrongthai et al. [51] reported that the overrun value increased with increased ice cream mix viscosity. The higher overrun with increasing viscosity could be explained by a more efficient breakdown of the incorporated air cells to a smaller air cell size of ice cream mix during freezing [51,52]. Similarly, the increase in the overrun value with the addition of CSOB in our study can be explained by the increase in the consistency index of the ice cream mix.

### 3.5.3. Sensory and Color Quality of Ice Cream

Sensory scores of ice cream samples are shown in Table 5. As can be seen, a significant difference was observed between the sensory scores of FF-IC and LF-IC. Whole-fat ice cream showed the highest value in all criteria. Low-fat ice cream containing CSOB (CBLF-IC), on the other hand, showed similar sensory properties to the full-fat sample (FF-IC),

except for the color and appearance criteria. With the addition of CSOB, the change in color values compared to the full-fat sample (FF-IC) is expected. There was no significant difference in the overall acceptability criteria of the CSOB-containing sample (CBLF-IC) and the full-fat control sample (FF-IC). This indicated that the color difference observed with the addition of CSOB could not adversely affect the consumability of ice cream. Eskandari and Akbari [53] and Akın et al. [54] reported that the addition of dietary fiber and other fat replacers did not cause a negative change in sensory scores of ice cream, similar to our study. In another study [51], the reduced fat ice cream prepared with inulin showed an acceptable sensory score, similar to our study. These results indicated that with the addition of CSOB, the tested quality properties of ice cream could be improved without adversely affecting the sensory properties and that low-fat ice cream could be produced in a similar way to achieve the quality properties of full-fat ice cream. *L\**, *a\** and *b\** color values of the samples are presented in Table 6. *L\**, *a\** and *b\** values of the samples were found as 77.08–83.42, 4.23–4.93 and 13.63–15.41, respectively. As can be seen, a significant difference was detected between the color values of the samples. The highest *L\** value was measured from the FF-IC sample, while the lowest *L\** value was obtained from the CBLF-IC sample. The high *L\** value of the FF-IC sample can be explained by the higher fat content than the other two samples. In addition, with the introduction of CSOB into the ice cream formulation, a decrease in *L\**, *a\** and *b\** values was observed. This result shows that CBLF-IC will cause a significant change in the color values of the ice cream samples.

**Table 5.** Sensory analysis of ice cream samples.


FF-IC: full-fat ice cream, LF-IC: low-fat ice cream, CBLF-IC: low-fat ice cream with chia seed oil by-product. Different letters in the same column indicate a statistical difference (*p* < 0.05).



FF-IC: full-fat ice cream, LF-IC: low-fat ice cream, CBLF-IC: low-fat ice cream with chia seed oil by-product. Different letters in the same column indicate a statistical difference (*p* < 0.05).
