MATERIALS AND METHODS

Materials: Skim milk powder (СJSC, Alekseevsky Dairy Canning Plant, Belgorod region, Russia), butter at 72.5% (OJSC, Rogachevsky Dairy Canning Plant, Belarus), commercial-grade granulated sucrose, wheat germ oil (Sampo Company, Russia) and vanilla favor (Boehringer Manheim GMB, Germany) purchased from the local market at Moscow, Russia. Stabilizer, Cremodan 334, was provided by DuPont-Danisco, Company Russia.

Methods: This study was conducted at Moscow State University of Food Production and All-Russian Scientific Research Institute of Refrigeration Industry (Moscow, Russia), during the period from October 2019 to January 2020.

Preparation of minor components extract: The minor components of wheat germ oil were extracted using hexane/methanol system, as described by Miraliakbari and Shahidi¹⁸. In 500 mL separator funnel, 20 g of oil was mixed with 200 mL of hexane, 100 mL of methanol was added to the mixture, sealed and then agitated for 5 min with periodic venting. The separator funnel was then sealed with nitrogen and stored at 4°C for 1 h. The methanol fraction was decanted into a 1 L round flask. The extraction process was repeated 4 times and the pooled methanol extracts were evaporated in a rotary vacuum evaporator (Rotavapor R110, Buchi, Switzerland) at 50°C. The wheat germ oil extract (WGOE) was stored at -80°C until used within a maximum period of one week. Some minor components, measured by HPLC, in the WGOE are shown in Table 1.

Ice cream making: Ice cream mixes were formulated separately in 2 kg batches to contain 10.0% fat, 15.0% sugar, 11.5% MSNF and 0.5% Cremodan 334 as a stabilizer.

Table 1:	Some minor components in the wheat germ oil extract

The WGOE was added to mixes at a rate of 0.5, 1.0, 1.5, 2.0 and 3.0% to create 5 experimental mixes. The control mix was formulated to contain the same amount of fat, sugar, MSNF and stabilizer but without WGOE. After blending the wet and dry ingredients, the mix was homogenized at 65°C using a laboratory homogenizer (Rannie, Copenhagen, Denmark) at 13.6 MPa and 3.5 MPa in a two-stage process, pasteurized at 81°C, cooled and aged overnight at 5°C. Just before freezing in a batch freezer (Carpigiani LABO 8 12 E Batch Freezers, Russia) for 12 min, vanilla was added to flavor the mix at a usage level of 0.1% (wt/wt). The resultant ice cream was poured into 250 mL PVC cups, covered and hardened in a deep freezer at -25°C for 24 hrs before analysis. Three replicates were done for each batch.

Analysis of ice cream mixes
Acidity and pH: Calibrated pH-meter (HANNA Instruments, pH 211, Italy), with a glass electrode, was used for directly measuring the pH value of the ice cream mixes. Acidity content, expressed as percentage lactic acid, was determined by using 0.10 N NaOH to the phenolphthalein end point¹⁹.

Dynamic viscosity: The dynamic viscosity of the ice cream mixes was measured using a Brookfield DV-II+ Pro Programmable Viscometer (Brookfield Engineering Laboratories, INC. Middleboro, MA 02346-1031, USA) with a measuring spindle SC4-31 at 4 rpm after aging. The ice cream mix was poured into a 10 cm³ metal cylinder and the viscosity was measured at 5°C. Each result was then recorded in triplicates in mPa.s after 30 sec rotation.

Overrun: Overrun was calculated for all ice cream samples using the weight-volume method as described by Daw and Hartel²⁰. A volume of ice cream mix was weighed before freezing and the same volume of ice cream was weighed after freezing. Overrun was calculated using the following equation:

Air cell size distribution: Air cell sizes were measured using an Olympus CX41 microscope (Japan) with a built-in camera according to the method of Chang and Hartel²¹. A temperature-controlled, refrigerated was set at (-6°C) for measurements. A small amount (0.2-0.5 mL) of ice cream was carefully placed in the depression on the slide and covered with a coverslip. Air bubbles were displayed at the surface of the coverslip. No dispersing liquid was needed. At least two slides of each sample were prepared and approximately four images were taken for air cells. The studies were carried out at an increase of 100 times. The research results were processed using the Image Scope M program.

Ice crystal size distribution: The dispersion of ice crystals was determined according to the method developed at the All-Russian Research Institute of Scientific Research²². For this, an Olympus Cx41 microscope connected to a PC with software was used.

Melting resistance: Melting resistance of ice cream samples was carried out according to the method of Goff and Hartel². Ice cream samples (250 mL) stored at -25°C were weight and prepared by carefully cutting the PVC cups and left to melt into a plastic container below a mesh screen at 20±1°C. After 60 min, the weight of the melted ice cream was recorded every 10 min for 120 min. Melting resistance was calculated as the weight of ice cream melt/weight of ice cream before melting x100. Melting rates were calculated by determining the equation for the linear portion of the melting data (from 60 to 120 min) according to Alvarez et al.²³

The dimensional stability: A sample of frozen ice cream was taken from a metal sample previously aged for at least 5 hrs at a temperature of -20°C, placed in a Petri dish and then in a thermostat with a temperature of 20±1°C. Immediately after installation in the thermostat, the sample was photographed every 10 min.

Sensory analysis: The sensory analysis of ice cream samples were carried out at the laboratory of All Russian Scientific Research Institute of Refrigeration Industry-Branch of V.M. Gorbatov, Research Center for Food Systems RAS 12, Moscow, Russian. Eleven trained judges (males and females) were selected to evaluate appearance, body & texture and aroma & taste on a scale of 1, 3 and 6 points, respectively²⁴. Ice cream samples of 25-30 g were served to the judges in a special ice cream cups at a serving temperature of 10°C.

Statistical analysis: The obtained data were performed in triplicate and were analyzed using the SAS software program version 9.1²⁵. Analysis of variance (One Way-ANOVA) and Duncan’s multiple range tests was used to determine the differences between treatment means. Statistical significance was tested at the 5% level.

RESULTS AND DISCUSSION

Ice cream mix properties: Acidity, pH and dynamic viscosity of ice cream mixes containing different concentrations of the wheat germ oil extract (WGOE), as bioactive minor components, are presented in Table 2. The addition of WGOE caused a slight (p>0.05) increase in acidity and a decrease in the pH of the ice cream mixes, due to the presence of some free fatty acids and interaction between phenolic compounds in the WGOE with milk proteins^12,26. Sagdic et al.¹¹ reported that the addition of some phenolic compounds caused a decrease in pH value due to the acidic nature of phenolic compounds. Fernandez-Orozco et al.²⁷ reported that the most abundant phenolic acids in wheat bran are derivatives of hydroxycinnamic acids, specifically ferulic acid, dehydrodimers and dehydrotrimers of ferulic acid and sinapic and p-coumaric acids. The decrease in pH value was significant (p>0.05) in ice cream mixes containing >1.0% WGOE compared to the control mix, however, pH values in mixes with different concentrations of WGOE showed similarity in between. A similar trend was found by Zhu et al.²⁸ in wheat starch-phenolic compound suspension. Some phenolic acids significantly lowered pH to 2.95-4.25, compared with control (pH 6.79). The acidity and pH of all ice cream mixes were also within the normal range reported by Bajad et al.²⁹, Gabbi et al.¹³ and Naeem et al.¹².

In general, all ice cream mixes containing WGOE were significantly more viscous (p<0.05) than the control mix (Table 2). The viscosity gradually increases (p<0.05) from 1057±61 to 2913±61 mPa.s, as the added WGOE increased from 0.0 to 3.0%. The high mix viscosities in WGOE mixes can be related to the presence of some minor components such as monoglycerides, diglycerides, phospholipids and phenolic compounds in the WGOE that can bind with water. Syed et al.³⁰ reported that binding of water with emulsifiers caused viscosity enhancement, which in turn increased the flow time. Additionally, the viscosity is more affected by the number and position of hydroxyl and methoxy groups of phenolic compounds. Similar, phenolic compounds increased rheological properties of both wheat starch and whey protein suspensions due to structural features of the phenolic compounds^28,31. Such an effect was also found by Guler-Akin et al.¹⁴ in mixes supplemented with 1.0% carob extract.

Ice cream properties
Overrun: Figure 1 shows the overrun percentage of the ice cream samples when supplemented with different concentrations of WGOE. The highest mean overrun percentage was determined in the ice cream sample containing 1.0% WGOE, followed by that containing 0.5% WGOE and control sample, but the differences among them were not significant statistically (p>0.05). The slight increase in the overrun could be due to the addition of WGOE, up to 1.0%, improving the viscosity of the mixture, which created stable foam¹². However, when adding the WGOE at high concentrations, >1.0%, a significant decrease in the percentage of overrun was observed (p<0.05). A similar trend was found by Salem et al.⁸ on a study about the red grape pomace extract added ice cream. The decrease in overrun at high concentrations of WGOE may be related to the high mix viscosity, which weakening air incorporation^29,32. The high mix viscosity also caused shear force increase during freezing, promoting fat globule interactions and fat coalescence³³.

Table 2:	Acidity, pH and dynamic viscosity of ice cream mix supplemented with different concentrations of wheat germ oil extract after aging at 5°C
Means (n = 3 ±SD) with same letters in the same column are not significantly different (p<0.05); WGOE, wheat germ oil extract

Fig. 1:	Mean overrun percentage of ice cream samples supplemented with different concentrations of the wheat germ oil extract (WGOE)

The raise of fat coalescence leads to the formation of thin lamellae between air cells, which may ultimately destabilize the film and break the air cells³⁴. Additionally, WGOE at high concentrations may increase the displacement of protein on the fat globule surface, accelerating fat destabilization and decrease the overrun².

Air cell size: The effect of adding different concentrations of WGOE on the mean air cell size of ice cream is shown in Fig. 2a-f and Table 3. In general, the mean air cell size was larger in ice cream samples containing WGOE (30-37 μm) than the control sample (26 μm).

Fig. 2(a-f):

Microscope images of air cell size of ice cream samples supplemented with different concentrations of the wheat germ oil extract (WGOE) after 1 day of storage at -25°C, (a) Control sample without WGOE, (b) Sample supplemented with 0.5% WGOE, (c) Sample supplemented with 1.0% WGOE, (d) Sample supplemented with 1.5% WGOE, (e) Sample supplemented with 2.0% WGOE, (f) Sample supplemented with 3.0% WGOE, →: Arow point to air cell

Table 3:	Mean air cell sizes of ice cream samples supplemented with different concentrations of the wheat germ oil extract
WGOE: Wheat germ oil extract

The mean air cell size of ice cream samples increasing from 26 to 37 μm as WGOE increased from 0 to 1.0%. Above 1.0% WGOE, the mean air cell size decreased to 30-32 μm on further addition, due to the highest viscosities and the highest shear stresses, which breaks down the air cells³⁵, but was still higher than the control sample. On the contrary, Sofjan and Hartel³⁶ and Warren and Hartel³⁷ found that increasing overrun decreased the mean air cell size. This trend was not seen in the ice cream sample containing WGOE. For example, ice cream samples containing 1.0% WGOE, which had the highest overrun, had also the highest mean air cell size (37 μm). Also, the mean air cell size of the ice cream sample containing 3.0% WGOE was higher (30 μm) than the control sample (26 μm), although it has a lower overrun. In this respect, it can be said that adding the WGOE at a low concentration (<1.0%) can act as an emulsifier, which helps to stabilize the air cell interface and prevent air cell breakdown³⁷. Abd El-Rahman et al.³⁸ reported that emulsifiers caused the agglomeration of fat which in turn finely dispersed and stabilized the air cells. Therefore, these results suggest that the breakdown or stabilization of air cells depends on more than one factor during freezing.

Over the storage period (-25°C) of 90 days, some air cells in ice cream samples appeared larger than those measured one day after hardening (Fig 3a-f). This finding is consistent with that found by Park et al.³⁹, who found that the air cell size increases with long storage time. Additionally, Sofjan and Hartel³⁶ reported that air cells tend to coalesce into larger cells during storage, especially at temperatures higher than -25°C. However, there was no much difference in the mean air cell size of ice cream samples (Table 3). The mean air cell size slightly decreased in the control sample and ice cream samples containing 0.5, 2.0 and 3.0% WGOE but was more stable in ice cream samples containing 1.0 and 1.5% WGOE as the storage of the period increased.

Ice crystal size: As seen in Table 3 and Fig. 4a-f, the mean ice crystal size ranged from 26.0 to 32.0 μm across different concentrations of WGOE. The mean size of the ice crystals was in the range mentioned by Muse and Hartel³⁵, but it was lower than that mentioned by Park et al.³⁹. The ice crystals in the control sample were the largest, while these in the ice cream sample containing 3.0% WGOE were the smallest. Ice crystal size gradually decreased with increasing the concentration of the WGOE. Increasing the concentration of the WGOE leads to an increase in dynamic viscosity, which in turn leads to a decrease in the growth of ice crystals^33,40. Schmidt and Smith⁴¹ mentioned that an increase in viscosity may inhibit the development of iciness in frozen dairy dessert. This owing to the concentration of the unfrozen phase in the lamellae may increase sufficiently to decrease the movement of water molecules and thus, limit ice crystal growth. Kamal-Eldin⁷ has reported that minor lipids can influence nucleation by increasing sites for nucleation, changing temperature at which nucleation occurs, or interfering with nuclei formation. Minor lipids can affect the rate of crystal growth, either retarding or accelerating it until the system reaches equilibrium. Therefore, WGOE, as bioactive minor components, are emerging as new options to limit ice crystal growth and recrystallization.

Melting resistance and dimensional stability: The melting resistance of ice cream as affected by the addition of WGOE is depicted in Fig. 5. In general, ice creams made with WGOE had the fastest melting rate (p<0.05) compared to control sample. The melting rate gradually increased as the concentration of the WGOE increase. Bajad et al.²⁹ and Muse and Hartel³⁵ reported that a decrease in the melting rate as an overrun increased. The larger volumes of air present in high overrun products caused a reduced rate of heat transfer, which resulted in slower melting rates³⁹. This trend was not seen in ice cream samples containing WGOE, up to 1.0%, which had an overrun >that of the control sample. However, this trend may compatible with ice cream samples containing >1.0% WGOE, which had the lowest overrun. Other authors, however, reported that as a viscosity of ice cream mix increased, the partial coalescence increases and the melting rate decreases^29,32,33. This same trend was also not seen in ice cream samples that had high viscosity. The dimensional stability of the control sample was more stable than that containing WGOE during the melting at 20°C for 100 mim; the dimensional stability decreased as the WGOE increases (Fig. 6a-d). These results confirm the above results of the meltdown test (Fig. 5). This means that adding the WGOE may change the physical properties of milk fat, e.g., reduce solid fat content.

Fig. 3(a-f):

Microscope images of air cell size of ice cream samples supplemented with different concentrations of the wheat germ oil extract (WGOE) after 90 days of storage at -25°C, (a) Control sample without WGOE, (b) Sample supplemented with 0.5% WGOE, (c) Sample supplemented with 1.0% WGOE, (d) Sample supplemented with 1.5% WGOE, (e) Sample supplemented with 2.0% WGOE, (f) Sample supplemented with 3.0% WGOE, →: Arow point to air cell

Abd El-Aziz et al.⁴² found that adding palm oil to butter oil resulted in a decrease in the solid fat content of the mixture at 0-10°C compared to both oils separately. Minor components when added to fat can affect crystallization; nucleation, crystal growth and polymorphism⁴³.

Fig. 4(a-f):

Microscope images ice crystal sizes of ice cream samples supplemented with different concentrations of the wheat germ oil extract (WGOE) after 90 days of storage at -25°C, (a) Control sample without WGOE, (b) Sample supplemented with 0.5% WGOE, (c) Sample supplemented with 1.0% WGOE, (d) Sample supplemented with 1.5% WGOE, (e) Sample supplemented with 2.0% WGOE, (f) Sample supplemented with 3.0% WGOE, →: Arow point to ice crystal

Although there are clear differences in the melting rate and dimensional stability, there are no clear differences in the properties of melted ice cream, such as smoothness, foam and fat clusters. Therefore, the components of the WGOE may have a negative effect on the melting rate more than the positive effect of the overrun, viscosity and partial coalescence of ice cream.

Fig. 5:	Meltdown of ice cream samples supplemented with different concentrations of the Wheat Germ Oil Extract (WGOE)

Fig. 6(a-d):	Dimensional stability images of ice cream samples supplemented with different concentrations of the wheat germ oil extract during holding for (a) zero time, (b) 30 min, (c) 60 min and (d) 100 min in thermostat at 20°C

Table 4:	Sensory properties of ice cream samples supplemented with different concentrations of the wheat germ oil extract
Means (n=3 ±SD) with same letters in the same column are not significantly different (P < 0.05); WGOE, wheat germ oil extract

Sensory properties: The effect of added WGOE on the sensory properties of ice cream samples is shown in Table 4. The WGOE did not affect the appearance of all ice cream samples at different concentrations. The texture and flavor were similar in the control sample and ice cream samples containing WGOE, up to 1.0%. Thereafter, they were decreased; the decrease was significant only at level > 1.5% WGOE. In general, the control sample and ice cream samples containing 0.5 and 1.0% WGOE showed similarity with each other for total scores: similar in sweetness, smoothness and meting properties scores into the mouth. However, the lowest overall total scores (P > 0.05) were given to samples containing 3.0% WGOE and 2.0% WGOE, respectively. Reduced structure scores may be related to a decrease in overrun and thus obtained a more compressed structure.

CONCLUSION

The final structure of ice cream depends on many different processing variables, ingredients and composition that tend to have a complex function to each other. The minor lipid components of plant oils can play an important role in the dairy industry, not only to improve their nutritional values and health benefits but also to improve their physical properties and microstructure. The addition of WGOE increased the mix viscosity and the air cell size of the ice cream while reducing the ice crystal size and the melting resistance. Additionally, the WGOE can be used to produce functional ice cream rich in bioactive minor components such as tocopherols, phospholipids, phenolic compounds and phytosterols that can increase the level of vitamin E and help prevent cholesterol absorption.

SIGNIFICANCE STATEMENT

This study has determined the rational dose of minor components extract from wheat germ oil for the production of ice cream that can be a positive influence on the functional and technological properties of both soft and hardened ice cream. The results of the research are leading contributions and provide the possibility to produce dairy products with higher quality. This study will help dairy researchers and laboratory practice for using minor components for manufacturing foodstuffs of functional nutrition, obtaining objective and comparable results for ice cream properties.