Response surface optimization of pudding formulation containing fish gelatin and clove (Syzygium aromaticum) and cinnamon (Cinnamomum verum) powder: Effect on color, physicochemical, and sensory attributes of the final pudding product

Abstract In this study, the response surface methodology (RSM) was used to optimize the pudding formulation ingredients including the fish/bovine gelatin ratio and cinnamon and clove powder and determine the color and physicochemical and sensory attributes’ change in final pudding product. Experiments were carried out based on a central composite design (CCD). The results showed that by increasing the ratio of fish gelatin to bovine gelatin (FG/BG) up to 3%, the moisture content increased slightly and then decreased significantly. Increasing the cinnamon powder to 0.5% reduced the moisture content. Increasing the FG/BG in the formulation of pudding samples reduced the protein content. The effects of cinnamon and clove powder on the protein content were increasing and decreasing, respectively. By increasing the FG/BG ratio, the samples syneresis showed a significant decrease, while the effects of cinnamon and clove powder on the syneresis were nonsignificant. As the level of cinnamon and clove powder increased, the L* value decreased. Cinnamon and clove powder had a linear effect, and the interaction of gelatins and clove powder had a significant effect on changes in redness. The effects of cinnamon and clove powder on b* value were significant. In terms of sensory evaluation, increasing the cinnamon powder concentration increased the appearance scores, while in the case of fish gelatin, this trend was downward. The linear effect of cinnamon powder on taste was significant, while other variables had no significant effect on the taste of the samples. The sample texture was significantly affected by fish gelatin and clove powder. Increasing FG/BG from 0% to 2.5% increased the texture score, but after this range, a decrease in the texture score was observed. The overall acceptance of samples was more affected by spice powder compared to gelatin. By increasing the cinnamon powder and FG/BG, the overall acceptance increased and decreased, respectively. In conclusion, the optimal FG/BG and cinnamon and clove powder were introduced 1.479%, 0.288%, and 0.619% respectively.


| INTRODUC TI ON
Functional food contains some health-promoting compounds beyond the traditional nutrients and plays an important role in enhancing human health (Sun et al., 2007). Dairy desserts are products of wide consumption. Their nutritional and organoleptic characteristics make them accepted by children and adults, but usually they have high energy density (Alimoradian et al., 2021;Tarrega & Costell, 2006). Pudding is one of the most important milk products, it is the semisolid milk protein, consisting of starch-based pastes and dairy desserts, which is a milk-based starch paste and has a typical semisolid food texture (Lim & Narsimhan, 2006). Pudding is dessert prepared by addition of suitable quantity of egg to whole milk or concentrated milk or condensed milk. If needed, any suitable sweetener and thickening materials could be added to the mixture (Sarker et al., 2016). Dairy desserts (milk puddings) are especially consumed by children and elderly people throughout the world. Pudding is characterized as a suspension of deformable particles (the swollen starch granules) dispersed in a continuous phase containing milk proteins and a gelling agent (Verbeken et al., 2006). Powdered and packaged forms of the pudding samples are available in the market . The formulation of the puddings is generally composed of vanilla, milk, sugar, starch, and gum (Ares et al., 2009) and the other ingredients such as cacao and fruit aromas, which are responsible for developing distinctive rich aroma of the puddings (Gurmeric et al., 2013). Due to these complex interactions among the different components of a pudding, it is likely that the substitution of milk with other dispersion media could cause drastic changes in the rheological behavior of the product. This aspect, however, has been little investigated in the literature (Lim & Narsimhan, 2006;Nunes et al., 2003). Besides ready-to-eat puddings, commercial powders are at consumers' disposal for the production of homemade desserts. Pudding powders are usually composed of starch, hydrocolloids, sugars, colorings, and aromas, and they are intended to be dissolved in milk (Vélez-Ruiz et al., 2006).
Gelatin from bone and connective tissue of pigs and cattle is traditionally used in the food industry as a gelling agent (Kouhi et al., 2020). However, the consumption of gelatin from these mammalian species contradicts ethnocultural and religious norms of a number of religions, and is also associated with the risk of contracting prion (Ahmed et al., 2020). In this regard, it seems relevant to look for alternative sources of food gelatin .
Such a source may be the connective tissue of fish (Lv et al., 2019), the industrial processing of which partially solves the problem of disposal and integrated use of waste from the fish processing industry (Uranga et al., 2020). There are several papers on the use of fish gelatin in food formulations (Giménez et al., 2013;Wangtueai et al., 2020) (Yasin et al., 2016). In our previous study, we successfully used fish gelatin instead of bovine gelatin in the formulation of replacement on pastille (Asgarzadeh et al., 2021).
Spices always play a prominent role in the kitchen as well as in certain medicinal activities like diuretic, eccoprotic, carminative aperients, expectorant, and many more. Spices have been used medicinally since early (Sachan et al., 2018). Utilization of spices in various forms like powder, extract, or essential oils has been well documented for inhibiting the growth of many spoilage bacteria and fungi in foods (Rajkumar & Berwal, 2003;Subbulakshmi & Naik, 2002).
Clove (Syzygium aromaticum) is one of the most valuable spices that have been used traditionally as a food preservative and for many therapeutic purposes. Clove is native to Indonesia, but it has also been cultured in several parts of the world (Hussain et al., 2017); it is a vital source of phenolic compounds such as flavonoids, hydroxycinnamic acids, and hydroxybenzoic acids. Eugenol is the main bioactive constituent of clove. With regard to the phenolic acids, gallic acid is found in higher concentration (783.50 mg/100 g fresh weight) (Shan et al., 2005). Clove and essential oils are reported to have antibacterial, antimycotic, yeast inhibitor, and Brownian enzyme inhibitor activity Daraei Garmakhany et al., 2017;Ghahfarrokhi et al., 2013). Such activity may be attributed to its 2-methoxy-4-(2-propenyl) phenol content.
Cinnamon (Cinnamomum verum) is one of the most well-known spices. It is used in the pharmaceutical and food industry as a powerful antiseptic and flavoring agent, respectively. It presents medicinal properties like digestive, stimulant, hypotensive, sedative, and vasodilator (Cemin, 2012). Cinnamon spice is one of the sources of effective antioxidants such as vanillic, caffeic, gallic, protocatechuic, p-hydroxybenzoic, p-coumaric, and p-hydroxybenzaldehyde (Muchuweti, 2007) and enhances the efficacy of other important antioxidants. The antioxidant activity of cinnamon is attributed to an array of flavonoid compounds that it contains. The essential oils present in cinnamon, including cinnamaldehyde, eugenol, and linalool, have been investigated in reference to peroxynitrite-induced nitration and lipid peroxidation (Iqbal et al., 2005).
Regarding dairy products, many studies have been published dealing with the effect of fat content on quality properties.
However, only a few papers deal with the quality characterization of commercial dairy desserts or pudding under the effect of gelling agents such as protein and fish gelatin (Fan et al., 2019;Yusof et al., 2019). Optimization of ingredients to obtain the desired quality of the product or optimization of the process to achieve desired results is performed by various optimization methods such as mixture design, response surface method (RSM), genetic algorithm optimal FG/BG and cinnamon and clove powder were introduced 1.479%, 0.288%, and 0.619% respectively.

K E Y W O R D S
cinnamon, clove, fish gelatin, pudding, response surface methodology (GA), and artificial neural network (ANN) (Daraei Ghahfarrokhi et al., 2013;Kashiri et al., 2012;Toker et al., 2013). This optimization method includes more than one ingredient or independent variables that their effect on the dependent variables or response was examined (Dutcosky et al., 2006). The relationship between independent factors and responses is explained by predictive mathematical models obtained from optimization approach (Flores et al., 2010). The aim of this study was to develop a pudding formulation containing clove and cinnamon powder and bovine gelatin accompanied by fish gelatin.

| Materials
Ingredients used for pudding were purchase from local markets. All other chemicals used in this study were of analytical grade and purchased from chemical suppliers.

| Preparation of the pudding samples
The formulation of the pudding includes 10 g sucrose and 9 g skimmed milk powder. The other ingredients include fish gelatin, bovine gelatin (0%, 2.5%, and 5%), and clove and cinnamon powder (0%, 0.5%, and 1%). The different treatment combinations used in the experimental design are shown in Table 1. Puddings were prepared by adding the solid mixture to 100 ml water slowly and mixing them with a magnetic stirrer (Yellow line, Germany). The dispersion was heated to 85°C for 20 min and stirred for 10 min at that temperature. The puddings were then cooled to room temperature (25°C) and then stored in a refrigerator (4-5°C) for 1 hr prior to the analysis. The experimental work was carried out in the Research Chemistry Laboratory, University of Tehran Science and Technology Park ( Figure 1).

| Moisture content
The moisture contents of different types of samples were determined by hot oven drying method according to AOAC (AOAC, 2003

| Syneresis percent
Ten grams of each sample were transferred to previously weighted centrifugation tubes and were held vertically at 4°C for 14 days.
Syneresis was quantified as the loss of weight of the sample after the removal of the exuded water (Aichinger et al., 2003;Azari-Anpar et al., 2017;Kokabi et al., 2021;Mehrinejad Choobari et al., 2021).  Kay et al. (2017) method was used to determine the viscosity. For this purpose, 600 ml of the pudding was transferred to a beaker immediately after mixing. Viscosity was measured by a Brookfield viscometer with spindle 4 at speeds of 20 rpm (Kay et al., 2017).

| Sensory evaluation
The sensory evaluation of prepared pudding samples with different treatments was conducted by a trained panel of 10 judges. Each panelist was given a set of pudding separately in isolated chamber and provided with a glass of fresh water to rinse their mouth before tasting the next sample. Each sample was evaluated for various quality attributes, such as appearance, color, texture, mouthfeel, taste, and overall (1) Syneresis ( % ) = exuded water (g)∕total weight of the sample × 100

Factor
Name Units

Maximum
Minimum Maximum TA B L E 1 Independent variable values of the process and their corresponding levels acceptance as per the prescribed proforma (Singh & David, 2017).
Panelists were asked to rate the samples on a prescribed sensory evaluation proforma with earlier stated attributes. Samples were assessed organoleptically using a 5-point hedonic scale, where 5 correspond to "like extremely" and 1 corresponds to "dislike extremely."

| The experiment design by RSM
In this study, the central composite design (CCD) was selected to optimize the process variables in two levels with 17 components, including three replicates at the central point for the estimation of the experimental error. The ranges and levels of independent variables are presented in Table 1. The statistical significance test was based on total error with 95% confidence level (p < .05). The independent variables of the process were included fish gelatin (A), cinnamon powder (B), and clove powder (C). According to Table 1, the ratio of fish gelatin to bovine gelatin varied between 0% and 5% of the sample weight, and the changes in cinnamon and clove powders were 0 to 1, which was calculated with alpha (α) equal to 1. The optimization of functional pudding production was done by using Design Expert software version 11.1.2.0 (Stat-Ease Inc., Minneapolis, MN, USA) ( Table 2). The multivariate model is an Equation (2). In the mentioned equation, Y is the predicted response, β0 is the constant coefficient, β 1 and β 2 are linear coefficients, β 11 and β 22 are square effects, and β 12 and β 21 are interaction effects.

| Process optimization
The RSM evaluated the effects and interactions of the fish gelatin, clove and cinnamon powder to increase protein and sensory attributes, decrease syneresis while other parameters were in range. For validation of the correlation, the responses were also experimentally analyzed and compared with predicted values from the regression equation.

| Moisture content
Jelly is one of the high moisture foods (Hartel et al., 2018). The moisture content is important to determine the shelf life and purity of the protein (Baziwane & He, 2003). Low moisture content increases the product's stability, thus increasing the shelf life of the product (Chukwu & Abdullahi, 2015). Regarding the moisture content of the treatments, according to Table 3, the proposed model was not significant (p > .05), and the independent variables in linear, binomial, and quadratic had no effect on the model (Table 3).
According to Figure 2a, with increasing the fish/bovine gelatin ratio to 3%, the moisture content increased slightly and then decreased significantly by increasing the fish/bovine gelatin ratio to 5%. Increasing the cinnamon powder to 0.5% reduced the moisture content, while the moisture changes in the samples containing clove powder were not significant (p > .05). In other words, these changes are constant and linear. However, the moisture content in all treatments was within the standard range (Khanum et al., 2001), so it was possible to keep them at refrigerator temperature. Hartel et al. (2018) suggested that foods with higher moisture content should be stored with relative humidity (RH) of 55%-65% to increase their shelf life (Hartel et al., 2018). The results are in accordance with the results of Yin et al (2021) regarding the effect of adding fish gelatin to yogurt.

| Protein content
It is well known that the physical and functional properties of gelatin depend not only on their amino acid composition but also on their Bloom index (Chou et al., 2016), molecular weight distribution, on the relative contents of α-, β-, and γ-chains, and on the presence of protein fragments of low molecular weight (da Trindade Alfaro et al., 2015). It should be noted that modified fish gelatin with anionic polysaccharides from seaweed κ-carrageenan and gellan gum can be a good alternative to mammalian gelatin (porcine or bovine) in the food industry (Sow et al., 2018). According to Table 3  this is the case with cinnamon and cloves due to their protein content, which is not comparable to gelatin. The average protein content in cinnamon and clove powder is 4 and 3.3 g/100 g, respectively (Khanum et al., 2001).

| Syneresis percent
Syneresis is defined as the extraction of a liquid from a gel that occurs due to slow gel contraction, and it is affected by time. The study results showed that the greater the syneresis value, the easier the gel formed to release water. Therefore, it is not preferred by the consumers (Draget et al., 2001). According to Subaryono et al. (2010), the product stored at low temperature (e.g., pudding) must have high gel strength and low syneresis (Subaryono, 2010).
It was observed that the experimental data of syneresis for the different formulations and batches did not satisfy equal variances and normality tests (Celeghin et al., 2016). This is a successful outcome because syneresis is a visual sensory attribute that strongly determines the acceptability of this type of products (Mleko & Gustaw, 2002).
According to Table 3

| Brightness (L * value)
Color is an important quality characteristic that contributes to the sensorial acceptability of food (Garcıá-Esteban et al., 2003).
However, color is affected by many factors such as spices added, packaging, or processing. According to Table 4  According to Figure 3a, with increasing the cinnamon and clove powder, the light intensity of the pudding samples was decreasing, but in cinnamon powder, this reduction had a more slope (Figure 3a).
The decreasing L * value shows that the specimen underwent a color change to a darker color. It is also possible to change the arrangement of milk caseins with retentate, which can create a denser struc-  properties of tannin, which range from clear yellowish to light brown (Anggono et al., 2018). In addition, cinnamon also contains 55%-65% cinnamaldehyde, which has a physical yellowish color. Thus, the content of cinnamaldehyde likely also affects the color of tooth enamel (Wijayanti et al., 2009). important methods in judging food quality (Djekic et al., 2021).

| Sensory evaluation
According to Table 5 and Equation 9, cinnamon powder had a significant effect on the appearance scores (p < .05).
In other words, cinnamon powder, more than fish gelatin and clove powder, was effective in changing the appearance of pudding samples. Figure  The effects of gelatin on the perception of flavor have been investigated in terms of the taste-aroma interaction (Cook et al., 2003).
According to Figure 4b, the effects of fish gelatin to bovine gelatin ratio and clove powder on taste scores were nonsignificant (p > .05) and with increasing the content of each variable from 2.5% and 0.5%, respectively, the taste score decreased. As can be seen from Figure 4b, by increasing the cinnamon powder from 0% to 1%, the taste score increased significantly (p < .05). As can be seen from Figure 4C, by increasing fish/bovine gelatin ratio from 0% to 3% the mouthfeel score increased, but decreased at higher than 3% fish/ bovine gelatin ratio. As can be seen, cinnamon powder incensement leads to a slight increase in the mouthfeel score, while clove powder addition decreased mouthfeel score slightly (p > .05).

| Texture
Texture is an important attribute in dairy desserts such as pudding, since it is highly correlated with consumer acceptance.
Gelatin with a higher gel strength will produce a harder jelly. The  Weenen et al., 2003).
As can be seen from Figure 4d, by increasing the ratio of fish gelatin to bovine gelatin from 0% to 2.5%, the texture scores increased, but after that, the scores decreased significantly. In the case of cinnamon powder, this trend was linear and concentration of 0.5% was introduced as the optimal point. Increasing the clove powder from 0% to 0.6%, increased the texture scores and then decreased. De  and Weenen et al. (2003) observed that in custard desserts texture is a multimodal attribute that can be affected not only by textural attributes (thick, smooth, fatty, rough, grainy) but also by some taste/flavor attributes such as the mucilage, polysaccharides, which are available in cinnamon.

| Overall acceptance
According to Equation 13, the linear effect of cinnamon powder (B) and the interaction effect of cinnamon-clove powder (BC) on the overall acceptance were significant (p < .05). Therefore, compared to gelatin, the overall acceptance was more affected by spices powder (Table 5). According to Figure 4E, with increasing the cinnamon powder, the overall acceptance also increased significantly (p < .05), while in the case of clove powder, the maximum effect on the overall acceptance was 0.5%. As the ratio of fish gelatin to bovine gelatin increased, the overall acceptance decreased significantly. Panelists

| CON CLUS IONS
These results indicate that the acceptability of the formulated samples may be improved simply by changing the concentrations of flavoring agents and fish gelatin. This study demonstrates that it is feasible to replace bovine gelatin by fish gelatin in puddings formulation, although it is necessary to improve the sensory profile to reach a better acceptability. In general, it should be noted that fish gelatin can be a good alternative to mammalian gelatin (porcine or bovine) in the food industry. Also, the use of spices such as cinnamon and clove powder can play an important role in improving quality characteristics, especially sensory scores.

| COMPLIAN CE WITH E THI C S REQUIREMENTS
This article does not contain any studies with human or animal subjects.

ACK N OWLED G M ENTS
The present study is related to research project, "The gelatin extraction of fish wastes and its application in production of dessert." The authors acknowledge the financial support of Kermanshah branch, Islamic Azad University, Kermanshah, Iran.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article.