1. Introduction

2. Materials and Methods

2.2. Methods

2.2.1. The making of beeswax oleogel

To prepare the oleogel, 5% of beeswax (BW) and refined sunflower oil (95%) were combined. The temperature of the mixture was raised to 80 °C to melt the wax. In order to guarantee a full and homogeneous distribution of the wax in the oil, the hot mixture was vigorously mixed to homogenize at 80 °C at 10000 x g using a T25 Ultra-Turrax model homogenizer (IKA, Germany). The samples were then ready for absorbing into the ice cream mix [20].

2.2.2. Ice cream manufacture and sampling

Ice creams were prepared in the laboratory of Atatürk University Faculty of Agriculture. In this study, twelve 12 different ice cream samples were produced using various fat and oil sources (milk fat and oleogel), stabilizers (salep and konjac gum), and emulsifiers [monoglyceride (MG), palsgaard (PG), and none emulsifier (NE)]. Ice cream samples were made with 1500g milk (3% milkfat or non-fat), 40g non-fat milk powder, 90g raw cream (50% milkfat) or oleogel, 300g sucrose, 15g stabilizers salep or konjac gum(KG)) and 8g emulsifiers monoglyceride(MG) or polsagaard (PG). At A, D, G, and J samples, emulsifiers were not used (NE).

Ice cream samples were made in an ice cream machine (Uğur Cooling Inc., Turkey) and hardened in a deep freezer at -20 °C. All analyses in the study were initiated in duplicate.

Table 1. The content of components used in ice cream mix formulations.

Table 1. The content of components used in ice cream mix formulations.

Samples	Fat or oil sources	Stabilizers	Emulsifiers	Milk	Milk powder	Raw cream	Sucrose
A	milk fat	Salep	NE	fat	+	+	+
B	milk fat	Salep	MG	fat	+	+	+
C	milk fat	Salep	PG	fat	+	+	+
D	milk fat	KG	NE	fat	+	+	+
E	milk fat	KG	MG	fat	+	+	+
F	milk fat	KG	PG	fat	+	+	+
G	oleogel	Salep	NE	non fat	+	_	+
H	Oleogel	Salep	MG	non fat	+	_	+
I	Oleogel	Salep	PG	non fat	+	_	+
J	Oleogel	KG	NE	non fat	+	_	+
K	Oleogel	KG	MG	non fat	+	_	+
L	Oleogel	KG	PG	non fat	+	_	+

2.2.3. Physicochemical analysis

The Physicochemical analysis of milk, cream, sunflower oil, and oleogel used in ice cream making

Dry matter, ash, fat and oil content and specific weights of milk, cream and oleogel were measured according to these methods [21]. Titratable acidity was determined by a titrimetric method. pH was measured with a pH meter (Seven Compact pH/Ion meter S220; Mettler Toledo, Switzerland)[22]. Milk, cream, and oleogel viscosity were measured at 4 °C using a viscometer (Model DV-II; Brookfield Engineering Laboratories, USA) at 20 and 50 rpm [23]. The amount of total oil in sunflower oil and oleogel was measured using the Soxhlet extractor model PBI (Buchi, Italy) according to AOAC (2000) Method No 30-25. [24].

Physicochemical analysis of ice cream samples

Dry matter, ash, fat and oil content and specific gravity were determined according to the methods given by the researchers. [22]. Titre edilebilir asitlik, titrimetrik yöntemle belirlendi ve pH, bir pH metre (Seven Compact pH/Ion meter S220; Mettler Toledo, İsviçre) [21] ile analiz edildi. Color determination in ice cream varieties was performed using a chromameter device (CR-300; KonicaMinolta, Japan) using a D65/10 standard light source, which corresponds to the daylight reading value according to the CIE system. L*, a*, and b* values of the samples were recorded in the reading made with the device. According to this, the L* value is brightness in daylight (0: black, 100: white), the a* value is green-red color (-80 to 0: green, 0 to +50: gray, +50 to +100: red), The b* value represents the blue-yellow color (from -50 to 0: blue, from 0 to +50: yellow)[25]. Mixture viscosity was measured at 4 °C using a viscometer (Model DV-II; Brookfield Engineering Laboratories, USA) at 20 and 50 rpm [23]. Also, overrun was determined using the method proposed by Jimenez-Florez et al. [26]; first dripping and complete melting times were detected using the method of using by Cotrell et al. [27]. A technique based on the process was used to calculate the fat destabilization index in ice cream and the melted ice cream samples [16].

2.3.4. Fatty acid analysis of ice cream samples

Fatty Acid analyses of ice cream samples were made using the methods proposed by Sukhija and Palmquist [28].

Lipid extraction: Removing fat and oil from ice cream sample

It was performed Lipid Extraction for free fatty acids (FFA) using the modified procedures by Deeth et al. [29]. Two grams of frozen sample were weighed into a 100 mL beaker, then 8 mL of methanol and 18 mL of chloroform were added and homogenized for 30 seconds. After homogenization, 9 mL of chloroform was added to the beaker and homogenized for another 30 seconds. Then 9 mL of zinc acetate (0.115 g of zinc acetate / 5 mL of deionized water) was added and homogenized for 30 seconds. The mixtures were then poured into a 125 mL separatory funnel and refrigerated until the two layers were separated. Once two layers were visible, the fat layer was transferred into a capped glass tube and stored in a freezer (-18°C) until fatty acid analysis.

Preparation of fatty acid methyl esters

Fatty acids methyl esters (FAME) were performed according to the (AOAC 996.01) (Satchithanandam et al., 2001) procedure [30] with some modifications. Four milliliters of 0.5 N methanolic sodium hydroxide were added to 0.1 gram of the extracted fat in a vial. The vial was then connected to a water-cooled condenser and its contents refluxed for 10 minutes. After refluxing, 5 mL of BF3-methanol reagent (Supleco Inc., Bellafonate, PA, USA) was added to the reaction flask through the concentrator and the contents of the flask were refluxed for another minute. After the vials were cooled, saturated NaCl solution was added to the bottle to float the pentane solution containing the methyl esters on the neck of the vial. Finally, five to seven milliliters of pentane solution containing methyl esters was transferred to a test tube and a small amount of anhydrous sodium sulfate was added to dry the pentane solution.

Analysis of fatty acid methyl esters in gas chromatography(GC)

It was analyzed using a Thermo Electronic (Austin, TX, USA) gas chromatograph-MS (Model TRACE GC Ultra) equipped with (Model AS-3000-Thermo Electronic Company) autosampler. A fused silica capillary column (0.25 mm id x 0.25 µm film thickness, x 60-m; SP-2380 Supelco, Inc., Bellefonte, PA, USA) was used to separate the methyl esters detected by a flame ionization detector (FID). The injection temperature was programmed between 10˚C and 220˚C at 2˚C / min. Helium was used as the carrier gas with a flow rate of 1.6 mL/min and a partition ratio of 30: 1. Identification of individual FAME from the sample was done by matching the retention time of unknown FAME with standard mixtures (Alltech Associates, Inc., Deerfield, IL, USA; Sigma-Aldrich Corp., Bellefonte, PA, USA). Fat acids analysis of ice cream samples was measured on a polar capillary column (30 m×0.32 mm) using a flame ionization detector. Analysis of the fatty acids profile was determined as fatty acid methyl esters. 300-μL of thawed and thoroughly mixed sample was taken in an 11-mL screw-capped test tube. Samples dissolved in 3mL of isooctane and 2mL of 1.5 N sodium methoxide and were then vortexed at a higher rate for exactly 3 min, allowed to separate for 5 min, and the supernatant was injected into the Gas Chromatograph.

Optical microscopy

The size and distribution of bubbles in the ice creams were observed and analyzed using a Light Microscope with Integrated Camera. After the samples were placed on a slide, a coverslip was covered and observed with an optical microscope (Zeiss Axio Scope A1, Carl Zeiss Co., Ltd., Jena, Germany) at 20x magnification. Images were transferred to a computer using a digital camera and analyzed with Image-Pro Plus 6.0 (Media Cybernetics Inc, Bethesda, USA) software [31].

Sensory assessment

Ice cream samples were placed in special 100 ml odor-free containers with glass lids and presented to the panelists in a randomly coded manner at regular intervals. While the panelists were performing sensory analysis, water was placed in 250 ml glass bottle containers to clean their mouths before moving on to the other sample. Sensory evaluations were made by considering color and appearance, texture and consistency, taste and smell, sweetness and gummy structure and general acceptability.Sensory evaluation was performed in a spaced seating arrangement in a room at an appropriate temperature (25 °C). Ice cream samples (–10 °C) were presented to the panelists. The samples were evaluated on the scoring tables from 1 to 9. 10 experts in the Department of Food Engineering (consisting of ten academicians, Ph.D., postgraduate, and undergraduate students) were evaluated by the panelists. Each panelist was experienced individuals trained and informed about sensory analysis methodology [32].

Statistical analysis

The trial was set up as 2 (adding of olojel and cream) x 2 (addition of stabilizer) x 3 (addition of emulsifier) according to a 2x2x3 fully random factorial trial plan. The experiment was set up according to a 2x2x3 full random factorial experiment plan, as 2 (addition of oleogel and cream) x 2 (addition of stabilizer) x 3 (addition of emulsifier). The experiment was carried out in three replications. Two-way analysis of variance (TWO-way ANOVA) was used for the determination the differences between the ice cream samples and the effects of storage. Accordingly, statistical analysis software SPSS version 15.0 (SPSS Inc. Chicago, Illinois) was used. The significant data as a result of analysis of variance (ANOVA) were tested according to the Duncan multiple comparison test at p <0.01 level.

3. Results and Discussion

3.2. The results of the physicochemical analysis of the mix and ice cream samples

The results of physicochemical analysis of the mix and ice cream samples were given in Table 3.

The dry matter and ash ratios of ice cream samples ranged from 29.82 to 33.13% and 0.72 to 0.97%, respectively. The milk fat ratio of the mixed samples ranged between 4.65 and 5.10%. Sulejmani and Demiri [33] made the ice cream samples with stevia and emulsifiers and found that drymater, ash and fat ratio ranged between 36.12 and 56.62%, 0.34 and 0.63% and 0.00 and 3.96%, respectively. The oil ratios ranged between 6.15 and 6.50. The oil ratio of samples with added oleogel was higher statistically (p < 0.01) than that of milk fat. The dreymater ratios by Sulejmani and Demiri were higher than that of this study but ash and fat ratios were lower. In parallel with the results of the researchers (0.16% and 0.22%), the acidity of the samples varied between 0.13% and 0.18%The pH of the samples ranged from 5.91 to 6.71. The results of the same research found that the pH of the ice cream samples was between 5.92 and 6.48 in parallel with this study[33] . The overrun ratios of the samples ranged from 6.50 to 42.10 %. It was found that the overrun ratio of ice cream samples containing milk fat was greater than that of oleogel (Figure 1). In a study, ice cream was made by adding 50 % and 100 %t sunflower oil instead of milk fat. It was discovered that it had the lowest overrun percentage and melt resistance among all ice creams and our results were parallel with the findings of this research [34].

Corradini and others found that when palm oil and coconut fat were added to the ice cream mix, the overrun of ice cream samples ranged between 48.8 and 50.4 % [5]. The findings of Corradini et al.[5] were higher than the findings of this study. Viscosity is one of the most important properties of an ice cream mix as it frequently contributes to ice cream's desirable body and texture. At 20rpm, the viscosity of the mix samples ranged from 10.06 Pa.s to 28.00 Pa.s. At 50 rpm, the viscosity ranged from 7.00 to 14.70 Pa.s. The viscosity of ice cream batters containing PG and oleogel was generally higher than that of other treatments (Figure 2). In a study, Mariano and Alamprese [35] added gelator at two different levels to the ice cream mix and found that mix viscosity of the samples with added gelator was statistically lower (p <0.01) than that of milk fat as different from this study. In a study by Ozdemir et al. [36], they found that at a sliding speed of 20 rpm the viscosity of the ice cream samples ranged between 7.03 and 9.28 Pa.s.This study's viscosity results were higher than those of Ozdemir et al. [36]. According to Minhas et al. the viscosity of ice cream samples ranged from 0.29 to 1.17 Pa.s.[37]. In this study, Minhas et al. [37] discovered viscosity values in ice cream that were lower than those of our findings. This kind of situation can be caused by various ice cream mix compositions as well as various stabilizers and emulsifiers.

Figure 1. The overrun ratios of ice cream samples added cream, oleogel, stabilizers, and emulsifiers.

Figure 1. The overrun ratios of ice cream samples added cream, oleogel, stabilizers, and emulsifiers.

Figure 2. The viscosity of the ice cream mix added cream, oleogel, and stabilizer.

Figure 2. The viscosity of the ice cream mix added cream, oleogel, and stabilizer.

3.6. The microscopic appearance of the samples of ice cream

The microscopic appearance of the samples of ice cream is given in Figure 10.

At the microscopic appearance of samples of oleogel added ice cream, while the oil and water particles were not dispersed as homogeneous (Figure 10A,C), at the samples of milk fat added ice cream components were dispersed as very homogenous (Figure 10B,D). The fat destabilization ratios of samples of oleogel added ice cream was lower than that of milk fat (Figure 4) as parallel to microscopic appearance.

Table 7. The correlations test results of ice cream samples.

Table 7. The correlations test results of ice cream samples.

	First Drop Time	Complete melting time	Melting Ratio	Dry matter	Fat or oleogel	pH	Overrun	L*	a*	b*	Fat Desatabili	Viscosity ( 20 rpm)	Viscosity ( 50 rpm)	C4:0	C6:0	C14:0	C16:0	C18:1	C18:2	C18:3
F.Drop.Time	1,00	,534**	-,580**	0,26	0,39	-0,09	-0,32	0,09	0,22	-0,37	-0,35	,809**	,856**	-0,38	-,439*	-0,39	-0,39	0,37	0,38	,419*
Co.Melt.Time	0.53**	1,00	-,752**	-0,05	,734**	-0,32	-,743**	-0,16	-0,17	-,467*	-,607**	0,27	0,20	-,803**	-,821**	-,754**	-,767**	,824**	,780**	,770**
Mel.Ratio	-,58**	-,752**	1,00	-0,23	-,439*	0,39	,549**	0,00	,408*	0,15	,513*	-0,36	-0,30	,510*	,625**	,472*	,553**	-,468*	-,482*	-,444*
Dry matter	0,26	-0,05	-0,23	1,00	-0,20	,569**	0,05	0,15	-0,37	0,36	0,40	0,34	0,25	0,09	-0,12	0,14	-0,13	0,15	-0,14	-0,07
Fat/oleogel	0,39	,734**	-,439*	-0,20	1,00	-0,37	-,751**	-0,12	-0,21	-0,39	-,702**	0,00	0,05	-,947**	-,809**	-,965**	-,775**	,554**	,967**	,951**
pH	-0,09	-0,32	0,39	,569**	-0,37	1,00	0,25	-0,25	0,02	0,14	,550**	0,28	0,18	0,31	0,19	0,33	0,19	0,02	-0,34	-0,23
Overrun	-0,32	-,743**	,549**	0,05	-,751**	0,25	1,00	0,26	0,31	0,27	0,30	0,07	0,11	,787**	,698**	,757**	,757**	-,715**	-,768**	-,748**
L*	0,09	-0,16	0,00	0,15	-0,12	-0,25	0,26	1,00	-0,03	0,32	0,07	0,06	0,08	0,17	0,07	0,21	0,04	-0,18	-0,18	-0,22
a*	0,22	-0,17	,408*	-0,37	-0,21	0,02	0,31	-0,03	1,00	-,490*	0,01	0,30	0,40	0,32	0,38	0,28	0,30	-0,06	-0,29	-0,20
b*	-0,37	-,467*	0,15	0,36	-0,39	0,14	0,27	0,32	-,490*	1,00	,412*	-0,35	-0,32	0,39	,409*	0,38	0,38	-0,31	-0,38	-,444*
Fat Destab.	-0,35	-,607**	,513*	0,40	-,702**	,550**	0,30	0,07	0,01	,412*	1,00	-0,16	-0,24	,610**	,528**	,656**	,431*	-0,26	-,655**	-,631**
Visc.(20)	,81**	0,27	-0,36	0,34	0,00	0,28	0,07	0,06	0,30	-0,35	-0,16	1,00	,953**	-0,01	-0,23	0,00	-0,15	0,16	-0,01	0,05
Visc.(50)	,85**	0,20	-0,30	0,25	0,05	0,18	0,11	0,08	0,40	-0,32	-0,24	,953**	1,00	-0,01	-0,12	-0,04	-0,03	0,06	0,02	0,09
C4:0	-0,38	-,803**	,510*	0,09	-,947**	0,31	,787**	0,17	0,32	0,39	,610**	-0,01	-0,01	1,00	,890**	,984**	,823**	-,627**	-,992**	-,954**
C6:0	-,44*	-,821**	,625**	-0,12	-,809**	0,19	,698**	0,07	0,38	,409*	,528**	-0,23	-0,12	,890**	1,00	,832**	,949**	-,646**	-,843**	-,818**
C14:0	-0,39	-,754**	,472*	0,14	-,965**	0,33	,757**	0,21	0,28	0,38	,656**	0,00	-0,04	,984**	,832**	1,00	,762**	-,558**	-,995**	-,955**
C16:0	-0,39	-,767**	,553**	-0,13	-,775**	0,19	,757**	0,04	0,30	0,38	,431*	-0,15	-0,03	,823**	,949**	,762**	1,00	-,703**	-,773**	-,760**
C18:1	0,37	,824**	-,468*	0,15	,554**	0,02	-,715**	-0,18	-0,06	-0,31	-0,26	0,16	0,06	-,627**	-,646**	-,558**	-,703**	1,00	,594**	,632**
C18:2	0,38	,780**	-,482*	-0,14	,967**	-0,34	-,768**	-0,18	-0,29	-0,38	-,655**	-0,01	0,02	-,992**	-,843**	-,995**	-,773**	,594**	1,00	,961**
C18:3	,42*	,770**	-,444*	-0,07	,951**	-0,23	-,748**	-0,22	-0,20	-,444*	-,631**	0,05	0,09	-,954**	-,818**	-,955**	-,760**	,632**	,961**	1,00

From the correlation test results (Table 7), there is a negative correlation between overrun and complete melting time (r=0.7, p <0.01). The complete melting time of the samples with high volume increase was shortened. Ice creams with low volume increase melt faster. It was determined that slower melting in ice cream samples was due to lower air transfer due to the large volume of air, but the main reason was low-fat stabilization [42] . There is a negative correlation between the overrun and fat ratio (milk fat/oleogel) (r=0.8, p <0.01). As the fat ratio increased, the volume increase decreased. But, Güven et al. [43] found that low-fat ice creams had low volume. In ice cream samples, as the ratio of saturated fatty acids (C:4, C:6, C:14, C:16) increased, the volume increase rate increased (positive correlation) too, and as the unsaturated fat ratio increased, the volume increase decreased. It is seen in Table 3 that the volume increase of ice cream mix added oleogels that are rich in unsaturated fatty acids were lower than that of milk fat ice cream. There is a positive correlation between the amount of milk fat or solidified oil and complete melting time (r=0.7, p <0.01). As the amount of fat increased, the completed melting time also increased (Table 3 and Table 4). The fat destabilization decreased as the fat ratio increased in the ice cream samples. Hatipoglu [44] found that as the fat content in ice cream increased, the volume increase rate also increased, but the viscosity of the ice cream mix samples decreased.

There was a positive correlation between pH level and fat destabilization (r=0.55, p <0.01). As the pH level increased, the fat destabilization level also increased. This suggests that , it is important to have a higher pH value for the fat destabilization value to be high in ice cream production. In ice cream samples with a high ratio of saturated fatty acids (C: 4, C:6, C:14, C:16), the complete melting time decreased (negative correlation), but as the ratio of unsaturated fatty acids increased, complete melting time decreased (p<0.01). In Table 4, it can be seen that the complete melting times of the samples added oleogel (G, H, F, J, K, L) are significantly (p <0.01) higher than the samples added milk fat as parallel correlation results. The first dripping time was positively correlated with viscosity results (r=0.81, p <0.01), but the differences between viscosity results did not affect the full melting time (Table 7).

3.7. Results of Sensory Analysis of Ice Cream Samples

Table 7. The results of sensory analysis of ice cream samples.

Table 7. The results of sensory analysis of ice cream samples.

Samples	Color Appearance	Texture Consistency	Gummy	Taste Smell	Sweetness	General Acceptability
A	8.50	8.00	8.00	8.00	7.50	8.00
B	9.00	8.00	7.00	7.50	7.50	7.00
C	8.50	8.50	8.00	9.00	8.50	9.00
D	8.00	8.00	7.00	7.50	6.50	6.50
E	8.00	8.00	7.00	7.00	7.00	7.00
F	9.00	8.00	8.00	7.50	7.50	7.50
G	8.00	7.00	7.00	5.00	6.00	5.00
H	7.50	7.50	7.00	7.00	7.50	7.00
I	7.50	7.50	7.50	6.50	7.00	6.50
J	8.50	8.00	8.00	7.50	7.50	7.50
K	8.50	7.50	6.50	7.00	7.50	7.00
L	9.00	8.00	8.00	7.00	7.50	6.50

Note: Items were scored as a maximum of 9 by panelists .

C samples with added milk fat, salep (stabilizer), and palsgaard (emulsifier) were preferred by panelists. Panelists scored the G sample with salep and oleogel lower than the I sample with salep, palsgaard, and oleogel.

4. Conclusions

Abbreviations List

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