Jujube mucilage as a potential stabilizer in stirred yogurt: Improvements in the physiochemical, rheological, and sensorial properties

Abstract Here, the mucilage of jujube was extracted and used as a natural stabilizer in the production of stirred yogurt. Yogurts were enriched with different concentrations of jujube mucilage (i.e., 0, 0.1, 0.15, and 0.2%), and their physical, chemical and sensory attributes were analyzed during 21 days of storage at 4°C. The results showed that the protein and fat contents of the yogurts were not significantly different compared with each other, while higher ash contents were obtained in yogurts which contained higher concentrations of the mucilage. The acidity and proteolysis of the stirred yogurts were enhanced in the presence of mucilage, and they exhibited lower concentrations of diacetyl and acetaldehyde, although the differences were not significant among the samples of different treatments. The storage time had adverse and direct effects on the amounts of acetaldehyde and diacetyl, respectively. The effects of storage time and the presence of jujube mucilage in yogurts caused a significant decrease in the percentage of syneresis, while their viscosity and WHC values increased. The magnitudes of dynamic moduli (G, G''), complex viscosity (η*), and loss tangent (tan δ) of stirred yogurts increased by increasing the concentration of jujube mucilage. The yogurts which had been enriched with mucilage were preferred slightly less by tasters during the storage period, but these differences did not amount to a statistical significance. Generally, the results of the present study showed that the jujube mucilage can be potentially used as a natural stabilizer in stirred yogurt.

Persian gum, carboxymethyl cellulose (CMC), guar gum, and xanthan in dairy products, and their applications have gained scientific developments (Dabestani, Kadkhodaee, Phillips, & Abbasi, 2018;Liu, Wang, Liu, Wu, & Zhang, 2018). These hydrocolloids can serve as gelling or thickening agents, thereby stabilizing the yogurt matrix and increasing the viscosity. The hydrocolloids inhibit syneresis, preserve the yogurt structure, and change the mouthfeel. Meanwhile, they are incorporated into dairy products in order to affect their rheological, structural, and sensorial characteristics.
One group of natural additives is the mucilage. As a heterogeneous polysaccharide complex, it can turn into slimy masses when water is added to it. Different forms of mucilage are obtained mainly from plant parts such as seeds in addition to specific microorganisms and marine algae. Generally, plant-derived types of mucilage are historically recognized for their medicinal applications. Food industries can take an advantage of them as water-retention agents, thickeners, emulsion stabilizers, suspending agents, gelling agents, film former binders, and sustained-release agents (Hassan et al., 2015).
Jujube (Ziziphus spp.) is a fruit which is widely cultivated in tropical and subtropical regions such as in southern and eastern Asia, Australia, and Europe. In traditional Chinese medicine, the Z. jujuba fruit has been considered as one of the five most valuable fruits.
Over the past several decades, Z. jujube has been recognized as a source of sedative compounds with hepatoprotective effects, and they are also characterized by antioxidant activities, compounds with immunological properties and anti-inflammatory effects (Ji et al., 2017;Wojdyło, Carbonell-Barrachina, Legua, & Hernández, 2016). The diverse array of its pharmacological effects has roots in the richness of its chemical ingredients, that is, phenolic acids (benzoic acids and hydroxycinnamic acids), vitamin C, flavonoids (flavonols and flavan-3-ols), nucleosides, triterpenic acids, and pigments (particularly anthocyanin compounds), along with an abundance of mucilage or polysaccharides that concern the interests of the current study (Ji et al., 2017;Wojdyło et al., 2016;Xie, Tang, Jin, Li, & Xie, 2016). The Z. jujube fruit is particularly rich in mucilage as it constitutes a large group of the biologically active compounds in the fruit (Ji et al., 2017). Mucilage can be considered as specific forms of complex polysaccharides which are mostly branched by l-rhamnose, l-arabinose, d-glucose, d-xylose, and galacturonic acid (Thanatcha & Pranee, 2011). Different varieties of the Z. jujuba fruits exhibit various molecular weights for their juice which range from 10 4 to 10 6 Da and can be recorded under diverse experimental conditions (Ji et al., 2017). Thanatcha and Pranee (2011) reported a comparative study on the characteristics of mucilage obtained from the jujube fruit and similar features of the guar and xanthan gum. It was reported that the jujube mucilage exhibited lower levels of water-holding capacity and emulsion capacity, but higher amounts of oil absorption, as compared to the guar and xanthan gum. This pseudoplastic hydrocolloid, that is, the jujube mucilage, can be employed to increase the stability and viscosity of food materials.
Due to the numerous benefits of jujube mucilage and its similar function with some types of gum, there has been more interest to study this mucilage as gum substitute in food products. Here, the aim is to evaluate the possibility of using the jujube mucilage as a stabilizer in stirred yogurt and to examine its functions in relation to the physicochemical, rheological, and sensory properties of yogurt.

| Extraction of mucilage from jujube powder
Jujube fruits were purchased from a local market. Impurities on the fruits were cleaned off. Then, the fruity pulp was separated, squashed, and mixed using a blender (IKA). The fruits were stored at 4°C before the extraction of mucilage which followed a method proposed by Thanatcha and Pranee (2011). To describe the procedure briefly, 200 ml of distilled water was added to 2.0 g of the jujube powder. The mixture was heated and rotated for 20 min (at 100°C and 400 rpm) with a magnetic stirrer (Heidolph). After the separation of waste materials by filtration, which was performed by a fine cloth, accompanied by a Büchner funnel and the process of centrifugation (Universal, Germany) at 3800g for 15 min, the mucilage solution or supernatant liquid was obtained and precipitated in ethanol (with a ratio of 1:4). The solution was then stored at 4°C for 24 hr. The obtained mucilage was filtered through a clean cotton cloth and dried into powder after 48 hr of storage at ambient temperature. The dried mucilage was stored at 4°C for further analysis and for use in the stirred yogurt.

| Yogurt production
The milk, which was used for the production of yogurt, contained natural amounts of fat and soluble solid content. It was mixed with the jujube mucilage powder at different concentrations, that is, 0% as the control (JM 0), 0.1% (JM 0.1), 0.15% (JM 0.15), and 0.2% (JM 0.2). Then, the milk was homogenized at 60°C and later pasteurized in a water bath at 90-95°C for 3-5 min. After cooling down to 40-45°C, the milk was inoculated with 2.5% (w/v) of the lyophilized mixed starter cultures (Lactobacillus bulgaricus and Streptococcus thermophiles, Mediterranea Biotecnologie srl., Termoli, Italy) (1:1) and incubated at 40-45°C to complete the coagulation. When the pH reached 4.6, the gel samples were stirred gently for 2 min. All of the stirred yogurts were stored at 4°C until the experiments were performed. The yogurts were analyzed at regular intervals, that is, after 1, 7, 14, and 21 days following the start of the experiment.

| Chemical analysis
After the first day of preparation, the stirred yogurts were analyzed for protein, fat, and ash contents (AOAC, 1995). The changes in pH of the stirred yogurts during storage were measured using a glass electrode laboratory pH meter (SOMET CZ, USA). The titratable acidity was determined (as lactic acid %) according to the titration procedure (AOAC, 1995). The extent of proteolysis in the yogurt samples during the storage was calculated according to the ratio of soluble nitrogen/total nitrogen (SN/TN) (Hassan et al., 2015). The fluctuations in the concentrations of acetaldehyde and diacetyl in the yogurt samples were measured using a spectrophotometer (NETZSCH) as described in the available literature (Hassan et al., 2015).

| Whey separation
The volume of whey on the surface of yogurt samples is generally considered as the wheying-off segment (ml/100 g yogurt), and here, it is measured according to the siphon method described by Amatayakul, Halmos, Sherkat, and Shah (2006). In order to quantify the whey separation, 25 g of yogurt samples was poured into a funnel lined with a Whatman filter paper number 41. The whey (ml) was collected after 1 hr of drainage at 4°C, and its quantity was expressed as an index of whey syneresis.

| Water-holding capacity (WHC)
To measure the water-holding capacity, 5 g of yogurt samples was poured into centrifuge tubes and the centrifugation was conducted at 3885 g for 30 min at 10°C. Then, the supernatant was separated and the sediment was weighed. Eventually, the WHC was calculated by the following equation (Sahan, Yasar, & Hayaloglu, 2008):

| Apparent viscosity
During storage, the apparent viscosities of the samples were measured by a rotational Brookfield digital viscometer (Model DV-II) equipped with a spindle-LV4. The experiments were conducted under stable conditions: a shear rate of 30 rpm at 30 s and at a temperature of 10°C.

| Dynamic rheological properties
The dynamic viscoelastic properties of the gel-like structure of yogurt samples were characterized on the first day of storage using a Physical MCP300 rheometer (Anton Paar), accompanied by a cone-plate geometry (50 mm diameter and 1.00 mm gap) at 15°C.
Strain sweeps (i.e., the amplitude tests) were performed in order to determine the linear viscoelastic region at a constant frequency of 1.0 Hz while the strain (γ) varied from 0.1% to 100%. The frequency sweeps were carried out at a constant strain of 1%, as the frequency increased from 0.1 to 12 rad/s (0.03 to 20 Hz) and led to the specification of the storage modulus (G'), the loss modulus (G''), the complex module (G*), and the loss tangent which is defined as tan δ = (G''/G'). Furthermore, the complex viscosity (η*) is an indicator of the stiffness of a material which could be achieved by the ratio of the complex module to the frequency (ω) (Ramirez-Santiago et al., 2010). All of the dynamic rheological measurements were conducted in triplicate, and the reported results were expressed as an average of the three measurements.
The following models specified the descriptions of the dynamic rheological curves of the yogurt samples: where the rheological behavior is characterized by a, b, c, and d as parameters.
where n* (or b) is the dynamic power law factor, k* (or a) is the dynamic consistency index (Pa sn*), ω is the radial frequency (s − 1), and η* is the dynamic viscosity (Pa sn*−1). The system is elastic when n* = 0, and the decrease in η* is parallel to the increase in the value of ω. A viscous system is described by n* = 1 and by the stability of the η* value. When the system is viscoelastic, the value of n* ranges from 0 to 1.

| Color
The evaluation of color or hues among the yogurt samples during storage involved the use of a colorimeter (Minolta, CR-300) which was initially calibrated by black and white surfaces. Based on the light reflection, the parameters of measurement were defined as L* (lightness), a* (redness and greenness), and b* (yellowness and blueness) which described each aspect of color.

| Sensory analysis
Sensory evaluation was conducted according to recommendations described in ISO 13,299:2003. The sensory panel consisted of 10 persons (five male and five female, aged from 25 to 45) who were trained how to evaluate the samples according to the 5-point hedonic test. The organoleptic properties of yogurt samples during storage were examined in terms of appearance, consistency, odor, flavor, and overall acceptance. The maximum score was considered as 5, which indicated the best possible quality of a yogurt sample, and the least score was defined as 1, representing the worst quality in a sample (Hosseini & Ansari, 2019).

| Statistical analysis
All experiments were performed in triplicate, and data analysis was conducted by the SPSS V.18 software. To study the variables and their effects, a complete randomized design was used. The analysis of variance (ANOVA) was performed, and the comparison of mean values was accomplished by Duncan's multiple range test (p ≤ .05).
(1) % WHC = 1 − weight of sediment obtained from centrifuge Initial weight of yogurt samples

| Chemical composition
The average chemical composition and the relevant variations in the stirred yogurt are presented in Table 1. Adding jujube mucilage was observed to have no significant effect on the fat and protein content of yogurt samples. However, the addition of high levels of jujube mucilage to yogurt increased the ash content, which can be due to the presence of minerals in the jujube mucilage. The results were in agreement with those reported by Hassan et al. (2015) and Sahan et al. (2008) where adding guar gum at a concentration of 0.1% and β-glucan at concentrations of 0.25 to 1% did not result in significant differences in the fat and protein contents. However, the ash content of free-fat yogurt samples increased along with the increase in the beta-glucan content. Lobato-Calleros, Ramírez-Santiago, Vernon-Carter, and Alvarez-Ramirez (2014) reported similar results for yogurt samples prepared with native and chemically modified maize starch, and tapioca starch.

| pH and titratable acidity
There were changes in the titratable acidity (%) and pH of yogurt during the 21 days of storage at 4°C ( Table 2). The addition of jujube mucilage to yogurts led to a significant increase in the titratable acidity. The yogurt samples with different concentrations of mucilage did not show significant differences in this regard (p> .05). In addition, as presented in Table 2, over time, either in the control sample or in the samples enriched with mucilage, titratable acidity was significantly increased. On the other hand, Table 2 shows that the trend of change in the pH was opposite to that of titratable acidity.
The pH of the samples decreased significantly by adding jujube mucilage. As shown in Table 2, on the first day of storage, there was no significant difference between the samples which contained 0.1 and 0.15% mucilage (p> .05). These changes were highlighted as the storage reached the seventh day, and thereafter, the changes in the pH values showed significant differences, regarding the 0 and 0.1% mucilage treatments. On the other hand, the control sample exhibited significant changes in the pH during storage just before the seventh day, and the pH values of the remaining days did not show significant differences (p> .05). However, for the yogurt sample with 0.1% mucilage, significant changes were observed in the pH throughout the 21 days of storage. Meanwhile, other concentrations of mucilage caused changes that were similar to the control sample, that is, with significant values before the seventh day (Table 2). Lactic acidification mainly determines the value of acidity in yogurts produced by conventional formulations. Lactic acidification proceeds the culture of the starter bacteria which progressively convert lactose to lactic acid during storage. However, the acidity of yogurts may increase slightly more than usual when other mechanisms are activated by the addition of the jujube mucilage. Indeed, it can be stated that jujube mucilage due to the polysaccharide structure as well as its prebiotic properties can increase the activity of yogurt starter and consequently increase acid production and decrease pH. Relevantly, a previous study claimed that probiotic bacteria can survive and grow for longer durations when fermented milks are enriched with citric fiber, thereby contributing to a faster conversion of lactose into lactic acid (Sendra et al., 2010). Another report suggested that the presence of a highly branched, acidic arabinoxylan in the psyllium husk gum (PHG) structure was a possible cause of the higher acidity observed in yogurts enriched with PHG (Ladjevardi, Gharibzahedi, & Mousavi, 2015). This is also confirmed by a previous study on the

| Flavor compounds
Acetaldehyde, diacetyl, acetone, and acetoin are the most common volatile compounds in yogurt. The typical aroma of yogurt is mostly caused by acetaldehyde, and Lactobacillus delbrueckii ssp. bulgaricus is the starter microorganism which creates this aromatic compound (Khalifa et al., 2011). Acetaldehyde is expected to have a concentration ranging from 23 to 41 mg/kg in yogurt, which generates the optimum flavor in yogurt. Furthermore, the researchers claimed that yogurt has a distinctive buttery or butterscotch taste of fermented milk that is caused by diacetyl.
The current research indicated the occurrence of changes in the acetaldehyde and diacetyl contents of yogurt samples during storage ( Table 2). As can be seen, in all yogurt treatments, the concentration of acetaldehyde has been reduced significantly (p < .05) during the storage time, as the highest and lowest concentrations were observed on the first and 21st days of storage, respectively. Acetaldehyde can evaporate or be converted to ethanol by the action of alcohol dehydrogenase which is produced by yogurt starters, and this can partly explain the decrease in the concentration of acetaldehyde during storage (Sahan et al., 2008). The decrease in the concentration of acetaldehyde during storage was found by Sahan et al. (2008) and Hassan et al., (2015) in yogurt enriched with plant polysaccharides, guar gum, and cress seed mucilage, respectively.
As presented in According to Gaafar (1992), who categorizes yogurts in terms of flavor, all the yogurts produced in the present study fall into the category of weak flavored yogurts due to containing < 4.00 ppm acetaldehyde.
As presented in Table 2, the amount of diacetyl in all samples, with or without mucilage, increased during the storage.
Accordingly, in each treatment, the highest diacetyl content was observed in any particular sample on day 21, and the lowest content of diacetyl was recorded in the sample on the first day. This  This can reduce the production of flavoring substances such as acetaldehyde and diacetyl.

| Proteolysis
The occurrence of changes in the proteolysis of yogurt showed various trends during storage (Table 2). It was observed that during the storage time, the proteolysis rate significantly increased (p < .05) within all samples (with or without jujube mucilage). The highest rate was observed on day 21, and the least was ascribed to day 1.
The increase in the proteolysis of the samples over time reflects the proteolytic activity of the lactic acid bacteria during the storage period. Plain set-type yogurt showed a similar trend of increase in the occurrence and extent of proteolysis during storage (Guzel-Seydim, Sezgin, & Seydim, 2005). Furthermore, low-fat yogurts which contain exopolysaccharide (EPS)-producing cultures and those which contain cress seed mucilage or guar gum are reportedly samples that are capable of higher levels of proteolysis (Hassan et al., 2015).
Moreover, it was revealed that the jujube mucilage was a cause for an increase in the amount of proteolysis. There was no significant difference between different treatments until the seventh day.
However, on days 14 and 21, the degrees of proteolysis in the plain sample (0% mucilage) and in the yogurt which contained 0.2% mucilage were minimum and maximum, which were statistically significant results (p < .05) ( Table 2). A similar observation was found by Hassan et al. (2015) and Abd El-Aziz et al. (2004) in yogurt containing cress seed mucilage or guar gum and free-fat yogurt containing waxy maize. Indeed, the polysaccharide structure of mucilage is reportedly capable of increasing the activity of the starter which, in turn, leads to an increase in the proteolytic activity and a decrease in pH (Donkor, Nilmini, Stolic, Vasiljevic, & Shah, 2007). As shown in Figure 1, a strong positive correlation was found between proteolysis and yogurt acidity in all samples (R 2 > 0.97). Accordingly, the increase in acidity indicates an increase in the activity of the starter and thus an increase in the rate of proteolysis.

| Syneresis and water-holding capacity (WHC)
Whey separation or syneresis is an unfavorable phenomenon which occurs in yogurt. The distinguishable separation of whey from the gel-like surface of set-type yogurts can regularly be defined as whey separation. The duration of storage is commonly associated with certain levels of syneresis (Lobato-Calleros et al., 2014). Supplementing the yogurt with some kind of hydrocolloids or mucilage, due to their ability to absorb and maintain water, can reduce this problem to a great extent (Basiri, Haidary, Shekarforoush, & Niakousari, 2018). As shown in Table 3, syneresis percentages of samples were significantly decreased during the storage period (p < .05). Actually, it should be noted that at a lower temperature, stronger bonds are able to be formed between the particles of the gel, and it is possible that their numbers increase. This can be assumed when swollen particles become linked to each other on a broader space. In addition, the inclusion of mucilage significantly reduced the syneresis in this study (p < .05) ( Table 3) ; Basiri et al., 2018). According to Sahan et al. (2008), the addition of β-glucan to nonfat yogurts reduced the degree of syneresis. However, there was no relationship between the β-glucan concentration and the syneresis value. In addition, the value of syneresis on the first day was more than the value on the 15th day.
A report suggests that high concentrations of PHG (psyllium husk gum) can be associated with the formation of a strong network structure which can ultimately increase the viscosity, in addition to a slower rate of syneresis in low-fat yogurt (Ladjevardi et al., 2015).    TA B L E 3 Physical characteristics of yogurt samples during 21 days of storage at 4°C (mean ± SD) * also the alteration of protein-protein connections which make new linkages with the mucilage (Donkor et al., 2007).

| Color
From the perspective of consumers, color is a decisive parameter of quality. It can represent the freshness, flavor, and the commercial value of a dairy product. Table 4

| Sensory analysis
Average scores for the flavor of stirred yogurt, either with or without mucilage, differed on day 1 and day 21 during storage (Table 5). It is known that the sensory and nutritional characteristics of yogurt, along with its general features, such as acidity, free fatty acid content, and the amount of flavor compounds, are affected by the initial chemical composition of the milk, the process conditions, the addition of flavorings, and the activity of the starter bacteria involved in fermentation (Bonczar, Wszołek, & Siuta, 2002). As shown in Table 5, the appearance of the samples was not significantly different on days 1 and 7. However, on the 14th day and 21st day, the samples which contained mucilage received a lower score from the sensory evaluators in relation to TA B L E 4 Changes in color of yogurt samples during 21 days of storage at 4°C (Mean ± SD) the appearance, compared with the control sample. No significant difference was observed in the consistency of yogurt samples by sensory evaluators. Moreover, the incorporation of jujube mucilage into yogurt reduced the consumers' desirability in terms of odor and taste, but these differences were not statistically significant (p> .05). The overall acceptance score of samples which contained mucilage was less than the control sample. However, the samples incorporated with jujube mucilage appeared to have acceptable sensory properties. In addition, all of the sensory indices in different yogurt samples decreased during the storage time, although this decrease was not significant compared with day 0 (p> .05). In line with our results, Staffolo et al. (2004) reported that the addition of inulin, wheat, and bamboo fibers had no significant effect on the sensory characteristics of yogurt. However, fiber-rich yogurts received a high score in terms of color, flavor, and texture. Noh, Seo, Lee, and Chang (2013) also demonstrated that the addition of CFE (2%-4%) to yogurt did not significantly affect the aroma, softness, taste, and overall liking scores, but the overall preference score significantly decreased in yogurt samples treated with 6% CFE.  Complex viscosity and the loss tangent of stirred yogurt samples, with or without mucilage, are provided in Figure 2(b, c). The complex viscosity, which is a measure of total rigidity, indicated higher values in samples with higher concentrations of mucilage.

| Rheological properties
The addition of mucilage to yogurts resulted in a stabilized gel structure, thereby increasing the integrity of the gel network and hence the complex viscosity. Consequently, the mucilage led to a high degree of firmness in yogurts. As reported by Sendra et al. (2010), the addition of orange fiber led to a stronger structure of gel-like structures in yogurts which could be attributed to a higher water absorption ability of included fiber, thereby increasing the complex viscosity. Another popular material function to describe the viscoelastic behavior is the loss tangent which indicates the superiority of one of the rheological properties (i.e., either viscose or elastic). This factor is defined as the ratio of the energy that is released (related to the viscose property of the material) to the stored energy (related to the elastic state). A higher value of loss tangent is likely to exhibit more of a liquid-like behavior than a solid-like behavior. In the present study, the samples containing mucilage were found to have a higher loss tangent than the control samples. However, the samples incorporated with 0.2% mucilage showed a lower loss tangent value than the samples containing 0.15% jujube mucilage. In fact, it could be stated that at low concentrations of mucilage, the yogurt becomes more inclined to display a quasi-liquid behavior, whereas higher concentrations of the mucilage can increase the viscose behavior of yogurt, thereby causing a more solid state. Table 6 indicates the values of a, b, c, and d, as adjusted by the rheological models (Equations 2 and 3). Due to the high value of R 2 obtained for G' (which was at least 0.92), and the one for G'' (at least 0.98), the models were able to fit well with the experimental data. Steffe (1996)   dynamic consistency index (a or k*) increased from 79.32 to 225.23.

| Modeling the rheological behavior
Moreover, the value of n* (or b*) varied from 0.9 to 0.92 among different samples, indicating the viscoelasticity of the above systems, although they were closer to viscose systems. In addition, by adding mucilage, the percentage of the linear viscoelastic region decreased and the yield stress was increased. In this regard, Keogh and O'kennedy (1998) studied the inclusion of milk fat, protein, and hydrocolloids in stirred yogurt. It was found that the starch did not affect the k*, whereas the gelatin and the xanthan/LBG mixture reduced the k* value. However, none of the variables showed a significant effect on the value of the n* parameter.

| CON CLUS ION
As a natural stabilizer, the jujube mucilage was incorporated into stirred yogurts. Chemical analyses revealed that using the jujube mucilage increased the acidity and proteolysis, but reduced the amounts of acetaldehyde and diacetyl compounds in stirred yogurts, as compared with the control sample. The assessment of physical parameters showed that the stirred yogurt which contained jujube mucilage can be capable of a lower level of syneresis, but a higher viscosity and WHC values. The treated samples showed significantly higher values of dynamic moduli, loss tangent, and complex viscosity, as compared with the control. The jujube mucilage had no significant effect on the sensory characteristics of yogurt, even though it caused yogurts to obtain slightly less scores in terms of overall acceptability. The jujube mucilage has been generally recognized for its beneficial effects on health, besides its function which was revealed here as an improver of physicochemical properties in yogurt. Further research is required to examine the capacities of its commercial applications on other dairy products.

ACK N OWLED G M ENT
The authors received no financial support for this study.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest in this paper.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.