Potential of date palm spikelet extract as an anti-oxidative agent in set-type yogurt during cold storage

ABSTRACT The study aimed to evaluate the effects of date palm spikelet extract (DPSE) from two cultivars on the chemical, antioxidants, and sensory properties of yogurt. The DPSE of Rziz (Rz) and Khalas (Kl) cultivars contains high levels of total phenolics and flavonoids as well as phenolic compounds with an antioxidant activity exceeded 90%. Increasing the DPSE concentration in yogurt improved the levels of total solids, protein, ash, total phenolic compounds, flavonoids, and antioxidant activity even during prolonged storage periods. The thiobarbituric acid reactive substances (TBARS) of yogurt was decreased by adding DPSE but increased with storage time. Throughout the storage period, the DPSE-formulated yogurt revealed better stability in terms of its physicochemical, antioxidative, and sensory characteristics compared to non-formulated yogurt. The findings of this study proved the antioxidant potential of 0.5–1% DPSE for both cultivars, with the superiority of the Rz extract as a functional additive to preserve yogurt.


Introduction
Yogurt and related fermented dairy products have a considerable global economic importance due to their high nutritional quality, such as compounds produced from the enzymatic breakdown of milk proteins (Capriotti et al., 2016). Yogurt has excellent health benefits and high nutritional quality because of its high protein, lipid, vitamin, and mineral contents (Sidira et al., 2017). Yogurt has significant therapeutic values and exerts beneficial health effects, such as preventing intestinal disorders and chronic diseases, decreasing cholesterol absorption, and reducing blood pressure, due to the good metabolic and probiotic potentials of lactic acid bacteria (Arief & Taufik, 2016). Although yogurt is one of the most consumed and widespread dairy products worldwide, it is considered poor in terms of antioxidant content (Raikos et al., 2019), which must be supported by fortifying it with natural antioxidants to raise its functional and nutritional efficiency. are characterized by their high phenolic compound, antioxidant, and flavonoid contents, as well as a small percentage of carotenoids; they have not been used or benefited from, and they become burdens to the environment (Abu Ayaneh et al., 2014). Utilization of these important waste products will not only add commercial value to them, but also help in reducing the harm they cause to the environment.
To improve the nutritional and functional properties as well as safety of yogurt, different research were conducted. Akpinar et al. (2020) reported that the use of enterococcus species in yoghurt production can positively affect the physical, chemical and rheological properties as well as the functional properties. Also Taghizadeh Moghaddam et al. (2020) showed that the use of L. plantarum and L. acidophilus decreased the level of bisphenol A as a harmful ingredient in yoghurt with the storage period. Moreover, fortifying yogurt with naturally sourced antioxidants can meet consumer demands and acceptability (Granato et al., 2017). The use of plants and agri-industrial waste for human consumption is also a viable approach to address environmental, economic, and sustainability challenges of the modern world. Therefore, this study was designed to investigate the effects of date palm spikelet extract on the chemical, antioxidative, and sensory properties of set-type yogurt

Materials
Fresh date palm spikelets of the Khalas and Rziz varieties were obtained from the date palm farm of the Date Palm Center, Alhufuf, Alhasa, Saudi Arabia during April 2019. The spikelets were manually separated from their inflorescence axis, washed three times with water, subjected to ovendrying at 45°C for 48 h, and then powdered using a Thomas Mill (Thomas Scientific, Swedesboro, NJ, USA) to pass a 0.5-mm sieve (Thomas Scientific). The dried samples were kept in sealed polyethylene bags at −20°C until further analysis and yogurt fortification. All chemicals used were of standard grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of spikelets extracts
The extracts were prepared using 50% ethanol as the extracting solvent. Moreover, 2 g of date spikelet powder was suspended in 200 mL of 50% ethanol and extracted using an ultrasound path (model 2800 CPX, Branson, USA) at 42.5°C for 20 min. The frequency was set to 40 kHz, and the constant power was 110 W. The extract was cooled to room temperature and then filtered using Whatman No. 1 filter paper. Then, the extract was freeze-dried and stored at −20°C for subsequent analyses.

Preparation of set-type yogurt
Set-type yogurt was prepared following the method previously described by Zhang et al. (2019), with slight modifications. Yogurt was prepared by adding skimmed milk powder (14%) and date palm spikelet extract (0.5 and 1%) to distilled water and the mixture was homogenized for 5 min and then pasteurized at 85°C for 30 min. The mixture was cooled down to 42°C and then inoculated with 2.5% (v/ v) starter culture of 1:1 Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus (YC-X11, Chr. Hansen, Denmark). After that, 14 g of the mixture was poured into 100-mL plastic cups and incubated at the same temperature until complete coagulation was achieved (pH 4.6; 4 h). After that, the yogurt cups were stored at 4°C for 1, 7, 14, and 21 d. Control yogurt without spikelet extract was prepared in the same way and the entire process was performed in triplicate.

Preparation of the yogurt extract
The yogurt supernatant extract was prepared as previously described by Zhang et al. (2019). Moreover, 10-g yogurt samples were poured into falcon tubes and centrifuged at 5000 ×g for 20 min at 4°C. The supernatants were collected and re-centrifuged at the same conditions. The clear supernatants were then stored at −80°C until further use.

Determination of approximate composition and total solid content of the DPSE and yogurt samples
The moisture, protein, and ash content of the spikelet extract and yogurt samples were determined using official standard methods (AOAC, 2005). The fat content of the spikelet extract was determined using the AOAC (2005) method and that of yogurt using the method of Badertscher et al. (2007). The total solid content was determined using the difference method as per the expression given below:

Determination of pH and titratable acidity of DPSE
The pH values of the extract samples were measured using a pH meter probe (Corning Scientific Products, New York, USA). Titratable acidity was measured for the sample after mixing 1 g of the sample with 10 ml of hot distilled water (90°C) and titration with 0.1 N of sodium hydroxide using three points of the phenolphthalein index (0.5%) until the onset of a pale pink coloration. It was calculated as per the expression: LA % = [10 × V NaOH × 0.009 × 0.1/W] × 100; where, 10 is the dilution factor, W is the weight (g) of the sample for titration, V NaOH is the volume of NaOH used to neutralize lactic acid, and 0.1 is the normality of NaOH.

Determination of total phenolic content (TPC)
The TPC of date spikelet powder extract (DPSE) and yogurt extract was determined using the Folin-Ciocalteu reagent method (Hernández-Carranza et al., 2016), with slight modifications. Moreover, DPSE (200 µL) was mixed with ten-fold diluted Folin-Ciocalteu reagent (400 µL), and the mixture was incubated at room temperature for 5 min. After that, 1 mL of 1 M Na 2 CO 3 was added, and the mixture was kept for 2 h at room temperature. After preparing the standard curve using gallic acid (0-1.0 mg/mL), the absorbance of DPSE, yogurt extract, and gallic acid were recorded at 765 nm using a spectrophotometer (Lambda EZ 150, PerkinElmer, USA). The total phenolic content was expressed as mg gallic acid equivalent per 100 g of extract (mg GAE/100 g).

Determination of total flavonoid content (TFC)
The TFC of DPSE and yogurt extract was determined using the method previously described by Hernández-Carranza et al. (2016). About 0.5 mL of an adequate dilution of extract was mixed with 0.5 mL of NaNO 2 (1.5% w/v) in a glass tube, the mixture was mixed and allowed to stand for 5 min. Thereafter, about 1 mL of AlCl 3 (3% w/v) was added and mixed for 1 min; then, 1 mL of NaOH (1 N) was added. The mixture was allowed to stand for 1 min and the absorbance was read at 490 nm using an UV-Vis spectrophotometer. To determine the total flavonoids a standard curve of quercetin was prepared. The result was expressed as mg of quercetin per 100 g of sample.

Determination of DPPH scavenging activity
Antioxidant activity of DPSE and yogurt extract was assessed using the DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity method (Hernández-Carranza et al., 2016), with slight modifications. Moreover, 1 mL of the extract was mixed with 2 mL of DPPH methanolic solution (0.25 mmol/L), and the mixture was incubated in the dark at room temperature (25°C) for 10 min. The control was prepared without the extracts, and the absorbance of both samples and control was measured at 517 nm using a spectrophotometer (Lambda EZ 150, PerkinElmer, USA). The DPPH radical scavenging activity was measured using the following equation: DPPH radical scavenging activity % inhibition ð Þ where, A sample is the absorbance of the sample, and A control is the absorbance of the control without sample.

Determination of ABTS + scavenging activity
The 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) radical scavenging activity of the DPSE and yogurt extract was determined according to the method previously described by Thaipong et al. (2006), with slight modifications. ABTS + regent was prepared by mixing it with potassium phosphate (pH 5). The working solutions were prepared by mixing 7.4 mM ABTS + stock solution and 2.6 mM of potassium persulfate stock solution (1:1, v/v), and the mixture was allowed to react in the dark at incubated room temperature for 16 h. For analysis, the solution was diluted by mixing 1 ml of ABTS + solution with 30 ml of methanol to obtain an absorbance of 1.049 ± 0.012 units at 743 nm using a spectrophotometer. Then, 200 μL of the extract was mixed with 1800 μL of ABTS + reagent diluted with methanol, and the decrease in absorbance was measured at 734 nm after 2 h. Antioxidant activity was evaluated as a percentage using the following equation: ABTS + scavenging activity (%) = [1 − (Abs sample/Abs control)] × 100.

Determination of phenolic compounds
The phenolic compounds were determined using HPLC (Shimadzu) equipped with a PDA detector and an Inertsil ODS-3 (5 µm; 4.6 × 250 mm) column. The mobile phase was a mixture of 0.05% acetic acid in water (A) and acetonitrile (B) with the flow rate of 1 ml/min at 30°C. The injection volume was 20 µl. The peaks were taken at 280 using a PDA detector. The elution programme was employed: 0-0.10 min 8% B; 0.10-2 min 10% B; 2-27 min 30% B; 27-37 min 56% B; 37-37.10 min 8% B; 37.10-45 min 8% B. The total running time per sample was 60 min.

Determination of TBARS
The thiobarbituric acid reactive substances (TBARS) of the yogurt extract were assessed by measuring malonaldehyde oxidation products (MDA) as previously described (Rosmini et al., 1996), with slight modifications. Moreover, 5 g of the yogurt samples was mixed with 20 mL of distilled water, homogenized, and filtered using a Whatman No.1 filter paper. Furthermore, 1 mL of the filtrate was added to 4 mL thiobarbituric acid (20 g/mL) and 100 μL butylated hydroxytoluene (10 g/100 mL) in a screw-cap tube. The sample was mixed vigorously for 10 min in a water bath at 95°C-100°C to facilitate the development of a pink color. Additionally, the sample was centrifuged at 5500 ×g for 25 min, and the absorbance of the supernatant was read at 532 nm. Then, the total amount of malonaldehyde was calculated (mg malonaldehyde/kg), according to the following equation: malonaldehyde (mg MDA/kg) = absorbance (532) × 7.8.

Sensory evaluation
The sensory evaluation of the yogurt product was performed according to the method previously described by Dantas et al. (2016), with the help of a panel of 37 male staff and students (20-35 years-old) from the College of Food and Agriculture, King Saud University. Prior to sample evaluation, three training sessions were carried out to acquaint the panelists with the sensory attributes that are to be evaluated. The panelists evaluated the sensory characteristics of the yogurt product throughout its storage period equal to that of the sensory evaluation, and the panel consisted of 12 trained and 25 semitrained members. Furthermore, attributes, such as taste, color, flavor, smell, texture, and general appearance, were evaluated using a 9-point hedonic scale test.

Statistical analysis
All data from three replicates were analyzed using one-way analysis of variance (ANOVA) through the IBM SPSS Statistics 23.0 software (SPSS Inc., USA). All data were presented as means ± standard deviation. Duncan′s multiple range test was used to determine the significance among means. Statistical significance was considered when p-value was less than 0.05 (p < .05). Principle component analysis (HJbiplot algorithm) was performed using MULTBIPLOT software as described in the instruction manual (Vicente-Villardón, 2010). HJ-biplot allows the analysis of all variables simultaneously and it achieves a high quality of representation of both samples and variables. In this analysis, a matrix of 20 rows (samples) and 16 columns (variables) was applied.
In the biplot, the distances between rows markers (samples) interpreted as similarity, in which, short distances indicate similarity, whereas long distances indicate dissimilarities. The length of column markers (vectors) indicate the standard deviations of the traits and the cosine of the angles between vectors indicate correlation between variables, in which, acute (< 90 °), obtuse or strait (˃ 90 ° or = 180°), and right (90 °) angles indicate positive, negative, and no correlations, respectively (Mutwali et al., 2016).  Zhang et al. (2019). The results also demonstrated that DPSE had high total flavonoid content (TFC), with Rz having a significantly higher value (1172.58 mg CQE/100 g) than Kl (1005.36 mg CQE/100 g). Additionally, DPSE expressed high antioxidant activity, especially the Rz spikelet extract, as determined by both ABTS and DPPH methods. The phenolic compounds of DPSE of Rz and Kl showed that the most abundant phenolic acid was syringic acid observed in Rz (43.55 mg/100 g) followed by protocatechuic acid. Moreover, among flavonols and flavan-3-ol, respectively rutin and (+)catechin were significantly (p ≤ 0.05) higher in Rz than Kl. However, resveratrol as a stilbene was significantly (p ≤ 0.05) higher in Kl than Rz. The results showed that most of the phenolic compounds were significantly (p ≤ 0.05) higher in Rz than Kl. These findings suggest great variations and abundance of phenolic compounds and flavonoids in the Rz spikelet extract, which possesses high antioxidant activity and indicates the suitability of this extract for different applications to extend the shelf life of animals' products. The abundance of phenolic and flavonoid compounds in DPSE may potentially bear antioxidant activities that are comparable with widely used synthetic antioxidants.

Chemical composition of yogurt formulated with DPSE during storage
The results of the evaluation of the chemical composition of yogurt formulated with different levels of the DPSE of two cultivars during storage are presented in Table 2. The addition of DPSE significantly (p ≤ 0.05) decreased moisture content, with a concomitant increase in the total solids of yogurt; whereas moisture content was significantly increased with storage period, with a concomitant decrease in total solids. The increase in total solids after DPSE incorporation may be responsible for the decrease in moisture content of yogurt, which indicated that DPSE had a high water-holding capacity. A similar increase in total solids and decrease in moisture content of yogurt containing medicinal plant extracts has been reported by Erturk and Demirkol (2014). The protein and ash contents of formulated yogurt were increased with DPSE addition in both cultivar extracts, with no observable effect during storage. The considerable amounts of protein, fat, and ash contents in DPSE of both cultivars can result in an increase of such constituents in the formulated yogurt. However, fat content was neither affected by the level of added DPSE nor the storage period, and this is mainly due to the fact that DPSE had a low fat content. In agreement with our findings, Jung et al. (2016) has reported an enhancement in protein and fat contents in yogurt fortified with red ginseng extract. Los valores se presentan como la media de muestras triplicadas (±DE) con base en los valores de peso seco. Las medias que no comparten un superíndice o superíndices en común a, b o c en una fila son significativamente diferentes en p ≤ 0.05, según la prueba de rango múltiple de Duncan.

Oxidative characteristics of yogurt formulated with DPSE
addition of DPSE and with storage may be attributed to the considerably high amount of TPC, TFC, and antioxidant activity of DPSE (Table 1)   Values are presented as means of triplicate samples (±SD) on wet weight basis. Means not sharing a common superscript(s) a, b, or c in a row, or A, B, or C a column are significantly different at p ≤ 0.05, as assessed using Duncan's multiple range test.
Los valores se presentan como medias de muestras triplicadas (±DE) con base en el peso húmedo. Las medias que no comparten un superíndice o superíndices en común a, b, o c en una fila, o A, B, o C en una columna son significativamente diferentes en p ≤ 0.05, según la prueba de rango múltiple de Duncan.
in TPC, TFC, and antioxidant activity of yogurt during cold storage may be due to the hydrolysis of high-molecular-weight and complex compounds, which prevent oxidation (Trigueros et al., 2014). Interestingly, the TPC, TFC, and antioxidant activity of the DPSE-containing yogurt were always higher than those of the untreated ones throughout the entire storage period, signifying that DPSE addition in yogurt overcomes the negative impact of storage and counteracts free radicals, thereby extending the shelf life of yogurt. Lipid peroxidation in terms of thiobarbituric acid reactive substance (TBARS) values of yogurt was decreased following the addition of different DPSE quantities. These findings suggested that DPSE brings about more lipid oxidation stability to formulated yogurt, compared with nonformulated ones. As storage period progressed, there were significant increases in the TBARS values of all samples with 1% DPSE-formulated yogurt, having the lowest amount of TBARS at the end of the storage period (21 d). The TBARS values of formulated and non-formulated yogurt were increased during storage, pointing to the continuous formation of aldehydes in the products. The incorporation of DPSE to yogurt abolished lipid oxidation and may prolong the shelf life of the product during cold storage. It has been reported that the stability of flavored phytosterol-enriched drinking yogurts during storage may have been due to the effective control of lipid oxidation (Semeniuc et al., 2016). Table 4 shows the results of sensory profile of yogurt during cold storage. Incorporation of DPSE had no effect on the sensory attributes of yogurt, compared with the control samples. Jung et al. (2016) fortified yogurt with ginseng extract and observed a decrease in its bitter taste, whereas other attributes did not show any significant change. Shokery et al. (2017) fortified yogurt with green tea and Moringa leaf extracts, and concluded that the appearance and smell of Moringa yogurt had excellent marks equal to that of the control yogurts. Moreover, both fortified yogurts had good scores, but overall acceptability was affected by the brightness of the product, as control yogurts had the highest score than freshly treated yogurt or those that were consumed after storage. Throughout the storage period, the sensory attributes of DPSE-formulated yogurt exhibited better stability, compared with the control group. The better retention of sensory attributes in DPSE-containing yogurt may be attributed to the protective role of DPSE against deteriorating factors, as DPSE carries constituents exhibiting antioxidant properties. Similar observations on the protective effect of the natural antioxidant content of green tea and Moringa leaf extract (Shokery et al., 2017), and jabuticaba extract (Pereira et al., 2016) on the sensory attributes of yogurt have been recently reported. Our results revealed that adding DPSE to yogurt can result in sensory quality retention and shelf life extension without affecting its organoleptic properties.

Principle component analysis (PCA)
The principle component analysis was conducted to profoundly observe the interactive effects of date palm spikelet extracts of two cultivars and storage conditions on the quality attributes of set-type yogurt. HJ-biplot showed high contribution of the principle components (PC1, and PC2) on the total variation (85.24%) of the plotted components ( Figure 1). In the biplot positive, negative and no correlations were evident as acute, obtuse or straight, and right angles were respectively found between the vectors of the chemical components, antioxidant activity, and sensory attributes of yogurt samples (Mutwali et al., 2016). Positive correlations were found between ABTS, DPPH, TPC, TFC, ash, and protein indicating the contribution of these chemical components to the measured antioxidant activity of yogurt samples . In addition, positive correlations were observed between fat, total solids and sensory attributes of yogurt samples suggesting the interaction between them in set-type yogurts. Interaction of total solids with milk fat globules is known to enhance the sensory attributes mainly the texture of yogurt (Aziznia et al., 2008). Moreover, positive correlation was seen between TBARS and moisture content indicating that increasing moisture content of yogurt sample might reduce its oxidative stability during cold storage. Three hierarchical clusters showing the interrelationship among the quality attributes of set-type yogurts as affected by DPSE and storage duration. Both DPSE and storage duration influenced the chemical composition, antioxidant activity, and sensory attributes of yogurt with the influence of storage time was more noticeable than DSPE in the clusters. Plain yogurt at first and seventh day of storage and those fortified with 0.5% of DPSE (Rz and Kl) at first day of storage were characterized by greater sensory attributes compared to other samples. In addition, yogurt samples fortified with 1% DPSE (Rz and Kl) at the first day of storage showed higher total solids and fat content compared to other samples. Increasing the storage time to 14, and 21 days negatively affected the sensory and oxidative stability of plain yogurt samples due to increased moisture and TBARS values. However, it is interesting to note that increasing the storage period improved the protein, ash, TPC, TFC, and antioxidant activity of DPSE-fortified yogurts compared to controls indicating positive impacts of DPSE on the nutritional and health qualities, and oxidative stability of yogurt during cold storage. Similarly, O'Sullivan et al. (2016) stated that seaweed extracts improved the yogurt stability by reducing TBARS value fortified yogurt compared controls during cold storage. In addition, Mohamed  reported that fortification of yogurt with Argel leaf extract significantly improved the physicochemical quality, oxidative stability, and sensory attributes of fortified yogurt compared to control. In all samples, yogurts fortified with DPSE of Ruziz cultivar (Rz) outscore that fortified with Khalas (Kl) DPSE and controls in all quality attributes. Overall, it is clear that incorporation of DPSE improved the nutritional values, antioxidative properties, and storability of yogurt without major influence on the sensory attributes of the product.

Conclusions
The present study concludes that DPSE contains substantial quantities of total phenolic compounds and flavonoids, has good antioxidant activity, and provides natural and healthy preservatives to yogurt during cold storage (4°C ± 1°C). Incorporation of different levels of DPSE (0, 0.5, and 1%) in yogurt enhanced its physicochemical properties and oxidation stability, without adverse effects on its organoleptic. Therefore, the application of 0.5% DPSE of either Rz or Kl varieties as a natural and healthy preservative to prolong the shelf life of yogurt is consistent with consumers' requirement and in accordance with the trend toward food products that contain natural, safe, and healthy ingredients.