Optimization of Extraction Parameters and Characterization of Tunisian Date Extract: A Scientific Approach Toward Their Utilization

Abstract

The response surface methodology (RSM) was used in order to select the extraction conditions of extract from Kentichi date powder; a by-product of the date-processing process. Powder/solvent ratio, extraction temperature, and extraction time all had an impact on sugar yield, and these model factors have quadratic effects influencing sugar yield. Optimal extraction was obtained with 300 g/L powder/solvent ratio, 32.7 °C extraction temperature, and 2.1 h extraction time. Under these conditions, Kentichi date powder's (KDP) sugar yield was 77.1%, which was close to the predicted value of the model (80.50%). The results of Kentichi date powder extract (KDPE) showed that the total sugar content is 160.09 g/L. However, the protein content is 10.31 g/L with a majority of the essential amino acids (essentially glutamic acid (28.39 mg/L) and aspartic acid (9.65 mg/L)). The determination of antioxidant activity of KDPE showed a high activity (DPPH IC₅₀ = 4.8 mg/mL, ABTS IC₅₀ = 3 mg/mL, FRAP = 4.70 μmol AAE/mL and, TAA = 18.04 μmol Fe(II)/mL). The results show also that the freeze-drying technique has a lot of potential for producing powder from KDPE with many desirable properties. The findings indicate that KDPE with a high nutritional value could be used as a component for the formulation of functional foods.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12355-022-01223-2.

Introduction

Nowadays, the COVID-19 pandemic has put humanity in jeopardy all over the world. Therefore, people must strengthen their immune systems in order to combat the virus (Galanakis et al. 2020). A diet rich in fruits and vegetables can help to boost the immune system (Moreb et al. 2021). Many studies have shown that fruits and vegetables are rich in fiber and phytochemicals and can prevent or reduce the risk of diseases including cardiovascular disease, diabetes, obesity, certain types of cancer, inflammation, stroke, and septic shock (Schauder et al. 2020).

Date palm (Phoenix dactylifera L.) belongs to the Arecaceae family (Angiosperms, monocots) which comprises 200 genera and more than 2 500 species (Al-Alawi et al. 2017). According to the literature, low-quality dates are used to feed animals and are often processed into date paste and date syrup, which are both widely used in the food industry by-products (Najjar et al. 2020). These by-products have health and nutritional benefits, including an immunostimulating effect on the reticuloendothelial system and strong antioxidant activity (Zerizer et al. 2014).

Several researchers showed that dates are rich in phenolic antioxidants, but their value varies depending on the variety of dates, agronomic, and environmental conditions (Alam et al. 2021). Furthermore, dates contain nutrients such as protein (essential amino acids), fiber, fat, minerals, and vitamins (Ibrahim et al. 2020). In addition, the most essential components of date fruits are carbohydrates including soluble sugars (glucose, fructose, and sucrose) and dietary fiber (cellulose, hemicelluloses, pectin, and fructans) (Kamal-Eldin et al. 2020). The date is an important nutrient and energy source due to its high carbohydrate content (Siddiqi et al. 2020). Thus, fresh dates contain about 157 cal per 100 g, whereas dry dates contain more than 300 cal per 100 g (Aljaloud et al. 2020).

Dates have the potential to contain a variety of bioactive phytochemicals, and dates have simple phenolic acids (gallic acid, vanillic acid, syringic acid), carotenoids (lutein, β-carotene), flavonoids and their derivatives (catechin, epicatechin, quercetin, apigenin), phytosterols (cholesterol, campesterol, -sitosterol), phenylpropanoids (caffeic acid, 5-O-caffeoylshikimic acid, ferulic acid) and anthocyanins (Zihad et al. 2021).

Due to these antioxidant components, dietary recommendations recommend increasing the consumption of antioxidant-rich foods. Date sugar can be one of those foods with extremely high antioxidant levels (Phillips et al. 2009). However, it became important to extract sugar from dates. Different unconventional extraction sugar procedures have been utilized in order to maximize the extraction yield of bioactive macromolecules from various plant sections (Nadar et al. 2018). Thus, water extraction usually requires a combination with other innovative techniques, such as ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), pressurized water extraction (PWE), and enzyme-assisted extraction (EAE), which facilitate polysaccharide dissolution via biodegradation or mechanical disintegration of plant cell walls (Chen et al. 2019).

Nevertheless, the extraction, isolation, and fractionation techniques change depending on the eventual purpose. For food application, aqueous extraction provides numerous advantages because water is not only economical and environmentally friendly, but it is also non-flammable and nontoxic, allowing for clean processing and pollution prevention (Filly et al. 2016). Therefore, the aqueous treatment approach for extracting sugar from Kentichi date powder appears to be a productive and promising method for the increased recovery of fruit sugars at relatively low temperatures and without the utilization of any toxic solvents.

Response Surface Methodology (RSM) is a method for developing and optimizing experimental data that are based on statistical and mathematical concepts. This modeling approach is beneficial when there are numerous input parameters that interact with one another and the interaction influences the system's output known as "response" (Peng et al. 2020). RSM utilizes an experimental design such as the central composite design (CCD) to fit a model using the least squares method, and the diagnostic testing tests offered by analysis of variance (ANOVA) are then used to show whether the suggested model is adequate. The response surface plots could be used to evaluate the surfaces and find the optimal values (Leili et al. 2020).

The RSM technique has been utilized in numerous areas and has demonstrated its efficacy as a numerical method by comparing it to experimental studies and other numerical research (Farouk et al. 2022). However, no scientific research is published to study the impacts of extraction time and temperature on sugar extract from date powder using simple technology, as well as its functional characteristics and antioxidant capacity.

The aims of this study were to maximize sugar yield of Tunisian date powder, employing RSM (a central composite rotary design), to characterize the optimized extract from Tunisian date powder and to assess its antioxidant capacity. An essay on preserving the extract through a freeze-drying method is also evaluated in order to increase its shelf life while keeping its nutritive value.

Material and Methods

Plant Material

The dates used in this work are Tunisian dates of "Kentichi" variety purchased at the central market (Tunis) in the Tamar stage (fully mature). The dates were cleaned, dried at 50 °C for 15 min, and ground to a powder with a mean particle size of 500 µm. The powder obtained was stored in airtight bottles in the refrigerator at 4 °C until later use (Yefsah-Idres et al. 2019).

Kentichi Date Powder Extract

The Kentichi date powder (KDP) was extracted with selected combinations of independent factors such as powder/solvent ratio, extraction time, and extraction temperature. To separate the extract from the insoluble residue, centrifugation at 6000 rpm for 10 min was performed to remove particles. The sugar yield was determined by the method of Dubois et al. (1956). Sugar ratio of the Kentichi date powder (KDPE) was expressed as the ratio of the extracted sugar in the total sugar content of the KDPE (Arrutia et al. 2020). The ratio of sugar extraction yield (%) was calculated as following Eq. 1:

$$\text{Sugar}\text{extraction}\text{yield}\left(\%\right)=\frac{M_{\text{se}}}{M_{\text{sp}}}\times 100$$ 1

where M_se is the concentration of sugar in the extract (g/100 g); M_sp is the concentration of sugar in the pulp (g/100 g). Optimized extract from Kentichi date powder (KDPE) was then employed for composition and antioxidant analyses.

Experimental Design

A centered and rotating compound plan (CCRD) with three variables and five levels was created. To obtain a second-order polynomial model that describes the sugar yield (Kentichi date powder extract (KDPE)) Y (dependent variable) as a function with three independent variables, the ratio between solvent and date powder (X₁) from 100 to 500 g/L, extraction temperature (X₂) from 20 to 60 °C, and extraction time (X₃) from 0 to 8 h (Table 1). Table S1 shows the coded and uncoded variables used in the design of surface response methodology.

Table 1.

Independent variables and their levels for optimizing the extraction of sugar from Kentichi date powder

Independent variables	Symbols	Coded factor levels
	Coded	Uncoded	− 1.68	− 1	0	1	1.68
Powder/solvent ratio (g/L)	X1	x1	100	200	300	400	500
Extraction temperature (°C)	X2	x2	20	30	40	50	60
Extraction time (h)	X3	x3	0	2	4	6	8

Second-order polynomial model, employed to describe the dependent variables Y as a function of the independent variables X_i, is presented in following Eq. 2:

$$\mathrm{Y}={\mathrm{\beta }}_0+\sum _{\mathrm{i}=1}^3{\mathrm{\beta }}_{\mathrm{i}}{\mathrm{X}}_{\mathrm{i}}+\sum _{\mathrm{i}=1}^3{\mathrm{\beta }}_{\mathrm{ii}}{\mathrm{X}}_{\mathrm{i}}^2+\sum \sum _{i<j=1}^3{\mathrm{\beta }}_{\mathrm{ij}}{\mathrm{X}}_{\mathrm{j}}+\mathrm{\epsilon }$$ 2

where X_i and X_j are the input factors that influence the response Y, while ${\mathrm{\beta }}_0,{\mathrm{\beta }}_{\mathrm{i}},{\mathrm{\beta }}_{\mathrm{ii}},$ and ${\mathrm{\beta }}_{\mathrm{ij}}$ (i ≠ j) were constant coefficients regression of the model, the coefficients β, which must be determined in the second-order model, are obtained by the least-squares method, and ε is the error. Data analyses were performed using Expert Design Software, version 7. The ANOVA procedure was used to perform variance analysis. The mean values were regarded to be significantly different at p ≤ 0.05.

Freeze-Drying

KDPE was placed on the stainless steel trays of the freeze-dryer and frozen at − 20 °C for 24 h before freeze-drying. The trays were then transmitted to the freeze-dryer, and the vacuum turned on, allowing some of the pulp's free water to be sublimed off. This procedure was carried out for 72 h at 0.0041 mbar chamber pressure. The temperature of the condenser was − 65 ± 1 °C. The freeze-dried powder (FDP) was stored in an airtight dark glass container until the analysis stage.

Analytical Methods

The pH and titratable acidity were measured for KDPE according to the standard analytical method (AOAC 1990). The total sugars content was determined for KDPE and FDP using the sulfuric acid and phenol method (Dubois et al. 1956). The DNS method was employed for the determination of reducing sugars content of KDPE (Miller 1959), with glucose as a standard for total and reducing sugars. The sucrose content of KDPE was determined with the formula established by Chafi et al. (2015) using following Eq. 3:

$$\text{Sucrose}\left(\%\right)=\left(\text{total sugars}\left(\%\right)-\text{reducing sugars}\left(\%\right)\right)$$ 3

The protein assay of KDPE and FDP is carried out by the method of Lowry (1951), using bovine serum albumin as a standard. All experiments were performed in triplicate.

Free Sugars Analysis

Fructose, glucose, sucrose, maltose, and lactose contents were analyzed employing HPLC technique. KDPE was filtered using a 0.45 µm filter. In equal volumes, the analytical standards and sample were injected separately (5 µL). HPLC analysis was carried out employing liquid chromatography in conjunction with a diode-array detector (DAD). Separation was performed with Shimadzu SHIMPACK VP-ODS 4.6 × 150 mm, 5 µm RP column. As the mobile phase for the chromatographic analysis, a mixture of methanol and distilled water (10:90 v/v) was used. A 5 µL volume was injected at a rate of 1.2 mL/min with a run time was 20 min. The temperature of the column was kept at 30 °C throughout the experiment (Alghamdi et al. 2020).

Amino Acid Analysis

High-performance liquid chromatography with post-column fluorescence derivatization (HPLC-FLD) was used to determine the concentration of free amino acids in KDPE, employing a C18 column with a particle dimension of 5 μm (250 × 4.6 mm) (Agilent 1200 series) and a flow rate of 1 mL/min. The excitation wavelength (λ_Ex) of 340 nm and the emission wavelength (λ_Em) of 440 nm were used in the fluorescence detection setup. The mobile phase of a binary gradient included solvent A: ACN, methanol, water (45:45:10), and solvent B: Na₂HPO₄ 2.75 g/L (pH 6.5), which were created according to Mechmeche et al. (2016). The amino acids were identified and quantified using a standard amino acid mixture (Sigma Chemical). The amino acid content was expressed as milligrams of amino acids per liter of KDPE.

Phytochemical Composition

Extract

The contents of active molecules were determined according to the protocol of Bigrali et al. (2008) with some modifications by stirring 100 mL of (KDPE and FDP) with 300 mL of methanol using a magnetic stirrer, for 24 h. The mixture was filtered using filter paper. The filtrate was centrifuged at 4000 rpm for 10 min. The supernatant was evaporated by rotary vacuum evaporator at 40 °C. The concentrated extracts are stored at 4 °C in dark glass bottles until use.

Total Phenolic Content

Total phenolic content (TP) was estimated by the colorimetric method using the Folin–Ciocalteu reagent (Waterhouse 2002). 100 µL of each extract is added to 500 µL of Folin reagent (diluted ten times with distilled water) and 1 mL of distilled water. After incubation for one minute, 1.5 mL of Na₂ CO₃ (20%) is added and mixed. Absorbance was measured after 2 h of incubation in the dark at 760 nm. The results are expressed in milligram gallic acid equivalents (GAE) per milliliter.

Total Flavonoid Content

Total flavonoid content (TF) was determined by the colorimetric method using the reagent of aluminum chloride and quercetin as standard according to the protocol described by Biglari et al. (2008). 1.5 mL of each extract was mixed with an equal volume of a 2% (AlCl₃, 6H₂O) solution. After incubation for 10 min at room temperature, the absorbance was measured at 367 nm. The results are expressed as milligram quercetin equivalents (QE) per milliliter.

Condensed Tannins Content

The method of Laouini et al. (2018) is employed to determine the condensed tannins content (CT), using catechin as a standard. A volume of extract 0.5 mL was mixed with 3 mL of the mixture of vanillin and methanol (4%), and 1.5 mL of hydrochloric acid is added and mixed thoroughly. The resulting mixture was allowed to stand for 15 min at 20 °C. The absorbance of each was measured at 500 nm. The results are expressed in milligram catechin equivalents (CE) per milliliter.

Antioxidant Activity

Antioxidant activity was determined by measuring the capability to scavenge different radicals by DPPH and ABTS method for KDPE and FDP, reducing power (FRAP), and phosphomolybdate method (TAA) for KDPE.

DPPH Radical Scavenging Activity

The potential to scavenge 2,2-diphenyl-2-picrylhydrazyl free radicals (DPPH) was determined by the Jan et al. (2013) method. 0.5 mL of each extract at different concentrations is added to 0.5 mL of the freshly prepared DPPH· solution (2 mg/50 mL of methanol). The mixture is stored in the dark for 15 min. The absorbance was measured at 517 nm against a blank which is methanol. The results are expressed as a percentage of inhibition; radical scavenging activity (%) was determined using following Eq. 4:

$$\text{DPPH scavenging activity}\left(\%\right)\left(\frac{{\text{A}}_{\text{control}}-{\text{A}}_{\text{sample}}}{{\text{A}}_{\text{control}}}\right)\times 100$$ 4

where A_control is the absorbance of DPPH· in methanol instead of samples. The antioxidant capacity of the various extracts was calculated graphically from the percentage of inhibition relative to the concentration of the extracts IC₅₀, which corresponds to the concentration required to reduce 50% of the DPPH· radical.

ABTS Radical Scavenging Activity

The capacity to scavenge free radicals (2,2'-Zinobis- (3 ethylbenzothiazoline)-6-sulfonic acid ammonium salt) radical ABTS was determined by the method of Lien et al. (1999). The preparation of ABTS·⁺ radical is carried out according to the method described by Jan et al. (2013). An aqueous solution of ABTS at a concentration of 7 mM prepared in ultrapure water is mixed with a solution of potassium persulfate at a concentration of 2.45 mM. The mixture is stored in the dark and at room temperature for 12 h to 16 h. ABTS·⁺ solution was diluted to have an absorbance of 0.750 ± 0.025 at 734 nm in ethanol. Once the concentrations were diluted, 1 mL aliquots of the extracts were mixed with 1 mL of the ABTS·⁺ solution after incubation for 6 min, the absorbance was measured at 734 nm against a blank which is ethanol. The results are expressed as a percentage of inhibition; radical scavenging activity (%) was calculated using following Eq. 5:

$$\mathrm{ABTS};\mathrm{scavenging};\mathrm{activity}\left(\%\right)=\left(\frac{{\mathrm{A}}_{\mathrm{control}}-{\mathrm{A}}_{\mathrm{sample}}}{{\mathrm{A}}_{\mathrm{control}}}\right)\times 100$$ 5

where A_control is the absorbance of ABTS·⁺ in ethanol instead of samples. The IC₅₀, which corresponds to the concentration required to eliminate 50% of the ABTS·⁺ radical, was determined graphically from the percentage of inhibition relative to the concentration of the extracts.

Antioxidant Activity by the FRAP Method (Ferric Reducing Antioxidant Power)

The reducing power was performed by the FRAP method described by Oyaizu (1986). The objective of this method is to measure the potential to reduce the ferric iron (Fe³⁺) present in the ferrous iron complex (Fe²⁺). Using iron sulfate (FeSO₄) as a standard, 1 mL of the extract was mixed with 2.5 mL of a 1% potassium ferrocyanide [(K₃Fe (CN₆)] solution and 2.5 mL of phosphate buffer (0.2 M; pH 6.6). Then, the mixture is incubated at 50 °C for 20 min. To stop the reaction, 2.5 mL of 10% trichloroacetic acid is added. After centrifugation at 6504 rpm/10 min, 2.5 mL of supernatant is added to 2.5 mL of distilled water and 0.5 mL of 0.1% iron chloride (FeCl₃). The absorbance of the reaction medium is measured at 700 nm. The results are expressed as micromoles of ferrous iron (Fe II) equivalents per milliliter.

Antioxidant Activity by the Phosphomolybdate Method

Total antioxidant activity (TAA) of sample in the phosphomolybdenum method was based on the reduction in molybdenum Mo (VI) to molybdenum Mo (V) in the presence of antioxidant compounds and therefore the formation of a green phosphate/complex Mo (V) at acidic pH (Laloo and Sahu 2011), using ascorbic acid as a standard. 0.3 mL of extract was mixed with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). This mixture is incubated in a water bath at 95 °C for 90 min then cooled at room temperature for 6 min. Absorbance was measured at 695 nm. Results are expressed as micromole of ascorbic acid equivalents per milliliter.

Results and Discussion

Kentichi Date Powder Extract

Response surface methodology (RSM) is a combination of mathematical and statistical methods for modeling and evaluating problems. The goal of this technique is to optimize the response surface, which is affected by various process parameters (Aslan and Cebeci 2007).

Predicted Model and Statistical Analysis

Extraction optimization for KDP extract is based on various combinations of variables, as described in the experimental design. The effect of three independent variables: powder/solvent ratio (X₁), extraction temperature (X₂), and extraction time (X₃), as well as their interactions, on yield sugar extraction in KDP (Y), was studied. Analysis of variance (ANOVA) was performed to study the relevance of the proposed models and to identify the most important factors. The p value and F test are used to confirm the significance of each coefficient and to determine the interaction of each parameter for the sample.

In the present study, the p value of the model is 0.0083 for sugars extraction from KDP; however, the coefficient of determination and the Adjusted coefficient of determination are (R² = 0.824) and (R²-Adj = 0.665) for KDP, respectively. They approved that the model was statistically significant (p < 0.05). The result implied that it was suitable for this experiment (Table 2). Another researcher has reported an R² coefficient of determination ranging from 94.26 to 98.66% (Appiah-Nkansah et al. 2016) for dried bagasse. The F values of the model indicate that they are not significant concerning the pure error as shown in Table 2. These results identify the accuracy and general availability of the polynomial model. The quadratic polynomial regression model proposed for sugars extraction was calculated using following Eq. 6:

$$\mathrm{Y}=80.45-3.93{\mathrm{X}}_1-0.78{\mathrm{X}}_2-0.66{\mathrm{X}}_3+3.42{\mathrm{X}}_{12}-0.69{\mathrm{X}}_{13}+0.32{\mathrm{X}}_{23}-6.11{\mathrm{X}}_1^2-14.40{\mathrm{X}}_2^2-11.44{\mathrm{X}}_3^2$$ 6

Table 2.

Analysis of variance (ANOVA) for the quadratic polynomial model of extraction of sugar from Kentichi date powder

Source	Degree of freedom	Sum of square	Mean square	F value	p value Prob > F
Model	9	4985.92	553.99	5.21	0.0083
A-(Powder/solvent) Ratio	1	211.09	211.09	1.98	0.1892
B-Extraction temperature	1	8.50	8.51	0.08	0.7831
C-Extraction time	1	6.05	6.05	0.06	0.8163
AB	1	93.71	93.71	0.88	0.3700
AC	1	3.84	3.84	0.04	0.8532
BC	1	0.82	0.82	0.01	0.9318
A2	1	538.62	538.62	5.06	0.0481
B2	1	2992.31	2992.31	28.14	0.0003
C2	1	1888.31	1888.31	17.76	0.0018
Residual	10	1063.41	106.34
Lack of Fit	5	1063.41	212.68
Pure error	5	0	0
Cor total	19	6049.33
Standard deviation	10.31	R2	0.82
Mean	58.62	R2-adj	0.66
Coefficient of variation	17.58	Predicted R2	− 0.35
Press	8205.00	Adequate precision	5.77

Indeed, the quadratic factors presented in the model of sugars extraction from Kentichi date powder (powder/solvent ratio, extraction temperature, and extraction time) (Table 2) are significantly different (p ≤ 0.05).

Study of Single-Factor Experimental Analysis

The effects of powder/solvent ratio on sugar yield of KDP are represented in Fig. 1a. The other extraction variables were set as follows: extraction time of 2 h, extraction temperature at 30 °C. According to the findings, sugar yield increased from 100 g/L to 300 g/L, where it maximized at 68.88% and then, decreased from 300 to 500 g/L. The diffusion of the solvent into cells was frequently enhanced by the liquid/material ratio, which also facilitated polysaccharide desorption from the cells (Chen et al. 2017).

Fig. 1.

Effects of different extraction parameters on sugar yield (a: extraction powder/solvent; b: extraction time; c: extraction temperature)

The effects of extraction time on the extraction yield of sugar are illustrated in Fig. 1b. Extraction time was from 0 to 8 h, while other extraction variables were set as follows: extraction temperature at 30 °C, and powder/solvent ratio of 300 g/L. Results demonstrated that maximum sugar yield (71.1%) was reached during a 2 h extraction interval, beyond 2 h, the sugar yield decreased, suggesting that the prolonged extraction process caused polysaccharide degradation (Prakash Maran et al. 2013). The use of aqueous extraction with the RSM method to extract sugar from date palm by-products has not been used in any other research. Iwassa et al. (2019) showed that the yields of soluble sugars increased gradually in the subcritical water extraction of asparagus by-products from 10 to 90 min and that these yields were stable for 120 min.

In Fig. 1c, we observed that the sugar production varied significantly from 20 °C to 30 °C. An increase in temperature favored the extraction of sugar. At this point, the following conditions were set: powder/solvent ratio, 300 g/L; and extraction time, 2 h. The extreme increase in temperature led to a lower extraction recovery (Setyaningsih et al. 2022).

Study of Independent Variables Interactions

The three-dimensional (3D) response surface and the two-dimensional (2D) contour projection of the regression equation are graphical representations that aid in the determination of the relationship between two variables and the identification of optimal experimental conditions when the third variable is set to zero levels (Li et al. 2020). Elliptical contour plots show a significant interaction between variables, whereas circular contour plots indicate non-significant interactions between variables (Sahu et al. 2020).

The effect of the powder/solvent ratio and extraction temperature on the sugar yield of KDP extract was determined, setting the extraction time to zero (Fig. 2). The sugar yield increased with increasing powder/solvent ratio, up to 300 g/L, and increasing extraction temperature, up to 30 °C. Beyond a powder/solvent ratio of 300 g/L or extraction temperature of 30 °C, there was a decrease in the sugar yield (Fig. 2a). In general, the increase in powder/solvent ratio facilitated sugar desorption from the cells. However, the reduction in sugar yield was caused by partial decomposition of sugar at high temperatures. The contour plots also had a circular shape, indicating that the interaction between powder/solvent ratio and extraction temperature is not significant (Fig. 2b). Additionally, setting the extraction temperature to zero, the effect of the powder/solvent ratio and extraction time on the sugar yield of KDP extract was studied (Fig. 2c). With the extraction time increased from 0 to 2 h or the powder/solvent ratio increased from 100 to 300 g/L, the yield increased at first and then, decreased when these two variables kept increasing thereafter. The contour plots showed circular shapes which justify that the effect of interaction between powder/solvent ratio and extraction time on sugar yield is not significant (Fig. 2d). In addition, Fig. 2e also shows the effect of temperature and extraction time on sugar yield when the powder/solvent ratio is set to zero. From these observations, we discovered that sugar output increased with temperature and time. Beyond an extraction time of 2 h or extraction temperature of 30 °C, there was no further increase in the sugar yield. The contour plots revealed a circular outline, indicating that there is no interaction between these two parameters and sugar yield (Fig. 2f).

Fig. 2.

Response surface and contour plots for sugar yield from Kentichi date powder showing the effects of powder/solvent ratio and extraction temperature (a, b), the effects of powder/solvent ratio, and extraction time (c, d), and the effects of extraction temperature and extraction time (e, f)

Moreover, the negative values for the quadratic term of powder/solvent ratio, temperature, and time (Eq. 6) indicate that the extraction conduction at high levels of this variable, i.e., to values over 300 g/L, 30 °C, and 2 h, respectively, can decrease the sugar yield.

Model Validity

To validate the established model, the optimized conditions were tested experimentally under the following conditions: powder/solvent ratio is 300 g/L, extraction temperature is 32.7 °C, and extraction time is 2.1 h. The fitted equation predicted a yield value of 80.50%. To confirm the model prediction, optimal extraction conditions were applied. An average value of about 77.1% of sugar content was obtained by three independent real experiments. There is not a significant difference (p > 0.05) between the experimental and theoretical values obtained from the model, which confirmed that the response model was adequate for optimization.

Extract Composition

KDP presents a concentration of 67.91 g/100 g total sugar, with a 77.1% extraction yield. As a result, KDPE is a cost-effective way to produce sugar. Furthermore, dry dates are one of the most abundant and inexpensive international fruits (Ghnimi et al. 2017).

The pH and titratable acidity values are 5.97–2.22% in KDPE, respectively (Table 3). The pH result is similar to this El-Nagga and Abd El-Tawab (2012) studied date syrup extraction by different methods. However, the acidity result is higher than our results. Likewise, the level of total sugars in KDPE is 160.09 g/L, of which 3.04 g/L are reducing sugars, and 157.05 g/L are sucrose (Table 3). The presence of reducing sugar in the waste explained the low content. These results are very far from those reported by El-Nagga and Abd El-Tawab (2012) studied a different variety of dates.

Table 3.

Physicochemical properties of Kentichi date powder extract

Parameters	KDPE
pH	5.97 ± 0.03
Titratable acidity (%)	2.22 ± 0.01
Total sugars (g/L)	160.09 ± 0.29
Reducing sugars (g/L)	3.04 ± 0.03
Sucrose (g/L)	157.05 ± 0.32
Free sugars1 (%)
Fructose	13.74
Glucose	14.26
Sucrose	72
Maltose	–
Lactose	–
Protein (g/L)	10.31 ± 0.07
Amino acids1 (mg/L KDPE)
Aspartic acid	9.65
Glutamic acid	28.59
Serine + Histidine + Glutamine	4.68
Glycine + Threonine + Arginine	6.27
Alanine	6.29
Tyrosine	5.03
Phenylalanine	1.81
Isoleucine	2.83
Leucine	0.96
Valine + Methionine	4.24
Total	70.35
TP (mg GAE2/mL)	3.13 ± 3.47
TF (mg QE2/mL)	0.25 ± 1.10
CT (mg CE2/mL)	0.16 ± 0.40

¹Contents of free sugars and amino acids were calculated by HPLC analyses

²Reference compound: GAE gallic acid equivalents, QE quercetin equivalents, CE catechin equivalents

Free sugars composition was evaluated by HPLC. One of the important components of KDPE is that sucrose is present in higher quantities than glucose and fructose. Approximately, about 72% is sucrose, 14.26% glucose, and 13.74% fructose; on the other hand, maltose and lactose are no longer present in KDPE (Table 3). These results are comparable to those obtained by Djaoud et al. (2020) analyzed the free sugars in syrup from a secondary date variety. Generally, soft date cultivars' fruits are dominated by inverted sugars (glucose and fructose) and constitute little or no sucrose, whereas dry date cultivars' fruits may contain a high proportion of sucrose (Ghnimi et al. 2017). Sugars, in addition to their primary role of sweetness, also play other functions in the food industry, such as preservation, fermentation, color, flavor, texture, solubility, hygroscopicity, crystallinity, and viscosity (Zaitoun et al. 2018).

In addition, the protein content is 10.31 g/L in KDPE. This result is much lower than a date fiber isolate given by Ben Yahmed et al. (2020). The amino acid contents were determined using HPLC and were also employed in the current study to evaluate the nutritional quality of protein in KDPE. Furthermore, the free amino acid content attends 70.35 mg/L essentially glutamic acid (28.39 mg/L) and aspartic acid (9.65 mg/L), but leucine (0.95 mg/L) has a lower content (Table 3). As a result, Kumar et al. (2021) reported that the optimal assimilation of proteins from various plant origins can provide enough necessary amino acids to meet human health requirements. The KDPE has low free amino acids content when compared to other protein isolates such as Moringa oleifera seed (Aderinola et al. 2018). Proteins seem to be well for their role in the physical structure of processed foods, aiding in the formation of a different number of gels, emulsions, and foams (Allen Foegeding 2015). The biochemical components of dates are affected by culture conditions including growth zone and produce period (fully mature stage), and it differs considerably between cultivars (Ben Yahmed et al. 2020), as well as different extraction methods (El-Nagga and Abd El–Tawab 2012).

KDPE contained 3.13 mg GAE/mL of total phenolic content (Table 3), indicating that it was a valuable source of phenolic antioxidants. These results are higher than those given by El-Nagga and Abd El-Tawab (2012). Then, the results showed that KDPE has total flavonoid contents that are 0.25 mg QE/mL. However, the condensed tannins content obtained is 0.16 mg CE/mL in KPDE (Table 3). KDPE contained a wide range of bioactive components capable of inhibiting the effect of reactive oxygen species implicated in human diseases such as cardiovascular disease and cancer. As a result, essential biological macromolecules may be protected from oxidation (Benmeddour et al. 2012).

Date varieties, geographic origin, fruit storage time, extraction conditions such as solvent used, plant material/solvent ratio, and extraction time are all factors that influence phenolic compounds (Masmoudi-Allouche et al. 2016).

Antioxidant Activity

The antioxidant activity of KDPE was determined using (DPPH, ABTS, FRAP, and TAA methods) with two aqueous and methanolic extracts, as shown in Fig. 3. The results indicate a significant difference between the two aqueous and methanolic extracts (p ≤ 0.05).

Fig. 3.

Antioxidant activity by DPPH, ABTS (a, b), and TAA, and FRAP (c, d) methods with two aqueous () and methanolic () extracts for Kentichi date powder extract (KDPE)

The KDPE aqueous extract was endowed with anti-free radical activity, an IC₅₀ concentration (DPPH) by about 4.8 mg/mL (Fig. 3a). On the other hand, the IC₅₀ value for the KDPE methanolic extract was found to be 14 mg/mL (Fig. 3b). Then, the ABTS test of KDPE aqueous extracts gives results whose IC₅₀ concentration is 3 mg/mL (Fig. 3a). However, the IC₅₀ value of methanolic extracts is about 7 mg/mL (Fig. 3b). Antioxidant activity assayed by FRAP showed that the KDPE aqueous extract is 4.7 µmol Fe(II)E/mL (Fig. 3c). On the other hand, the methanolic extract gives a low level of antioxidant activity (3.37 µmol Fe(II)E/mL) (Fig. 3d). However, the TAA of KDPE is 18.04 and 15.86 µmol AAE/mL for the aqueous and methanolic extracts, respectively. It should be noted that aqueous extracts have a higher antioxidant capacity than methanolic extracts (Fig. 3), showing that antioxidants in the KDPE are mostly polar.

As a result, extract can be thought a low-cost source of active molecules with natural antioxidants, implying that the extract's improved antioxidant activity was mostly attributable to the active components of KDP that are readily soluble in water. Additionally, the migration of the active component to the food simulant is affected by the polarity of the migrant and simulating media. Also, the sum of glucose, fructose, sucrose, and fructans is termed “water soluble carbohydrate” (Al-Sheikh Ahmed et al. 2020). Phillips et al. (2009) confirmed this result by comparing the antioxidant activity content among natural sweeteners (fruit sugars, e.g., date sugar) versus refined sugar and found that date sugar has the highest antioxidant activity of the natural sweeteners studied. Faraji and Lindsay (2004) demonstrated that antioxidant activity for fructose, sucrose, raffinose, sorbitol, or mannitol was confirmed when integrated at 16% of the aqueous phase in model emulsions of fish oil in water.

The date-fruit syrup waste extract (DSWE) contained larger hydrophilic phenolic molecules that could rapidly migrate to the aqueous phase, resulting in a high TPC release profile in the water medium (Saleh 2011). As a result, Rangaraj et al. (2021) showed that the antioxidant activity of films containing DSWE was higher in the water medium than in the 95% ethanol medium. On the other hand, Hu et al. (2016) proved that the antioxidant activities observed for complex carbohydrates correlate with the presence of phenolic and/or protein components. KDPE contains a concentration of aspartic acid and glutamic acid, both of which have high antioxidant properties due to the presence of additional electrons that can be produced when free radicals interact with them (He et al. 2013).

Effect of Freeze-Drying Process on Physicochemical Properties of Kentichi Date Powder Extract

Fruits and their extracts have a limited shelf life because of their high water content (Silva-Espinoza et al. 2020). The pulp drying for the production of fruit powders allows can be preserved for a long time, enabling it to be employed in the production of instant beverages and other industrial applications (Cordeiro 2020). Freeze-drying is the process of producing high-quality products by sublimating a frozen sample at reduced pressure while avoiding high temperatures. The preservation of taste, flavor, and thermo-sensitive compounds with biological activity are among the advantages of freeze-drying (Uscanga et al. 2021). Table 4 shows the total sugar content, protein content, and antioxidant activity of the lyophilized powder of KDPE.

Table 4.

Physicochemical properties of freeze-dried powder of Kentichi date powder extract

Parameters	FDP
Total sugars (mg/g DM1)	742.8 ± 0.31
Protein (mg/g DM1)	88.8 ± 0.08
DPPH IC50 (mg/g)	3.2 ± 0.13
ABTS IC50 (mg/g)	2.8 ± 0.18

¹Dry matter

The FDP results show that the total sugars and protein content are 742.8–88.8 mg/g dry weight, respectively; in other words, the increase after drying is 41.84 and 158.6%, respectively. ABTS and DPPH tests showed that FDP has an increase of 6.67–33.34%, with IC₅₀ values for ABTS and DPPH of 2.8–3.2 mg/g, respectively. A similar drying trend was also shown by Assefa and Keum (2016), and Shonte et al. (2020), who found an increase in antioxidant activity and protein content of freeze-dried yuzu and stinging nettle powders. Due to water removal, the relative increases in mineral content, total acidity, carbohydrates, and total sugars in the dried fruits were significantly higher than in the fresh samples (Radojcin et al. 2021). On the other hand, proteins in most foods retain their nutritional value and digestibility when dried (Guiné 2018).

Numerous researchers have indicated the benefits of freeze-drying over other drying methods in terms of chemical and nutritional properties (Radojcin et al. 2021).

Conclusion

In this study, aqueous extraction was used to extract sugar from Kentichi date powder. In order to determine the optimal treatment conditions, the Box–Behnken (BBK) design for response surface methodology (RSM) was utilized. It was demonstrated that optimal values of powder/solvent ratio, extraction temperature, and extraction time were 300 g/L, 32.7 °C, and 2.1 h, respectively. The sugar yield reached 77.1% under the above-optimized conditions. The Kentichi date powder extract (KDPE) findings revealed a total sugar content of 160.09 g/L. Aspartic acid (9.65 mg/L) and glutamic acid (28.39 mg/L) make up the majority of the 10.31 g/L of protein. KDPE showed exhibited excellent antioxidant activity in a dose-dependent manner in various in vitro models such as DPPH radical scavenging activity, ABTS + radical scavenging activity, FRAP method, and Total antioxidant activity TAA. The freeze-drying of extract appears to be a viable option for preserving nutritional quality for a long time. Overall, in terms of extraction from fruit, aqueous extraction had the benefits of efficiency, low cost, and being environmentally friendly. However, the product of KDPE is a matrix that could be used as a basic ingredient in the formulation of functional foods. Therefore, this research makes an important addition to the literature, considering the lack of reports on the extraction of the date palm by-product using a simple technique.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors Contribution

NM wrote the main manuscript text of the thesis; MM contributed to statistic analysis; KS contributed to protein analysis; ZT contributed to antioxidant activity analysis; MH reviewed the manuscript; FK supervised the work. All authors reviewed the manuscript.

Funding

The authors have no relevant financial or non-financial interests to disclose.

Declarations

Conflict of interest

The authors have no conflict of interest.

Ethical Approval

This article does not contain any studies with human Participants or animals performed by any of the authors.