Antioxidant activities, functional properties, and application of a novel Lepidium sativum polysaccharide in the formulation of cake

Abstract A novel heteropolysaccharide, named cress water soluble polysaccharide (CWSP), was purified from Lepidium sativum seeds. Antioxidant activities and functional properties were characterized thermally using thermal gravimetric analysis (TGA), and the differential scanning calorimeter (DSC) results of CWSP were evaluated. The total antioxidant capacity and the metal chelating activities of CWSP at 3 mg/ml were equivalent to 116.34 µg ascorbic acid and 62.57%, respectively. As for the CWSP that was used for the production of cakes, it was thermally stable, and it presented high water (WHC) and oil holding (OHC) capacities and good emulsion properties. The samples were prepared with different levels of CWSP (0.1. 0.3, and 0.5%) and analyzed during 15 days of storage at room temperature. The obtained results indicated that the addition of CWSP had a significant effect on the texture profile, leading to the increase in all parameters in terms of hardness, springiness, cohesiveness, adhesiveness, and chewiness. Moreover, the reformulation samples presented higher a* and lower L* and b* than the control sample. The sensory evaluation showed that the formulation of cake with 0.3% of CWSP was the most acceptable. Therefore, CWSP was shown to be a new alternative for improving the quality attributes, indicating potent antioxidant activities on the shelf life during the storage of bakery foods.

of Europe. It is a seed that contains extremely nutritious substances with a pleasant aroma and flavor along with important minerals, vitamins, and metabolites, such as polysaccharides and glycoprotein complexes (Arezoo & Milad, 2018).
Polysaccharides have largely been added to food packaging (Yanming et al., 2021) and foods, as bakery, meat, and dairy products to improve their quality and provide a high sensory quality (Ben Slima et al., 2018;Xi et al., 2020). In fact, polysaccharides are used to change the textural and physicochemical properties of foods even with a very low concentration (Rongbin & Fan, 2021). Moreover, food polysaccharides usually show remarkable rheological properties, such as thickening, stabilizing, gelling, binding, and emulsifying properties of food products (Arezoo & Milad, 2018). Polysaccharides have also been successfully used to improve food shelf life via preventing settling or creaming, influencing water crystallization, retaining moisture, retarding staling, and preventing syneresis or the retro-gradation of starch products (Xi et al., 2020). Polysaccharides extracted from byproducts can improve the cost-effectiveness of the overall production process, and hence the final product (Ben Salah et al., 2011).
The purpose of this study was to explore the water-soluble polysaccharide from cress seeds (CWSP) to highlight its antioxidant activities and functional properties and to evaluate its effect on cake formulations during 15 days of storage.

| Materials and reagents
L. sativum seeds were purchased from the market of Sfax city in Tunisia. They were crushed by a Moulinex blender LM 241, and then sieved with a 0.2 mm mesh size. The powder was kept in a wellsealed amber glass container to protect it from sunlight. The cupcake ingredients were obtained from Gourmandise (Sfax-Tunisia).
2.2 | Extraction of water-soluble polysaccharide from L. sativum (CWSP) Water-soluble polysaccharide from cress seeds (CWSP) was purified according to the method of Ben Slima et al. (2018). Briefly, ethanol was used to pre-extract and remove cress seed powder pigments.
The extract was obtained after 20 volumes of deionized water addition at 90°C while being stirred for 4 hr. The obtained filtrates were evaporated under vacuum. The concentrated liquid was then precipitated at 4°C for 24 hr using 95% (v/v) ethanol. The final purified polysaccharide (CWSP) was later on lyophilized and stored at 4°C.

| DSC measurements
The phase transition was determined using DSC analysis. The thermal analysis of CWSP was tested with a differential scanning calorimeter from −2.77 to 250°C (Mettler Toledo 181 Star) at a rate of 5°C/min.

| TGA Thermo-gravimetry analysis
Thermo-gravimetric analyzer (Mettler Toledo TGA/SDTA 8951E) was used to determine the thermal gravimetric analysis (TGA). Five milligrams of the sample was introduced into the sample pan and heated from 30 to 400°C under nitrogen atmosphere. The gas flow rate was 40 ml/min.

| Water-holding capacity
The water-holding capacity (WHC) was recorded by the method of Trigui et al. (2018). One gram of CWSP was dispersed in distilled water (25 ml). The suspensions were held at different temperatures (25, 50, or 75°C) for 1 hr. Besides, the excess water was drained at 50°C for 25 min. The WHC was calculated according to the following formula: The WHC was expressed as g of the absorbed water per g of the sample.

| Oil-holding capacity
The oil-holding capacity (OHC) was determined following Trigui et al. (2018). The samples (0.5 g) were poured in volumes of 10 ml of soybean oil. Then, the suspensions were kept at different temperatures (25, 50, or 75°C) for 1 hr. The obtained supernatant was removed and the excess oil was drained. OHC was calculated as the weight of the tube contents after dividing draining by the weight of WHC = weight of the tube content after draining weight of the dried SWPS the dried CWSP. OHC was expressed as g of the absorbed oil per g of sample.

| Emulsifying activities
Determination of emulsifying activities CWSP emulsifying activities were assayed as reported by Ben Slima et al. (2018). Soybean oil was added to the CWSP solution (0.3%) at a ratio of 3:2 (v/v) and stirred in the vortex for 2 min at 2400 rpm.
After 1, 24, and 168 hr, emulsification indices E1, E24, and E168 were calculated as follows: where he (mm) is the emulsion layer height and ht (mm) is the mixture overall height after t hours.

Microscopic assessment of emulsions
After 24 hr of emulsion storage, a light microscope (NIKON ECLIPSE E200) with a 10 × objective lens was used to examine the emulsion samples. For sample preparation, a pin-tip amount of the emulsion was smeared on the microscope glass slide and then quickly covered with a coverslip.

| Total antioxidant activity
The total antioxidant activity of CWSP was determined by the phosphate molybdate method (Prieto et al., 1999)

| Preparation of cakes
The cakes consisted of the following ingredients: wheat flour, sugar, egg yolk, baking powder, and butter. All ingredients were obtained from Souad Gourmandise society, Sfax-Tunisia. Wheat flour supplemented with CWSP was prepared. After mixing the ingredients, the dough samples were placed on aluminum trays and baked at 180°C for 30 min and then allowed to cool. Then, the samples were stored at room temperature under natural relative humidity conditions in a closed bag for storage studies (0-15 days) until analysis. Each analysis was performed on three independent experiments which were evaluated in triplicate.

| Antioxidant activities of cake
DPPH•radical-scavenging, ABTS, and reducing power were measured in cakes as previously described (Msaddak et al., 2015). Five grams of bread sample was homogenized with 25 ml ethanol for 2 hr at 25°C using an orbital shaker at a stirring speed of 200 rpm. After centrifugation at 8000 rpm for 30 min, the supernatant was recovered. The DPPH radical-scavenging activity (%), the ABTS radicalscavenging activity (%), and the reducing power (absorbance at 700 nm) of cakes were measured as previously described (Msaddak et al., 2015). The determination of antioxidant activities was carried out in triplicate.

| Thiobarbitric acid assay (TBA)
TBA was based on a method adapted from Bajaj et al. (2016). Briefly, 10 g of ground cookies samples was mixed in 25 ml of distilled water and 10% trichloroacetic acid. To 1 ml of filtrate, an amount of 3 ml of TBA solution (0.67%) and 0.05 N of H 2 SO 4 was added and the mixture was heated on a water bath at 95℃ for 30 min. After cooling, n-butanol (4 ml) was added and centrifuged at 1500 rpm for 10 min. The organic layer was pipetted out and the absorbance was measured at 532 nm.
The determination of TBA was carried out in triplicate.

| Texture profile analysis (TPA)
The crumb texture was evaluated for cakes stored at 25°C for 24 hr and 72 hr using a Texture Analyzer (Model TA.XTplus, Stable Microsystems,). The texture profile analysis (TPA) for cakes was EI = (he∕ht) * 100 determined by a texture analyzer (TA-XT2i, Stable Micro Systems Ltd.,) and performed at room temperature (Moza & Gujral, 2017).
Texture parameters (hardness, springiness, cohesiveness, and chewiness) were quantified during 10 days of storage for 20 mm-thick cake slices using the TPA double compression test. Actually, the samples were compressed to 50% compression at a speed of 10 mm/ sec and a 5 s delay between the first and second compression. The determination of TPA was carried out in triplicate.

| Determination of color of cakes
The color parameters of crumb cakes (lightness L*, redness a*, and yellowness b*) were determined with a Color Flex spectrocolorimeter (Hunter Associates Laboratory Inc.,) for 10 days of storage at room temperature . The determination of color was carried out in triplicate.

| Sensory evaluation
The sensory properties (texture, color, odor, taste, and overall acceptability) of freshly prepared cakes were evaluated by 60 panelists.
A nine-point hedonic scale with 1 (dislike extremely), 5 (neither like nor dislike), and 9 (like extremely) was used. Water was provided to rinse the mouth between evaluations.

| Statistical analysis
One-way analysis of variance (ANOVA) was performed using the SPSS program (V17.0) to determine the significant differences between treatments. The results were expressed as mean values and standard errors from three replications. All tests were performed at the 0.05 level of significance.

| DSC measurement
Differential scanning calorimetry (DSC) is considered a powerful technique to examine chemical and physical changes allowing to determine the kinetics of polysaccharide polarizing during thermal processing. As shown in Figure 1a, an obvious endothermic peak at 87.83°C in DSC curve was detected, indicating the dehydroxylation F I G U R E 1 Thermal analysis: of CWSP: (a) DSC; (b) TGA reaction and the loss or the dehydration of peripheral polysaccharide chains (Jalaleldeen et al., 2020). Besides, an exothermic peak was observed at 274.96°C, indicating the level of cross-linking of polymers (Matheus, 2016). A similar curve was observed in the case of tamarind seed polysaccharide, which seems to behave completely differently; its endothermic peaks were indicated at 85.29°C and 270.78°C and it was followed by an exothermic peak at 318°C (Ktari et al., 2020). Other studies reported that polysaccharides ex-

| Thermo-gravimetry analysis
TGA is a simple tool to study the thermal behavior and the decomposition pattern of polymers. In fact, it can provide the mass change measurement in materials related to decomposition pattern, dehydration, and oxidation of polymers with temperature and time.
As shown in Figure 1b, the initial weight loss at the temperature range 28.33-175°C was observed, which could be attributed to the loss of bound and free water (Kittur et al., 2002). The weight loss of CWSP was shown in the temperature range 175.00-566.03°C, which might be due to the CWSP decomposition. The results suggested that CWSP was relatively stable below 566°C. Hence, the thermal stability of CWSP may be an important functional property in some food applications.

| Water-holding capacity of CWSP
The WHC represents the amount of water held, used to assess the stability, sensory, and texture of sample. As shown in  (Trigui et al., 2018). It was reported that WHC could be attributed to pore size, polysaccharide source, and the capillarity of the molecule conformational structure (Shen et al., 2019).

| Oil-holding capacity of CWSP
The OHC of CWSP was investigated at 25, 50, and 75°C. As shown in Table 1, the highest OHC value was detected at 50°C, which recorded 1.31 g oil/g sample. This value was lower than that of polysaccharides from Lilium lancifolium Thunb (9.25 g oil/g sample) (Gao et al., 2015), and higher than durian seed gum (0.44-1.29 g oil/g sample) (Mirhosseini & Amid, 2012). Although OHC is partially linked to chemical composition, it is closely related to fiber structure porosity and the affinity of the fiber molecule to oil (Shen et al., 2019).

Effects of pH, temperature, and ionic strength on emulsion stability
Emulsifiers are frequently exposed to extreme ionic strength, pH, and temperature in various industrial processes. Therefore, CWSP  it is reasonable to suggest that the CWSP stability of emulsions is due to gel-like network formation (Shao et al., 2017). Generally, Na + had a larger protecting effect on the polymer chains by collapsing down the side chains (Shao et al., 2015). For emulsions prepared with corn oil, the increase in NaCl concentrations from 0.1 to 2 M caused an E24 decrease from 70.27% to 51.42%, respectively. It is worth noting that the ionic concentration up to 0.8 M and droplet coalescence were observed. This could emanate from the decrease of electrostatic repulsions between particles, due to the decrement of the original surface charge of particles (Xiao et al., 2016).
As depicted in Figure 2b, emulsions could be influenced by pH value differences altering polysaccharides' molecular con- biopolymers is most possibly attributable to their diverse structural and chemical compositions . In this study, at acidic conditions, polysaccharide molecules adsorbed onto the surfaces of the emulsion droplets were less dissociated, and therefore had a compact conformation, demonstrating its good emulsifying capacity. This is favorable for preventing emulsion

| Chelating ability of CWSP
Ferrous ion is generally considered as the most potent pro-oxidant that accelerates the reaction oxidation through Fenton reaction (Heng et al., 2019). The chelating ability of CWSP at different concentrations is presented in Figure 3b.

| Antioxidant activities in cake
The DPPH radical scavenging activities of the cake samples are shown in Figure 4a. Cakes prepared with 0.5% of CWSP presented the highest DPPH radical scavenging activities with 77.07% on day 1 and 70.26% on day 15. Comparable trends were also noticed for ABTS ( Figure 4b) and ferric-reducing power assay (Figure 4c).
In fact, the ABTS for control cake was 56.  (Bhat et al., 2020). The most important reduction of antioxidant activity was observed in the control cake, which could affect the storage stability of the product. Interestingly, CWSP-

| Lipid peroxidation
Lipid peroxide formation is accompanied with a secondary endproduct, malondialdehyde (MDA), formation. As shown in Figure 4d, TBA value for reformulated cake samples (F1, F2, and F3) was lower than that for the control sample. Remarkably, CWSP decreased TBA values in comparison with those of the control throughout the storage. TBA value that is less than 0.6 mg/kg MDA for cake samples is said to be nonrancid, while values ranging between 0.65 and 1.44 mg/kg MDA are considered as rancid but still acceptable. As for the values that are higher than 1.5 mg/kg sample, they are considered rancid and unacceptable (Alshima 2012). On the 15th day of storage, the control sample was rancid but still acceptable. The control sample showed the highest TBA value compared to other reformulated cake samples, which indicated that the polysaccharide addition as antioxidant ingredient, can extend the shelf life of cake by retarding oxidation reactions.

| Determination of color of cake formulated with CWSP
Since cake color has an important effect on consumer perception, it is necessary to determine the modification of the sample that affects the values of color parameters. The results presented in Table 2 reveal that cake incorporated with CWSP had a significant influence on color parameters. The values of a*, which ranged between 4.13 and 6.48, in the samples with CWSP (F1, F2, and F3) were significantly higher in comparison with those of the control sample. However, the results showed that the lightness (L*) and b* values decreased for the reformulated cake samples, which were found to be darker than

| Texture analysis of cake formulated with CWSP
The results of texture analyses of reformulated and control cakes during the storage period at room temperature are illustrated in Table 3. They revealed that CWSP addition at different levels in-   Grigelmo-Miguel et al. (1999) reported an increase in chewiness in muffins with dietetic fibers.
Chewiness modifications could be related to the viscosity of added compounds (Moza & Gujral, 2017). Furthermore, CWSP incorporation increased adhesiveness. Hence, owing to the functional properties of polysaccharides, CWSP could improve the texture profile and stability of bakery products.

| Sensory evaluations
The sensory evaluation of cakes was evaluated in terms of their color, texture, odor, taste, and general acceptability using the nine-point

TA B L E 4 Sensorial properties of cakes
F I G U R E 5 Images of the different samples of cakes formulated hedonic scale (Table 4). Among the cakes, the control sample was the lowest in all sensorial attributes. Nonetheless, cake with 0.3% of CWSP (F2) was more acceptable according to panelists compared with other formulations with a better taste ( Figure 5). Korus et al. (2015) demonstrated that flaxseed mucilage can be a tangible solution to improve the sensory properties and preserve the dough quality. However, Fan et al. (2007) suggested that Auricularia auriculara polysaccharide flour added in the baked products does not affect the sensory evaluation of the final product.

| CON CLUS ION
Polysaccharides extracted from cress seeds showed potent antioxidant activities and important functional properties. The obtained results showed that CWSP was used as a readily accessible source of natural antioxidants as determined in three assay models and as food additives to improve cake quality in terms of color and texture. Sensorial analysis proved that cakes with 0.3% of CWSP represented the best acceptability by the panelists. This novel polysaccharide can be widely applied to develop bakery food with good quality.

ACK N OWLED G M ENT
The authors thank Mrs Leila Mahfoudhi, Emeritus teacher of English in the Faculty of Sciences of Sfax, for proofreading and polishing the language of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. Formal analysis (equal); Validation (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
A data available on request of authors.