Elaboration of an organic beverage based on grape juice with positive nutritional properties

Abstract The present study aimed to develop a natural beverage with interesting phytochemical composition and biological activity based on grape juice without added sugars or artificial additives. Two groups of blends were made by diluting concentrate grape juice with a sugar content of 65 °Brix with two different mineral waters (BA: Bezoya with low mineralization; BB: Solan de Cabras with high mineralization). Lemon juice was used for pH correction, and mixtures of extractions of hop with tea and hop with mint were used to increase aroma. Samples were stored under refrigeration (4°C) then subjected to physicochemical and sensory analysis. The results demonstrated that malvidin‐3‐O‐glucoside pigment was the predominant pigment with a concentration ranging from 75.71 ± 12.49 to 84.87 ± 1.70 mg/L. The levels of sugars ranged from 79.90 ± 1.37 to 82.37 ± 0.55 g/L and total soluble solids were between 5.47 ± 0.12 and 5.77 ± 0.06 °Brix. Total acids presented a significant difference, ranging from 1.40 ± 0.00 to 1.43 ± 0.06 g/L in BA samples and from 1.10 ± 0.10 to 1.20 ± 0.00 g/L in BB samples. For 20 days, the color increased in all beverages. However, BA drinks presented higher acidity and higher red color intensity than BB drinks, so the type of water and pH influenced the color of beverages. The sensory evaluation showed that the beverage made with low mineral water and flavored with a mixture of hop with tea was preferred.

and antiviral effects (Butu & Rodino, 2019). Nowadays, researchers are interested in the production of foods with lower sugar or alternative sources of sweeteners (Moldovan & David, 2020). Fruit juices could replace sugar sweeteners and contribute to preventing pathologies associated with their consumption (Agulló et al., 2021), such as obesity, diabetes, and cardiovascular disease (Styburski et al., 2020).
In addition, among challenges facing the food industry is the expansion in the creation of new products and persuasive consumers to buy them. In this case, it is essential to recognize the needs and the consumer's demands while developing new and innovative products (Świtalski & Rybowska, 2021). To respond to consumer demand related to the consumption of natural and healthy products, several studies focused on the development of new and healthy beverages based on fruit juice (Bhalerao et al., 2020;, 2013, 2016bGonzález-Molina et al., 2012;Shams Najafabadi et al., 2021;Tiencheu et al., 2021). During physical exercise, oxidative stress is induced because of an imbalance between the production of reactive oxygen species and antioxidant capacity in the body, leading to an increase in inflammatory markers, muscle damage, and gastrointestinal dysfunction (AbuMoh'd, 2020;Elejalde et al., 2021;Martins et al., 2020). Grape and its derivatives could be beneficial facing oxidative damage because of the presence of phenolic compounds (De Oliveira et al., 2021). Its carbohydrate content is necessary for glycogen deposition and improvement of practice during long-term exercise (Martins et al., 2020). Concentrated grape juice is a product obtained by physical methods for removing water and increasing the content of soluble solids present in the respective total juice by at least 50%. By diluting the concentration or dried juice to the initial concentration based on °Brix as reconstitution parameter, reconstituted grape juice is obtained (Dutra et al., 2021).
Grape juices consist of water (81%-86%) and a high concentration of sugars (glucose and fructose), with high acidity owing to the existence of organic acids that balance the sweet and sour tastes.
The new drink prepared from grape juice is a natural product in which the use of artificial colorants and flavorings was avoided. No sugars are added, only those of the fruit juice and with great nutritional value and biological activity. Lemon juice is added to the beverage to reduce the pH and provide a pleasant flavor. Lemon is one of the citrus fruits that are characterized by their content of flavanones, vitamin C, minerals, and citric acid, which provide nutritional value in a beverage (Agulló et al., 2021;Gironés-Vilaplana et al., 2013;González-Molina et al., 2012). Anthocyanins have a high potential for utilization as natural colorants to replace synthetic dyes in food systems owing to their attractive colors, water solubility, and health benefits (Brenes et al., 2005;Gérar et al., 2019;Tan et al., 2021). Their stability depends on the pH, lack of vitamin C, high concentration of sugar (Cosme et al., 2018), oxygen, light, temperature presence of ascorbic acid, and metal ions (Moldovan & David, 2020;Vidana Gamage et al., 2022).
Among challenges for academic and industrial investigation is the production of natural flavors, which determine the sensory characteristics of beverages and other food products because of the growing preference of consumers for sustainable and natural products (Vilela et al., 2019). Since the prepared drink is an organic product without artificial ingredients, natural flavors extracted from spices and herbs (hop, tea, and mint) are used to enhance the sensory properties and increase the aroma of the beverage.

| Raw materials
Concentrated red grape juice was used with a sugar content of 65 °Brix, pH 3.5, and SO 2 <40 ppm (Vinos y Bodegas). Dilutions were performed with mineral waters with different mineralization: Bezoya (Calidad Pascual) and Solan de Cabras. (Mercadona), with 40 mg/L of C vitamin and 10 mg/L of sodium.
The aroma was improved by infusion extractions from organic red tea (Cafetearte), organic dried mint (Soria Natural), and hop (Summit).

| Extraction
To obtain the extracts, 4 g of each herb or spice (hop, tea, and mint) were weighed, crushed in the mortar, and mixed with 30 ml of diluted grape juice ( Figure 1). The extraction of the aromas and flavors from the three mixtures was performed using ultrasounds (3300EP SONICA) for 10 min followed by centrifugation (Eppendorf,5430 R) at 6000 rpm at 20°C for 10 min. Finally, the extracts were filtered using filter papers and kept refrigerated at 4°C until being added to the beverages.

| Beverage preparation
For the preparation of the beverages, preliminary experiments were carried out, aimed to obtain a product with a suitable sensory profile, in terms of acidity, color, and flavors. Several drinks were prepared with the two types of water previously described (different mineralization) and flavored with different herbs and spices (cardamom, hop, fresh mint, dried mint, black tea, red tea, and green tea).
Based on sensory analysis, the appropriate ingredients identified for the final formulation of a natural drink flavored with a mixture of hop--tea and hop-mint.
The natural drink was formulated by diluting grape juice (47 ml) with water (453 ml) to obtain 50 g/L of sugar. Using a bottle of 500 ml for each sample (Table 2), two groups of beverages were prepared ( Figure 1), the first group "A" using natural mineral water with low mineralization (Bezoya) and the second group "B" using natural mineral water with high mineralization (Solan de Cabras

| Physicochemical analysis
The Crison brand pH meter GLP 21 model was used for the pH measurements of each sample for 20 days. Sugar concentration (glucose and fructose), total soluble solids (TSS), total acidity, organic acids, and other parameters were identified with OenoFoss ™ equipment (FOSS Iberia) using Fourier transform infrared spectroscopy.

| Color parameters analyzed by UV-visible spectrophotometry
Since the drink was prepared with grape juice, the same wavelengths (280, 420, 520, and 620 nm) were selected for the measurement.
For 20 days, the absorbance was determined using an Agilent 8453 F I G U R E 1 Schematic of flavor extraction and beverage preparation spectrophotometer (Agilent Technologies S.L.) and a 1 mm optical path glass cuvette. The color intensity (CI), the amount of color present in the juices (CI), was obtained by the sum of absorbances at 420, 520, and 620 nm. The tonality (T) was calculated by the quotient between the absorbance values at 420 and 520 nm (Burin et al., 2010). Total phenolic content was the absorbance at 280 nm (Milella et al., 2019).

| Determination of anthocyanins
Anthocyanin determination was according to Escott et al. (2017).
The anthocyanins were identified and quantified with a series 1200 high-performance liquid chromatograph (HPLC), equipped with a diode array detector. Twenty-microliter samples of previously filtered 0.45 µm membrane were injected into the HPLC apparatus.

| Sensory analysis
The sensory evaluation test was carried out in the tasting room of the

| Evolution in pH and color
Analysis of pH values of beverages ( Figure 2)  Regarding the absorbance at 520 nm, the length at which anthocyanins absorb, Figure 3 shows an increase in absorbance at 520 nm in all beverages during the storage period. However, the influence of the type of water and acidity on the absorbance of anthocyanins was observed. Drinks with lower pH values and a lower degree of mineralization (BA) presented higher absorbance values than drinks with higher pH values and a higher degree of mineralization (BB).

| Physicochemical analysis
The results of the physicochemical analysis using the FOSS analyzer are presented in Table 4. Samples present a significant difference between the two groups except for malic acid, ammonia, and density. It observed that the samples made with weak mineral water and presented a lower pH were the ones that contained the highest level of sugar content (glucose/fructose) and total acids. The levels of sugars in the beverages ranged from 79.90 ± 1.37 to 82.37 ± 0.55 g/L.

| Sensory analysis
The results of the sensory analysis on sample preference are presented in Figure 6. Flavored drinks were compared sensorially with the control drink in both groups. In general, in cases of an appreciable difference between flavored drinks and control drinks, tasters preferred flavored drinks. As shown in Figure 6, BAHT, BAHM, BBHT, and BBHM presented higher average scores than the control drinks (BAN and BBN). On the other hand, tasters identified a difference between drinks with water A or B. Concerning CI, BA drinks were rated slightly higher, with a maximum value of 3.75 ± 0.89 in BAHT.
These results agree with spectrophotometric colors measurements ( Figure 3). In terms of aromatic intensity and quality, results showed significant differences between samples. BBHM (4.00 ± 0.76) was the most preferred, followed by BAHT (3.63 ± 1.19).
However, samples were slightly herbaceous without significant differences. Fruity and floral parameters showed significant differ-

| DISCUSS ION
The samples prepared were subjected to physicochemical and sensory analysis to determine their different properties. Results demonstrated that samples had low pH values (Figure 2). The pH values differed slightly among the samples, although the same quantity of lemon juice was added during beverages preparation. The pH was lower for beverages prepared with low-mineralized water (BA) compared to beverages prepared with high-mineralized water (BB). The trend with total acids (Table 4) was the same: BA samples had higher levels of total acids (1.43 ± 0.06 g/L) compared with BB samples.
This high acidity in sample drinks could be due to the low pH of the concentrate grape juice (pH = 3.5) used for the preparation of beverages, the presence of tartaric, malic, and citric acids in the grape juice composition (Cosme et al., 2018), and the use of lemon juice, which is characterized by its content of ascorbic acid (vitamin C) (Hooshyar et al., 2020) and citric acid . It should be noted that these values ensure the safety of the beverage by promoting resistance to microbial deterioration (Hani et al., 2019) including Clostridium botulinium (Porfírio et al., 2020). Furthermore, an acidic pH lower than 3.5 is important to obtain the required red color and the stability of anthocyanins (Hani et al., 2019). According to our results (Figure 3), the absorbance at 520 nm (an increase during 20 days) was higher in the BA sample where the acidity was higher ( Figure 2). The same trend with CI and total phenolic content with higher values were marked in BA samples (Table 3). These results clearly show that the degree of mineralization of waters influenced the acidity of grape juice beverages and made a significant difference in the absorbance at 520 nm and color between the two groups of beverages. The color characteristic depends on the anthocyanins content, which are responsible for the red color of grape juice and present the most important indicator of grape juice quality (Burin et al., 2010;Dıblan & Özkan, 2021). The stability of anthocyanins is affected by different factors such as the chemical structure, pH, temperature, oxygen, concentration, light, enzymes, presence of co-pigments, and food matrix composition (proteins, carbohydrates, ascorbic acids, minerals, salts, and sugars) (Morata et al., 2019;Ren & Giusti, 2021;Vidana Gamage et al., 2022) reported that the stability, CI, and absorption wavelength of anthocyanins depend on pH of the medium. In lower pH solutions, the predominant form of anthocyanins is the flavylium cation which shows an intense red color.
In strong acidic media (pH 1-2), CI increases strongly. When pH increases, this form turns to uncolored carbinol pseudobase. The color becomes blue-violet in a basic pH because of the transformation to a quinoidal base form. Consequently, anthocyanins are stable in low pH values. Additionally, the color change based on pH conditions is also influenced by the type of anthocyanins, cyanidin shows red color at pH < 3, violet color at pH 7 and 8, and blue color at pH > 11.
However, peonidin has higher stability at high pH than other anthocyanidins; at acidic conditions, it shows red color to cherry, and at pH 8 shows a deep blue color (Chandra et al., 2021). Moreover, color stability also depends on the water concentration as the decrease in the concentration of water improves the deprotonation rate of the flavylium, lowering the stability of color (Chandra et al., 2021).
According to Hooshyar et al. (2020), the glucoside forms of malvidin, delphinidin, cyanidin, pelargonidin, peonidin, and petunidin are the main abundant anthocyanins in red grapes. Our results (Table 5) present the same types of anthocyanins with different concentrations among samples and with a predominance of malvidin pigment in all samples, which is the main anthocyanin found in red grape juice among the six monomeric anthocyanins (Cosme et al., 2018). The concentration of anthocyanins in grape juices depends on cultivars, raw material, processing technology, and heat treatment (Cosme et al., 2018). Dıblan & Özkan (2021)  Finally, results of the sensory analysis show that flavored beverages were preferred over control beverages ( Figure 6). Panelists gave BAHT the highest mean score, including overall acceptance.
This could be due to the preferred sour taste of the formulation, which was positively correlated with the highest level of total acid (1.43 ± 0.06 g/L) (Table 4) among beverages. Tasters greatly accepted the good taste and aroma of the mixture hop-tea (BAHT) added to grape juice, which was characterized by a strong aroma, flavor, and a pleasant taste due to the abundant content of vitamin C in grapes (Ayoub et al., 2020). In addition, color is an important sensory property when choosing a food product, especially in beverage products. In this case, grape juice was preferred as a source of natural colorant due to the high concentration of phenolic compounds that provide sensory characteristics (color, taste, and flavor), namely anthocyanins, which are responsible for the color of grape juice (Cosme et al., 2018). Attractive colors (red, orange, and purple) and water solubility of anthocyanins allow their incorporation into aqueous food systems as natural colorants (Morata et al., 2019).
In addition, color is used by consumers to determine the quality of agricultural and food products because of the strong correlation between color and flavor. It has been noticed that the identification of flavor decreases when the colors of food products are different from the expectations of consumers (Chandra et al., 2021). On the other hand, besides their pleasant flavors and aroma, spices have helpful effects on human health and act as natural preservatives (Ivanišová et al., 2005;Potortì et al., 2019;Souza et al., 2020). According to Moghaddam et al. (2018), fruits, vegetables, and herbs beverages are considered as health-promoting agents because of their content agents, and organic acids. This encourages the combination of good sensory properties (color and pleasant aroma) from natural sources and health benefits because of contents rich in phenolic compounds of grape juices and herbs in one product, which could be acceptable to consumers increasingly searching for products free of artificial additives.

| CON CLUS ION
In this study, new natural beverages were formulated from a combination of concentrated grape juice with two different mineral waters (Bezoya and Solan de Cabras), using lemon juice for pH correction, naturally flavored with herbs and spices extracts. The results of color measurement and pH indicated that beverages prepared with low mineral water (BA) present high acidity and high CI. On the other hand, the most interesting in terms of sensory analysis is the beverage prepared with low mineral water and flavored with a mixture of hop and tea (BAHT). The most abundant anthocyanin in prepared drinks was malvidin-3-O-glucoside, followed by peonidin-3glucoside. Moreover, grape juice, lemon juice, and plant extracts have great potential in the development of a healthy fruit drink due to the antioxidant activity provided by their polyphenolic compounds. This is presented as an alternative for consumers looking for drinks that are less artificial and more beneficial to their health. Additionally, the beverages prepared contribute to sustainable development as they are produced under organic production conditions.

ACK N OWLED G EM ENTS
The authors acknowledge the Algerian government for the scholarship offered to Yasmina Bendaali giving an opportunity to carry out this project. Authors also thank Fernando Boned and Fernando Chivite (in memoriam) for their full support in this project.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

CO M PLI A N CE WITH E TH I C S R EQ U I R E M E NT S
This study does not contain any studies with human or animal subjects.

DATA AVA I L A B I L I T Y S TAT E M E N T
Supplementary data are available upon reasonable request.