Incorporation of omega‐3 fatty acid‐rich grape seed oil in yoghurt: Response surface optimization of physicochemical, textural, and sensory attributes during refrigerated storage

Abstract The demand for consuming low‐fat or nonfat dairy products, especially fat‐free yoghurt, has increased considerably because of the effects of high‐fat diet on human health during the two past decades. Generally, consumers prefer low‐fat products to the same high‐fat products. For this reason, manufacturers are looking for an ideal source for replacing fat substitute. In this research, the effect of grape seed oil (GSO) as a fat replacement on different quality attributes of the produced set yoghurt was determined. The effect of diverse ratios (3:0, 1.5:1.5, and 0.5:3%) of milk fat and GSO on the change in the quality attributes of the set yoghurt for up to 22 days of refrigeration period (4 ± 1°C) was investigated. Statistical analysis revealed that increase in GSO concentration leads to a significant increase (p < .05) in viscosity, acidity, and water‐holding capacity (WHC), whereas syneresis and pH value decreased during the storage time. Furthermore, increasing the proportion of fat replacement to 3% (w/w) in set yoghurt increased the samples hardness while in case of cohesiveness; negative effect was observed because of the action of fat globules within the protein system. Result of fatty acid analysis revealed that the yoghurt samples containing GSO have higher unsaturated fatty acid content than the control yoghurt sample. In conclusion, the best fat replacement concentration of GSO in producing low‐fat yoghurt was found in 1.5%, which also had the highest overall acceptance score between different yoghurt samples containing different levels of GSO.

products, especially fat-free yoghurt, has increased considerably because of the effects of highly fat diet on human health. Generally, consumers prefer low-fat products to the same high-fat products (Astrup et al., 2019). For this reason, manufacturers are looking for an ideal source for replacing fat substitute. Fat replacement are compounds that affect the properties of the product, such as taste, mouth feel, texture, viscosity, and other organoleptic properties (Cheng et al., 2008). There are many sources that can be produced, and many researches have been done in this regard from various sources (Adapa et al., 2000;Sharma et al., 2017). The grape seed oil (GSO) has a very mild flavor and consists mainly of triglycerides.
The GSO extraction processed usually by cold press or by use of nonpenetrating solvent such as hexane in a conventional soxhlet extraction or superheated fluid method (Chougui et al., 2013). During last year, the published reports about the application of palm oil in dairy products (especially in yoghurt) increased consumer concern for the consuming of dairy products. There are two main reasons for this concern, including food adulteration (replacement of milk fat with palm oil without any awareness to the consumers) and high saturated fatty acid content in palm oil compared to milk fat that may be lead to increase heart diseases in consumers. Therefore, in this research, the application of GSO as a functional oil with unsaturated fatty acids instead of milk fat for set yoghurt producing was evaluated. The effect of these substitutions on the different physicochemical attributes (viscosity, acidity, water-holding capacity (WHC), syneresis, pH value), textural properties, and sensory characteristics (appearance, flavor, hardness, and overall acceptability) was measured during the 22 days of refrigeration period. After measurement of these physicochemical attributes, response surface methodology (RSM) was used for optimizing the production conditions of low-fat set yoghurt.

| Yoghurt preparation procedure
For preparing yoghurt samples, 2% of nonfat dry milk powder was added to three cow's milk treatments. After homogenization and pasteurization process in 90°C for 3 min, they cooled to 45°C. The GSO was added to the formulation (according to  Figure 1 shows the sample obtained pick for fatty acid analysis of GSO by gas chromatography.

| Syneresis
For measuring yoghurt sample syneresis, 25 g of different samples drained with filter paper (no. 589/2, S&S, Dassel, Germany) for 2 hr at 7°C. The obtained filtrate liquid was weighted and the syneresis expressed as the percentage of filtrated liquid (Wu et al., 2000).

| Water-holding capacity (WHC)
The WHC was calculated as the obtained pellet weight after 60-min centrifugation (13,500 g for 60 min, 10°C) of yoghurt sample relative to the original weight of yoghurt sample according to the Equation 1 ( Parnell-Clunies et al., 1986): where w1 is the original weight and w2 is the obtained weight of the yoghurt sample after 60-min centrifugation (13,500 g for 60 min, 10°C).

| Hardness
The force required for penetrating a cylindrical probe (
The sample obtained picks for fatty acid analysis of grape seed oil by gas chromatography

| Yoghurt sample sensory characteristics
A 9-point hedonic scale method was performed with nine trained panelists in the age range of 20-40 years, for measuring different sensory attributes including sample appearance, texture, flavor, and overall acceptance. The obtained quality scores were changed to quantity scores for parametric statistical analysis as 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like or dislike, 4 = dislike slightly, 3 = dislike, 2 = dislike very much, and 1 = dislike extremely.

| Statistical analysis and experimental design
This research was conducted based on a completely randomized design (CRD). For statistical analysis, the samples were selected randomly and multirange Duncan test was used to compare the mean values of the results at a confidence level of 95%. All the experiments were conducted in three replications, and SPSS Version18.0 (SPSS Inc.) was used to perform the statistical analysis.
The optimization of GSO enriched set yoghurt production was done by using Design Expert software version 6.0.2. (Stat-Ease Inc.).
For experiments designing, the central composite design (CCD) was used. Independent variables, including GSO concentration (X 1 ) and storage time (X 2 ) at three different coded levels: low (−1), medium (0), and high (+1), were investigated. The ranges of independent variables were determined from preliminary experiments. The range of GSO concentration and storage time was 0-3%, and 0 to 22 days, respectively ( Table 2).
The full quadratic equation of the response variables for yoghurt was derived by using RSM as Equation 2: where Y is the response, βo is constant, β 1 and β 2 are the linear coefficients, β 11 and β 22 are the quadratic coefficients, and β 12 is the interaction coefficient. X 1 and X 2 are GSO concentration and storage time, respectively.

| Optimization
In order to find the optimal condition for GSO enriched set yoghurt production, independent variable, that is, GSO concentration, put up in the chosen range and storage time was chosen maximum and responses, that is, water-holding capacity, viscosity, hardness and pH value, were considered at maximum level, while syneresis and titratable acidity were considered at minimum level.

| pH and acidity
The physicochemical properties of set yoghurt samples are illustrated in Table 3. As it can be seen from Table 3  Syneresis introduced as one of the main parameters, represent the quality and consumer's acceptability of yoghurt sample during

| Water-holding capacity
Water-holding capacity (WHC) is defined as the ability of food to maintain its own or added water during different processing condition (force, pressure, centrifugation, or heating). The amount of WHC of all yoghurt samples containing GSO was higher than the control sample during refrigeration period (p < .05). As can be seen from

| Fatty acid composition
The yoghurt samples fatty acid profile is shown in

| Hardness
Hardness is the most important parameter for evaluating yoghurt texture, which equal to the required force to create a certain deformation and considered as a measure of the yoghurt firmness.

| Apparent viscosity
Different parameters including the number and strength structure and spatial distribution of casein micelles bonds between yoghurt affect the apparent viscosity of yoghurt sample (Izadi et al., 2015). Table 5 shows the apparent viscosity of yoghurt samples during 22 days of cold storage (4°C).
As it can be seen from

| Sensory evaluation
The effect of GSO as a fat replacer on the organoleptical attributes of set yoghurt is illustrated in Figure 2.
Sample T 2 with 1.5% (w/v) GSO, obtained the highest overall acceptance score on the first day of storage period. Color plays an important function in product quality, especially in sample like yoghurt and has a direct effect on appearance. As it can be seen all yoghurt samples obtained the appearance score above 4 ( Figure 2). The difference between appearance scores of set Small and capital letters indicate a significant difference in the columns (the difference between the samples in a day of storage) and the rows (difference of one sample during storage) at level of 5%.   and hardness (measured instrumentally) were observed for yoghurts containing 3% GSO (Figure 2). The overall acceptance results showed that the total sensory score of all samples decreased over the time, which could be due to the growth of acid producing bacteria.

| Response surface optimization results
The design matrix of CCD and as well experimental findings for the responses were shown in Table 6. Thirteen experiments were done according to design with 2 factors and 3 levels for each variable. Different quantitative responses such as pH, titratable acidity percentage, syneresis, water-holding capacity (WHC), hardness, and viscosity value were considered for optimization.
The analysis of variance (ANOVA) and lack of fit test were considered for the significance of models of regression equations (Table 7). As can be seen from  As can be seen, the yoghurt samples pH, increased by increasing the GSO concentration while decreased during storage period.
The inverse trend was observed for acidity value ( Figure 3b). As  is the process by which water is released or extracted from a gel due to gel shrinkage. This parameter is not pleasant in yoghurt, and it has a negative effect on the product's acceptability to consumers. The increase of syneresis during the storage period may be due to the increase of yoghurt samples acidity which result to formation of high and stronger casein bands, lead to increase yoghurt sample syneresis during the storage period (p < .05). Similar studies showed that the water-holding capacity is inversely proportional to yoghurt sample acidity. Therefore, the reduction of acidity increased the gel network strength, which results in the maintenance of high water retention capacity. Bierzuńska et al.

Sum of squares
(2019) also reported, gel network stability, prevents free water release and also increases the WHC is due to more water molecules binding to proteins in the yoghurt structure during storage period (Bierzuńska et al., 2019).
Storage period has a significant effect on the yoghurt samples hardness. Figure 3e shows that yoghurt samples hardness, increased during storage period and GSO concentration has no significant effect on this quality attribute (p > .05). As can be seen from Figure 3f the yoghurt samples viscosity was increased by increase of GSO The correlation coefficient (R 2 ) between actual data obtained by experiments versus predicted data obtained by regression models and their response equations are listed in Table 8. Figure 4 shows the actual versus predicted data graph for different quality attributes of grape seed oil (GSO) enriched low-fat yoghurt samples. As it can be seen from Figure 4 and Table 8, the high correlation coefficient (R 2 ) obtained showed the suitability of the developed equations by regression models for predicting the quality attributes of final yoghurt samples under the effect of GSO concentration and storage period.

| Optimized conditions
Response surface method with CCD was used to determine the optimum conditions for producing GSO enriched low-fat yoghurt.
Thirteen experiments were analyzed, according to the design, with two independent factors at three levels for each variable. The results for the different yoghurt samples showed that the optimum levels of GSO concentration and storage period as independent variables were 3% and 22 days, respectively. The optimal levels for the pH, acidity, syneresis, WHC, hardness, and viscosity with a desirability of 75.4% are shown in Figure 5.

| CON CLUS ION
The findings of this study indicated that GSO has a good potential as an alternative source of fat in set yoghurt; therefore, set yoghurt containing different amount of GSO was made and analyzed. The use of GSO affected the textural properties and further increased the hardness in set yoghurt containing 3% (w/w) fat replacement. The yoghurt sample viscosity was strongly affected by the total solids content and GSO concentration. Increasing the amount of GSO led to an increase in the total solids content and apparent viscosity enhancement. The rheological properties of yoghurt are highly dependent on its total solids content. During the storage period of yoghurt, WHC, acidity, and hardness all increased, and pH value and syneresis decreased. It can be concluded that by incorporating the optimum levels of GSO and milk fat into the formulation of low-fat set yoghurt; it is possible to develop a low-fat foods with similar textural properties to its full-fat yoghurt. Based on the results, the best fat replacement level for producing low-fat yoghurt was found to be 1.5%, which also had the highest overall acceptance score between different yoghurt samples containing different levels of GSO. In the optimum production condition (GSO concentration 3% and 22th days of storage period) the optimal levels for the pH, Acidity, syneresis, WHC, hardness and viscosity obtained as 4.3, 1.22, 6.63%, 50. 478%, 0.57 N and 11,750 (cP), respectively with a desirability of 75.4%.

TA B L E 8
The developed regression model for predicting physicochemical properties of grape seed oil (GSO) enriched low-fat yoghurt

F I G U R E 4
The actual data (experimental) versus predicted data obtained by regression models for different quality attributes of grape seed oil (GSO) enriched low-fat yoghurt