Phenolic composition and encapsulation of Brazilian grape seed extracts: evaluating color stabilizing capacity in grape juices

Abstract

The color of grape juice is an important acceptance attribute by consumers, but it suffers losses during storage. The use of commercial antioxidants has limitations because the concept of a “100% natural drink” of Brazilian legislation. This work characterized Brazilian grape seeds, and the cultivar extract with the greatest potential was encapsulated in arabic-gum (encapsulated extract-EE) to evaluate the color stabilizing capacity. The EE used in the grape juice was compared with the commercial antioxidants sulphite and enological tannin during storage (150 days). The BRS Magna and BRS Violeta grape seeds had the highest phenolic content, and the EE showed high catechin (4108 mg/kg), epicatechin (1161 mg/kg) and procyanidin-B2 (905 mg/kg) values. Sulfite was found to be the best color stabilizer. The use of EE (0.5 g/L) in grape juice improved color stability and anthocyanin stability. It was demonstrated that encapsulated grape seed extract has color stabilizing potential and that Brazilian grape seeds are a raw material of high technological value.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-024-05956-8.

Introduction

Interest in recovering bioactive phenolic compounds from grape by-products has gained prominence recently. The main residues from grape processing are composed of skins and seeds, representing about 25% of the total grapes used in processing juices and wines worldwide (Machado et al. 2021). Most of the phenolic content is found in the grape residue, such as anthocyanins, flavanols, tannins and phenolic acids (Capanoglu et al. 2013; Sirohi et al. 2020), which can be applied by pharmaceutical and food industries (Teixeira et al. 2014; Machado et al. 2021).

The grape-based beverage industry has been looking for new technologies to implement to preserve the color stability of its products, as process conditions such as heating, oxygen dissolution, fruit oxidative enzymes and commercial pectinases interfere with color stability (García-Estévez et al. 2017; Prado et al. 2019; Ntuli et al. 2020). In addition to grape juice process steps, storage time also causes deterioration of anthocyanins and loss of color (Ntuli et al. 2020). The color of grape juice is an important quality attribute, making this drink attractive to consumers (Granato et al. 2016), which makes studies on color stability relevant.

An alternative commonly used in the juice and wine industry to delay color degradation during processing and storage is the use of the antioxidant sulfur dioxide (sulphite) (Ntuli et al. 2020). Another option used to improve the color stability of grape juices and wines has been the use of enological tannins extracted from wood, which are basically composed of phenolic catechins, procyanidins, gallocatechins and ellagitannins (García-Estévez et al. 2017; Ntuli et al. 2020). However, according to Brazilian legislation (Brasil 2018), the use of sulphites or substances exogenous to grapes in whole juices prevents industries from using the expression “100% natural juice” on their labels, which is a designation desired by consumers. This fact has reinforced the search for natural antioxidants to be used in this beverage.

Studies have shown that grape seed phenolic compounds are an alternative for increasing the natural antioxidant content in foods and beverages (Toaldo et al. 2013; Yadav et al. 2018; Carpes et al. 2020; Silva et al. 2020; Silva et al. 2022). However, phenolic compounds extracted from residues can be rapidly oxidized in the presence of heat, light and oxygen, limiting their application in industry; and the encapsulation technique has been successful in protecting grape seed extracts (Estévez et al. 2019; Pedrali et al. 2020).

Natural stabilizers obtained by the enzymatic hydrolysis of grape seeds by endoproteases have already been used to stabilize the color of wines, and the process for obtaining the stabilizer is patented (Machado et al. 2021). However, the antioxidant power of grape extracts varies depending on several factors, mainly between different species and cultivars (Teixeira et al. 2014; Machado et al. 2021). New Brazilian grape cultivars (hybrids of Vitis vinifera x Vitis labrusca) created for juice production, such as BRS Violeta, Isabel Precoce, BRS Cora, BRS Magna and BRS Carmem, have high phenolic compound content in the peel and seed fractions (Camargo et al. 2014; Padilha et al. 2019), and may be a potential raw material for recovering phenolic antioxidants with industrial application. Previous studies have applied encapsulated grape seed extracts to increase the antioxidant compound content in milk/yogurt (Yadav et al. 2018), and to obtain antioxidant microcapsules (Boschetto et al. 2013; Estévez et al. 2019; Pedrali et al. 2020). In addition to potential applications in food processing, phenolic compounds in seeds are associated with several health benefits for consumers, being used in formulating food supplements and commercial drugs (Teixeira et al. 2014; Machado et al. 2021).

In this context, the present study aimed to evaluate the phenolic content (HPLC–DAD) and antioxidant capacity of Brazilian grape seed extracts, encapsulate the extract and verify its color stabilizing capacity in whole grape juices during storage compared to commercial antioxidants.

Materials and methods

External standards and reagents

Ethyl alcohol, potassium persulphate, Folin–Ciocalteu reagent were obtained from Merck (Darmstadt, Germany). Gum Arabic was obtained from Vetec Química Pura (Brazil). Trolox (6-hydroxy-2,5,7,8-tetramethylchromate-2-carboxylic acid), TPTZ (2,4,6-Tri(2-pyridyl)-s-triazine), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The external standards of phenolic compounds for HPLC: (−)-epicatechin, kaempferol-3-O-glucoside and monoglucosidic anthocyanins malvidin, delphinidin, peonidin, cyanidin and pelargonidin were obtained from Extrasynthesis (Genay, France). Trans-resveratrol and cis-resveratrol stilbenes were obtained from the Cayman Chemical Company (Michigan, USA). Hesperidin, naringenin, (+)-catechin, (−)-epigallocatechin, (−)-epigallocatechin gallate, procyanidin B1, procyanidin B2, rutin, quercetin-3-O-glucoside, malvidin 3,5-diglucoside, cyanidin 3,5-diglucoside, pelargonidin-3,5-diglucoside, and caffeic, chlorogenic, coumaric, syringic, trans-caftaric and gallic acids were all from Sigma Aldrich (St. Louis, MO, USA).

Seed collection, phenolic extraction and encapsulation

The residues from processing monovarietal grape juices were provided by the Queiroz Galvão Alimentos S/A company, Fazenda Timbaúba, Petrolina, PE, Brazil. Isabel Precoce, BRS Violeta, BRS Cora, BRS Magna and BRS Carmem cultivar Brazilian grape (red) seeds were separated from the residues, dehydrated in an electric dryer at 60 °C until constant weight, vacuum packed in polyethylene bags kept in a freezer at − 18 °C until the analysis moment. The dehydrated seeds were crushed and sieved to obtain a powder with a granulometry ≤ 16 mesh (1.18 mm).

The phenolic content was extracted from the seed powders using ultrasound (USC-1400 model, Merse, Brazil) at a frequency of 36 kHz for 120 min at 35 °C. The solvent used was ethanol 50% in the proportion 1:10 m/v (seed powder/solvent) (Silva-Junior et al. 2023). After maceration, the ethanol extract was centrifuged at 3000g for 5 min (EEQ-9004/B centrifuge model, Edutec, Brazil) and the supernatant was stored in an amber bottle at − 18 °C until performing the chemical analysis.

The cultivar seed extract that presented the highest phenolic content and antioxidant capacity was used to be encapsulated. The ethanolic extract was added to an arabic gum encapsulant in a 1:6 ratio (reactive substances to Folin–Ciocalteu/arabic gum) and homogenized in an ultrasound device at a frequency of 36 kHz for 20 min at 35 °C. The spray drying conditions were: temperature and air velocity at 140 °C and 25 m/s, respectively, with the sample injection volume being 250 mL/h. The spray nozzle was 1.0 mm, and the air volume in the nozzle was maintained at 35 L/min (Silva-Junior et al. 2023). The equipment used was a “Spray Dryer” MSDi 1.0 model, manufactured by Labmaq (Brazil). The outlet air temperature was 80 °C. The obtained powder was collected, stored in an airtight glass bottle, and kept at − 18 °C until analysis and application in grape juices. The spray drying process parameters such as encapsulation efficiency and yield were calculated by the methodology described by Pedrali et al. (2020) using the Folin–Ciocalteu reducing substance content of gallic acid equivalent (GAE) as a variable. Water activity was obtained using an Aqualab digital device, model 4TEV (Brazil).

Preparation of grape juices, addition of antioxidants and experimental model

The BRS Magna (red) cultivar was used to elaborate the whole juice, for which the grapes were harvested at the ripening stage used for juices in the sub-middle São Francisco Valley, Brazil (18 degrees Brix, and titratable acidity 0.7%). The juice was prepared by the “Hot press” process (Silva et al. 2019) with 100 kg of grapes being destemmed and crushed, heated to 60 °C, with pectinase (Endozym Pectrofruit PR, AEB Bioquímica, Brazil) added at a dose of 4 mL/100 kg of grapes, and then remaining under maceration for 60 min (depectinization). The grapes were pressed and the drained juice was fractionated to add antioxidants/color stabilizers and then pasteurized at 85 °C for 3 min, cooled by immersion in cold water until a temperature < 40 °C. The juice presented basic analytical characteristics of degree Brix = 18.1, titratable acidity 0.7% and pH = 3.64.

The treatments consisted of: whole juice without the addition of antioxidant (control); juice with 80 mg/L of the potassium metabisulphite antioxidant added (40 ppm of free sulphur dioxide), maximum dose allowed by Brazilian legislation; juice with 100 mg/L Red Supertan enological tannin (Coccitech, Italy) added as a natural stabilizer (dose recommended by the manufacturer for red wines); juice with microencapsulated grape seed extract added at a dose of 0.5 g/L (100 mg/L of Folin–Ciocalteu reducing compounds); and juice with microencapsulated grape seed extract added at a dose of 1.0 g/L (200 mg/L of Folin–Ciocalteu reducing compounds).

Thus, fifteen colorless bottles of 300 mL were filled for each evaluated treatment, and 3 bottles were analyzed at each storage time at room temperature (26 ± 3 °C) kept protected from light in a corrugated cardboard box. The times evaluated were 30, 60, 90, 120 and 150 days after filling. The bottles were opened at the defined times and immediately analyzed for color, antioxidant capacity and phenolic compounds in HPLC.

Color analysis of the juices

The color intensity was measured by the sum of the absorbances at 420, 520 and 620 nm, corresponding to yellow, red and violet colors, respectively. The darkening index (tonality) was obtained by dividing the absorbances between 420 and 520 nm. The analysis was performed in a UV 2000A model UV–Visible spectrophotometer (Instrutherm, Brazil) using glass cuvettes with a 0.1 cm optical path and the absorbances converted to a 1 cm optical path, following the methodology described by the International Grape and Wine Organization (2011).

In vitro antioxidant capacity

First, 0.5 g of microencapsulated grape seed extract was resuspended in 5 mL of 70% methanol and the mixture was sonicated at 40 kHz/20 min at 27 °C (UNIQUE USC-1400A model, SP, Brazil) to analyze antioxidant capacity. The sonicated mixture was centrifuged at 3000 turns for 5 min, and the supernatant collected and destined for analysis (Wanderley et al. 2023). Grape juices were diluted in ultrapure water for analysis.

The in vitro antioxidant capacity was determined using free radical scavenging methods with DPPH (Kim et al. 2002) and iron chelation power (FRAP) (Rufino et al. 2006). Trolox analytical standard was used to construct the analytical curve and the results were expressed as Trolox equivalents per kg of grape seed powder (mmol TE kg⁻¹) in the DPPH method, and in mmol of Fe²⁺ per kg of powder in the FRAP method. The Folin–Ciocalteu reducing capacity was determined according to Singleton and Rossi (1965). The absorbance readings were performed in a UV 2000A model UV–Visible spectrophotometer (Instrutherm, Brasil).

The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical activity was measured by quenching the absorption maximum at 517 nm. The method consisted of mixing 100 µL of the sample with 2.90 mL of 100 mmol ethanolic solution of the DPPH radical, and incubating it in the dark for 30 min.

The FRAP reagent was prepared by mixing 25 mL of acetate buffer solution (300 mmol; pH 3.6), 2.5 mL of TPTZ solution (10 mmol TPTZ in 40 mmol HCl) and 2.5 mL of FeCl₃ (20 mmol) in aqueous solution. Next, 90 μL of the previously diluted juices/extracts and 270 μL of water were added to 2.7 mL of the FRAP reagent (kept at 37 °C for 30 min in an IT2002 model AAKER thermoreactor, Brazil). Absorbance was measured at 595 nm in the spectrophotometer.

The Folin–Ciocalteu reducing capacity was determined using 0.10 mL of the sample, 7.90 mL of water and 0.50 mL of the Folin–Ciocalteu reagent. The absorbance at 765 nm was read and the results were expressed in mg kg⁻¹ of powder or mg/L of juice, compared to a 25–500 mg/L gallic acid calibration curve (GAE).

Quantification of individual phenolic compounds by HPLC

Liquid chromatography analyzes were performed on an Agilent 1260 Infinity LC System chromatograph (Agilent Technologies, Santa Clara, USA) coupled to a diode array detector (DAD) (G1315D model). Data were processed using the OpenLAB CDS ChemStation Edition software program (Agilent Technologies, Santa Clara, USA).

Extracts to be injected were prepared as previously described in section "In vitro antioxidant capacity". The individual phenolic profile was determined in RP-HPLC/DAD following the method validated by Padilha et al. (2017) with adaptations by Dutra et al. (2018). An Eclipse Plus RP-C18 Zorbax (100 × 4.6 mm, 3.5 μm) column was used, and the pre-column was C18 (12.6 × 4.6 mm, 5 μm) (Zorbax, USA). The oven temperature was 35 °C and the injection volume was 20 µL of the sample previously filtered through a 0.45 µm membrane (MillexMillipore, Barueri, SP, Brazil). The solvent flow was 0.8 mL min⁻¹. The gradient used in the separation was 0–5 min: 5% B; 5–14 min: 23% B; 14–30 min: 50% B; 30–33 min: 80% B, in which solvent A was a phosphoric acid solution (pH 2.0) and solvent B was methanol acidified with 0.5% H₃PO₄. The compounds were detected by comparison with external standards (R² > 0.995). The detection limits for all analyzed compounds were LOQ < 1.41 mg/L and LOD < 0.17 mg/L.

Statistical analyses

The results obtained from the analyzes were tabulated, submitted to analysis (ANOVA), and compared by the Tukey’s test at 5% error probability using the SPSS version 20.0 program for Windows (IBM, NY, USA). Principal component analysis (PCA) was also performed using the Past version 4.03 software program (USA).

Results and discussion

Phenolic compounds and antioxidant capacity of the Brazilian grape cultivar seed powders

The in vitro antioxidant capacity and the HPLC phenolic profile of the grape seed powders are shown in Table 1. There was a significant difference (p < 0.05) in the phenolic content of the studied grape cultivar seeds. In relation to the total phenolic compounds quantified in HPLC, the BRS Violeta cultivar seeds had the highest values (9881 mg/kg), followed by BRS Magna (4666 mg/kg), Isabel Precoce (2123 mg/kg), BRS Cora (2112 mg/kg) and BRS Carmem (1090 mg/kg). BRS Violeta presented the highest individual phenolic content, especially catechin (7,435 mg/kg), followed by procyanidin B2 (817 mg/kg), epicatechin (675 mg/kg) and procyanidin B1 (646 mg/kg). BRS magna was the second cultivar in terms of amount of individual phenolics, in which the main phenolic quantified was also catechin (2669 mg/kg), followed by procyanidin B2 (765 mg/kg), epicatechin (716 mg/kg) and procyanidin B1 (352 mg/kg). In addition to monomeric flavanols (catechins) and proanthocyanidins, the seeds also showed lower amounts of chlorogenic acid (59–116 mg/kg) and quercetin 3-glucoside (10–18 mg/kg).

Table 1.

Phenolic compounds and antioxidant capacity of grape seed powder from the juice process residue in the sub-middle of the São Francisco Valley, Brazil

Phenolic compounds mg/kg DW	Seeds
	Isabel Precoce	BRS Cora	BRS Violeta	BRS Magna	BRS Carmem
Catechin	1082 ± 180 c	850 ± 10 c	7435 ± 778 a	2669 ± 746 b	475 ± 101 d
Epicatechin	320 ± 51 cd	466 ± 2 bc	675 ± 92 ab	716 ± 173 a	186 ± 38 d
Epigallocatechin Gallate	56 ± 7 c	38 ± 24 b	173 ± 21 a	ND	23 ± 12 d
Procyanidin B1	215 ± 31 bc	113 ± 4 c	646 ± 85 a	352 ± 74 b	75 ± 14 c
Procyanidin B2	390 ± 56 cd	525 ± 19 bc	817 ± 87 a	765 ± 183 ab	246 ± 45 d
Quercetin 3-glucoside	ND	11 ± 3 b	18 ± 1 a	17 ± 1 a	10 ± 2 b
Rutin	ND	ND	ND	3 ± 1	ND
Chlorogenic acid	59 ± 38 c	109 ± 19 abc	116 ± 12 ab	143 ± 2a	75 ± 10 bc
∑ quantified phenolics	2123 c	2112 c	9881 a	4666 b	1090 c
In vitro antioxidant capacity
DPPH mmol TE/kg	2302 ± 184 b	2153 ± 294 b	5102 ± 159 a	5195 ± 7 a	1775 ± 180 c
FRAP mmol Fe2+/kg	11,510 ± 918 b	10,764 ± 1470 b	25,508 ± 794 a	25,978 ± 36 a	7872 ± 899 c
Folin-Ciocalteu mg/g GAE	93 ± 8 bc	87 ± 17 b	162 ± 11 a	183 ± 11 a	58 ± 7 c

Results presented as mean ± standard deviation (n = 3). Means followed by different letters on the line differ from each other by the Tukey’s test (p < 0.05). ND = Not detected or not quantified

Previous works which quantified individual phenolics in grape seeds and their extracts for encapsulation mentioned catechin, epicatechin, procyanidin B1, procyanidin B2, quercetin 3-glucoside and gallic acid as the main compounds present (Yadav et al. 2018; Pedrali et al. 2020), corroborating the phenolic profile obtained in the present study for Brazilian grape seeds.

Grape seeds used for extraction and encapsulation of phenolic compounds in the study by Pedrali et al. (2020) showed catechin (589 mg/kg dw), epicatechin (326 mg/kg dw), procyanidin B1 (62.5 mg/kg dw), procyanidin B2 (159 mg/kg dw) and quercetin 3-glucoside (24.7 mg/kg dw) as the main antioxidant substances. Based on these previous values, the phenolic content found in the BRS Violeta and BRS Magna seeds in the present work were considered high.

The values obtained in the seeds regarding the antioxidant capacity (AOX) also differed from each other by the Tukey’s test (P < 0.05) among the studied cultivars. According to Granato et al. (2018), it is necessary to use different antioxidant systems in studies to screen the antioxidant potential of foods and beverages, and to elucidate the compounds associated with the activity measured. Thus, free radical scavenging methods (DPPH), Folin–Ciocalteu (FC) reducing power and iron chelation power (FRAP) were used in the present study.

The seeds which showed the highest antioxidant capacities were BRS Magna and BRS Violeta, followed by Isabel Precoce and BRS Cora, and BRS Carmem. The antioxidant capacities obtained for BRS Magna were 5195 mmol TE/kg (DPPH), 25,978 mmol Fe²⁺/kg (FRAP) and 183 mg/g of gallic acid (FC). BRS Violeta presented values similar to BRS Magna, not differing statistically (Tukey’s test, p < 0.05).

The antioxidant capacity of the seeds studied was generally considered high when compared to the antioxidant activities of the juices of the respective grapes (Lima et al. 2014; Dutra et al. 2018). These results can be explained by the fact that most of the grape antioxidant compounds are present in the residues that are removed during processing (Camargo et al. 2014; Capanoglu et al. 2013). It is also noteworthy that catechins and procyanidins present in the seed have the highest in vitro antioxidant activities among the main phenolic compounds found in grapes and derivatives (Muselik et al. 2007).

The supplementary figure S1 presents the “Biplot” principal components analysis between the cultivars and the chemical profile of the seeds. Principal Components 1 and 2 (PC1 and PC2, respectively) explained 90.5% of the variance of the experiment, in which PC1 (71.5% of variance) grouped the BRS Violet and BRS Magna cultivars in the positive part, associated with the highest quantified phenolic compound values and antioxidant capacity. PC2 with the lowest weight of variance (19%) separated BRS Magna from BRS Carmem, mainly due to the higher catechin, procyanidin B1, epigallocatechin gallate and total phenolic compound contents quantified in HPLC (TPQ). The DPPH, FRAP and FC antioxidant capacities were grouped in the positive part of PC1, strongly correlated with catechin, epicatechin, and procyanidins B1 and B2, which are the phenolic compounds responsible for the antioxidant capacity of the Brazilian grape seeds evaluated.

Based on the characterization results of the grape seed in the present study, it is evident that the BRS Magna and BRS Violeta cultivars have the greatest potential for extracting phenolic antioxidant compounds. Thus, the BRS Magna grape seed was chosen to encapsulate the extract and apply it in the study to evaluate the color stability of the whole juice due to having harvests/processing in all months of the year by the partner company, which would facilitate availability of raw material for bioactive compound recovery industries.

Phenolic profile and antioxidant capacity of encapsulated BRS Magna grape seed extract

The encapsulation process parameters, phenolic profile and antioxidant capacity of the arabic gum encapsulated extract (EE) are shown in Table 2. The encapsulation process showed an efficiency of 76.2% (Folin–Ciocalteu reducing substances) and process yield of 50.4%, resulting in a powder with a water activity of 0.23. The Folin–Ciocalteu reagent reducing capacity of the EE presented an average value of 203 mg/g GAE of powder. The total of phenolics quantified in HPLC was 6.65 mg/g of powder, mainly represented by catechin, epicatechin and procyanidin B2 flavanols (Supplementary Figure S2), which were also the main compounds present in the seed characterization (Table 1). The EE antioxidant capacity evaluated by the DPPH method was 1.05 mmol TE/g, and presented 5.27 mmol Fe²⁺/g with FRAP. Previous works that encapsulated grape seed ethanol extracts with different encapsulants and in different techniques mentioned that the best efficiency results ranged from 77.4 to 92.3%, where the encapsulated extracts obtained showed Folin–Ciocalteu reducing agent values ranging from 1.2–39.2 mg/g, and the main individual phenolics found were catechin, epicatechin, procyanidin B1 and procyanidin B2 (Yadav et al. 2018; Estévez et al 2019; Pedrali et al. 2020). Based on this, we consider that the encapsulation process of the BRS Magna grape seed extract in the present study is adequate for the proposed purpose, and with a high concentration of phenolic antioxidants, thus constituting a potential raw material for recovering bioactive phenolic compounds.

Table 2.

Process parameters, phenolic composition and antioxidant capacity of encapsulated BRS Magna grape seed extract

Encapsulated grape seed extract	Mean ± deviation
Encapsulation process parameters
Efficiency %	72.6 ± 2.2
Yield %	50.4 ± 1.2
Aw	0.23 ± 0.01
Individual phenolics in mg/kg of powder
Catechin	4108 ± 196
Epicatechin	1161 ± 67
Procyanidin B1	392 ± 4
Procyanidin B2	905 ± 98
Gallic acid	63 ± 1
Quercetin 3-glucoside	21 ± 2
Rutin	4 ± 1
Total quantified in HPLC	6654 ± 370
Antioxidant capacity (powder)
Folin-Ciocalteu mg/g GAE	203 ± 2
DPPH mmol TE/kg	1055 ± 4
FRAP mmol Fe2+/kg	5274 ± 18

Legend: GAE = Gallic acid equivalent activity

Color stability and individual anthocyanins in grape juices with different antioxidants

The juices without (control) and with the addition of antioxidants were stored in corrugated cardboard boxes at room temperature (26 ± 2 °C) and analyzed at 30, 60, 90, 120 and 150 days. The treatments were denominated as: juices that did not receive the addition of antioxidants (control), and juices that received sulphur dioxide (Sulphite), enological tannin, encapsulated extracts of grape seed at doses of 0.5 g/L (D1) and 1.0 g/L (D2).

The evolution of the color and the main anthocyanins (quantified in HPLC) are presented in terms of quantity in Fig. 1. The data from the color evolution curves and anthocyanins were applied in the linear model (y = bx + a) and obtained good fits (R² ≥ 0.78; p < 0.000), except for sulphite regarding hue (R² = 0.56; p < 0.002) (Table 3). Next, the parameters were obtained with the fitting of all variables associated with the juice color in the linear model (y = bx + a): slope (b) which represents the speed of the reactions, and the intercept (a) which represents the initial values of the variables studied.

Fig. 1.

Evolution of color and individual anthocyanins of grape juices with different treatments during 150 days of storage. Legend: anthocyanins (y axis = mg/L; x axis = day); color parameters (y axis = color index; x axis = day)

Table 3.

Parameters obtained from the evolution of color and individual anthocyanins in grape juice treated with different antioxidants during 150 days of storage

Color intensity	Model ($y=bx+a$)		R2	Sig	Tukey p < 0.05
	$b$	$a$			Times n = 5 (30–150)
Control	− 0.14	47.4	0.95	0.000	a b c c d
Tannin	− 0.16	49.7	0.96	0.000	a b c d e
Encapsulated D1	− 0.10	42.6	0.85	0.000	a b bc c d
Encapsulated D2	− 0.12	48.7	0.82	0.000	a a ab b c
Sulphite	− 0.10	40.9	0.80	0.000	a ab ab b c
Browning index (tonality)
Control	0.0008	0.28	0.78	0.000	a a a b c
Tannin	0.0005	0.33	0.79	0.000	a a ab b c
Encapsulated D1	0.0006	0.34	0.94	0.000	a a b c c
Encapsulated D2	0.0006	0.34	0.91	0.000	a a b bc c
Sulphite	0.0003	0.33	0.56	0.002	a a a a b
Petunidin-3-glucoside
Control	− 1.41	74,251	0.86	0.000	a b b c c
Tannin	− 2.38	814.1	0.94	0.000	a b c d d
Encapsulated D1	− 1.86	790.3	0.89	0.000	a a b c c
Encapsulated D2	− 1.99	777.3	0.96	0.000	a b c d e
Sulphite	− 1.20	759.1	0.93	0.000	a b c cd d
Cyanidin-3,5-diglucoside mg/L
Control	− 0.08	41.77	0.86	0.000	a b b c c
Tannin	− 0.12	44.12	0.94	0.000	a b c d d
Encapsulated D1	− 0.10	43.76	0.86	0.000	a b c c c
Encapsulated D2	− 0.11	44.35	0.96	0.000	a b c d d
Sulphite	− 0.09	44.13	0.88	0.000	a b c c c
Delphinidin-3-glucoside
Control	− 0.10	24.11	0.93	0.000	a b c d e
Tannin	− 0.11	25.02	0.94	0.000	a b c d d
Encapsulated D1	− 0.10	24.38	0.94	0.000	a b c d d
Encapsulated D2	− 0.11	24.64	0.95	0.000	a b c d d
Sulphite	− 0.10	25.72	0.94	0.000	a b c d e
Malvidin-3,5-diglucoside
Control	− 1.25	512.3	0.92	0.000	a b b c c
Tannin	− 1.72	539.6	0.95	0.000	a b c d d
Encapsulated D1	− 1.51	537.6	0.91	0.000	a b c cd d
Encapsulated D2	− 1.58	540.6	0.97	0.000	a b c d e
Sulphite	− 1.28	534.1	0.93	0.000	a b c cd d
Cyanidin-3-glucoside
Control	− 0.023	8.01	0.91	0.000	a b b c d
Tannin	− 0.028	8.33	0.94	0.000	a b c d d
Encapsulated D1	− 0.026	8.22	0.92	0.000	a b c cd d
Encapsulated D2	− 0.028	8.35	0.95	0.000	a b c d d
Sulphur Dioxide	− 0.023	8.39	0.92	0.000	a b c cd d

The color intensity (CI) decreased significantly (Tukey’s test; p < 0.05) for all juices in the 150 days of storage, and there was an increase in tonality (relation between red/yellow colors). The increase in tonality shows that there was a decrease in the red color and an increase in the yellow color of the juices, which is an expected change during the shelf life. However, when it happens quickly, it is undesirable for the quality of the grape juice (Prado et al. 2019; Ntuli et al. 2020).

The increase in juice hue and decrease in color intensity was confirmed by the significant decrease (p < 0.05) for the malvidin 3,5-diglucoside, cyanidin 3,5-digluside, petunidin 3-glucoside and delphinidin 3-glucoside anthocyanins, which were the main anthocyanins quantified in the juices, and are responsible for the red/violet color (Fig. 2). According to the values obtained for parameter b, the use of sulphite was considered the best antioxidant treatment of color, followed by encapsulation of grape seed extract; delaying the decrease in color intensity, the increase in hue and the degradation of petunidin 3-glucoside, which was the main anthocyanin present in terms of quantity in the whole juice (see Table 4).

Fig. 2.

PCA of the phenolic profile of grape juices with different color stabilizing treatments during storage. Caption: C = Control; T = enological tannin; E1 = 0.5 g/L encapsulated seed extract; E2 = 1.0 g/L encapsulated seed extract; 30, 90 and 150 = storage days

Table 4.

Evolution of the phenolic profile and antioxidant capacity of BRS Magna grape juice treated with different antioxidants

Variables	Treatments/day
	Control			Tannin				Encapsulated D1				Encapsulated D2			Sulphite
	30	90	150	30	90	150	30		90	150	30		90	150	30	90	150
Phenolic acids
Gallic acid	9.7 ± 0cde	8.6 ± 0ef	9.2 ± 1.3def	28.3 ± 0.2a	26.9 ± 0.2a	27.7 ± 1a	9.1 ± 0def		7.8 ± 0.3f	10 ± 1bcde	9.8 ± 1bcde		10.6 ± 1bc	11 ± 1bcd	9.1 ± 0def	7.5 ± 0f	12 ± 0.1b
Syringic acid	14.4 ± 1ab	14 ± 0abcd	13.7 ± 0abcde	14.4 ± 0ab	13.5 ± 0bcde	13.2 ± 1cde	14.7 ± 0a		13.1 ± 1de	14.3 ± 1abc	14 ± 0abcd		13.2 ± 1cde	13.8 ± 2abcde	13.8 ± 1abcde	12 ± 1de	14 ± 0abcd
Caftaric acid	129 ± 1bcd	133 ± 1bc	136 ± 9ab	134 ± 1bc	128 ± 1bcd	127 ± 2bcd	129 ± 1bcd		121 ± 9d	137 ± 3ab	126 ± 1bcd		122 ± 1cd	132 ± 2bcd	130 ± 6bcd	126 ± 4bcd	146 ± 1a
Chlorogenic acid	4.6 ± 0f	6 ± 0e	7.4 ± 0.4cd	4 ± 0f	5.5 ± 0e	6.9 ± 0.1cd	4.4 ± 0.1f		5.5 ± 0.4e	7.5 ± 0cd	14.2 ± 0a		5.6 ± 0e	8 ± 0.1cd	14.1 ± 1a	5.9 ± 0e	9.2 ± 0b
p-Coumaric acid	3.3 ± 0bc	3.4 ± 0b	2.9 ± 1cdef	2.6 ± 0ef	2.5 ± 0ef	2.7 ± 0def	2.7 ± 0def		2.4 ± 0.1f	3 ± 0bcde	2.8 ± 0def		2.9 ± 0cdef	3 ± 0bcde	3 ± 0bcd	3 ± 0.1bcd	3.9 ± 0a
Stilbene
trans-resveratrol	1.1 ± 0a	1.1 ± 0a	1.1 ± 0a	1.2 ± 0 a	1 ± 0.4a	1.2 ± 0a	1.2 ± 0.1a		1.1 ± 0a	0.9 ± 0a	1 ± 0a		1 ± 0a	1 ± 0a	1.1 ± 1a	1.2 ± 0a	1.2 ± 0a
Flavanols
Catechin	1.8 ± 0f	1.9 ± 0e	1.4 ± 0h	2 ± 0c	2.3 ± 0b	1.4 ± 0h	1.9 ± 0de		1.3 ± 0.0i	2.6 ± 0a	1.4 ± 0i		1.3 ± 0i	1.2 ± 0h	1.5 ± 0g	1.8 ± 0f	2 ± 0cd
Epicatechin	46 ± 0abc	43 ± 0bcd	36 ± 1.3fg	46 ± 1ab	41 ± 1de	30 ± 1i	46 ± 0ab		40 ± 3def	35 ± 2gh	46±abc		41 ± 1de	32 ± 1hi	47 ± 2a	42 ± 1.5cd	38 ± 1efg
Epigallocatechin gallate	2.4 ± 1a	1.9 ± 0b	1.8 ± 0bc	2.5 ± 0a	1.9 ± 0b	1.6 ± 0c	2.5 ± 0a		1.9 ± 0.1b	1.7 ± 0bc	2.4 ± 0a		1.8 ± 0bc	1.7 ± 0bc	2.4 ± 0.1a	1.9 ± 0.0b	2 ± 0.0b
Procyanidin B1	4.3 ± 0gh	5.2 ± 0cd	5.3 ± 0cd	4.3 ± 0h	4.5 ± 0gh	4.6 ± 0fgh	4.3 ± 0fg		4.9 ± 0.2ef	5.5 ± 0bc	4.7 ± 0h		5.4 ± 0.0bcd	5.7 ± 0ab	4.4 ± 0.1gh	5.1 ± 0.1de	5.9 ± 0a
Procyanidin B2	78 ± 1a	64 ± 1b	54 ± 2def	79 ± 1a	62 ± 1bc	50 ± 1f	78 ± 1a		60 ± 5bcd	54 ± 1def	81 ± 1a		62 ± 1bc	52 ± 0ef	80 ± 3.9a	64 ± 2b	57 ± 1cde
Flavonols
Quercetin 3-glucoside	6.4 ± 0abc	6.3 ± 0.1abcd	6.2 ± 0.3bcde	6.6 ± 0ab	6 ± 0abcde	5.8 ± 0de	6.4 ± 0.1abc		5.8 ± 0.4e	6 ± 0abcde	6.4 ± 0abc		5.9 ± 0cde	5.9 ± 0cde	6.3 ± 0abcd	6.2 ± 0bcde	6.8 ± 0a
Rutin	30.5 ± 1a	26.8 ± 1bc	22.8 ± 1de	31.7 ± 0a	26 ± 0.2bc	20.8 ± 0e	31 ± 0a		24.6 ± 2cd	23 ± 0de	30.2 ± 1a		25 ± 1bcd	21 ± 1e	31 ± 1a	27 ± 1b	25 ± 1bcd
Kaempferol 3-glucoside	14.1 ± 1e	19.9 ± 0a	16.5 ± 1d	14.5 ± 0e	19.5 ± 0ab	14.9 ± 0e	14.4 ± 0e		18.3 ± 2bc	16.5 ± 1d	14.1 ± 1d		18.1 ± 1bc	15 ± 1e	14.5 ± 1e	20.2 ± 1a	18 ± 0cd
Anthocyanins
Cyanidin 3,5-diglucoside	40.8 ± 1a	35.2 ± 1b	30.6 ± 1def	41.4 ± 1a	33.5 ± 1bcd	27.4 ± 0.4g	42 ± 0.2a		33 ± 3bcde	30.5 ± 1ef	41.8 ± 1a		33.6 ± 1bcd	29 ± 1fg	42 ± 2a	35 ± 1bc	32 ± 1cde
Delphinidin 3-glucoside	23 ± 0.2ab	15 ± 0b	10 ± 0.1b	23 ± 0.2ab	14 ± 0b	9.3 ± 0.1b	22 ± 0.1ab		14 ± 1b	55 ± 44a	23 ± 0ab		14 ± 0.1b	9.6 ± 0.1b	24 ± 1ab	16 ± 1b	12 ± 0b
Malvidin 3,5-diglucoside	492 ± 5a	402 ± 1b	334 ± 13cd	501 ± 5a	384 ± 4bc	301 ± 5e	503 ± 1a		373 ± 29bc	331 ± 6de	503 ± 8a		383 ± 4bc	318 ± 5de	506 ± 23a	400 ± 14b	351 ± 2.7cd
Cyanidin 3-glucoside	7.7 ± 0a	5.9 ± 0.0bc	4.7 ± 0.1ef	7.7 ± 0a	5.7 ± 0bcd	4.4 ± 0f	7.7 ± 0a		5.5 ± 0cd	4.6 ± 0f	7.7 ± 0a		6 ± 0bcd	4.5 ± 0f	7.9 ± 0.3a	6 ± 0.2b	5.2 ± 0de
Petunidin 3-glucoside	727 ± 7a	635 ± 6b	535 ± 23de	752 ± 8a	607 ± 6bc	487 ± 7e	735 ± 11a		588 ± 49bc	540 ± 2de	718 ± 11a		580 ± 7cd	496 ± 8e	738 ± 23a	637 ± 27b	586 ± 3bcd
Antioxidant Capacity
DPPH mM TE/kg	16 ± 1a	18 ± 1.2a	19.1 ± 1a	18.3 ± 1a	18 ± 1a	19.3 ± 1.1a	16.8 ± 1a		18 ± 0.3a	19.2 ± 2a	18 ± 1a		18.2 ± 1a	17 ± 1a	17 ± 0.2a	16 ± 2a	17 ± 3a
FRAP mM Fe2+/ kg	43 ± 4b	48.2 ± 1a	48.6 ± 2a	49.1 ± 1bc	47.7 ± 2a	49.2 ± 0bc	47.4 ± 2bc		45.2 ± 2bc	44.5 ± 1bc	49.8 ± 3a		48 ± 2bc	48.6 ± 0bc	46 ± 2bc	46 ± 2bc	47 ± 2bc
Color evolution
Colour Intensity	44 ± 0ab	33.8 ± 1ef	24.7 ± 0gh	44.3 ± 0a	33.4 ± 1f	25.7 ± 0gh	39.1 ± 1cd		35.9 ± 1def	26.9 ± 1gh	43.6 ± 1a		40 ± 3bc	28 ± 1g	37 ± 0.2cde	33.1 ± 3f	24 ± 1h
Browning index (tonality)	0.31 ± 0h	0.32 ± 0gh	0.41 ± 0bc	0.35 ± 0f	0.36 ± 0ef	0.41 ± 0ab	0.36 ± 0ef		0.39 ± 0cd	0.42 ± 0ab	0.36 ± 0ef		0.41 ± 0bc	0.43 ± 0a	0.34 ± 0fg	0.35 ± 0.0f	0.38 ± 0de

Results presented as mean ± standard deviation (n = 3). Means followed by different letters on the line differ from each other by the Tukey’s test (p < 0.05)

Sulphite, followed by the encapsulated grape seed extract were the best color stabilizing treatments in the evolution kinetics (parameter b), and confirmed by the Tukey’s test (p < 0.05) applied in the five storage times (30–150 days), as can be seen in Table 3. These results show that the encapsulated BRS Magna grape seed extract has potential as a color stabilizing agent in whole grape juice, being a natural component recovered from the residue of the grape itself. The enological Red Supertan tannin (Coccitech, Italy) at a dose of 100 mg/L obtained inferior results regarding color stabilization to the encapsulated grape seed extract.

The potential for color stabilization of cv. Tempranillo (Vitis vinifera L.) red wine with the addition of 200 mg/L of VR Color enological tannin (Laffort, Rentería, Spain) during 8 months of storage was evaluated in the study by García-Estévez et al. (2017), concluding that the wine which received the enological tannin better preserved its color and anthocyanin content when compared to the control. According to these authors, the high formation of anthocyanin-derived pigments such as vitisins A and B, and/or flavanol–anthocyanin condensation products were responsible for color protection. Several factors can influence the color stability of a wine or grape juice, mainly the phenolic composition, and the phenolic profile of the studied Tempranillo wine was not presented in the study by García-Estévez et al. (2017). However, the authors mention that the use of oenological tannin is recommended for wines which are deficient in tannins. In contrast, the use of commercial grape seed tannin (1000 mg/L equivalent to gallic acid) for color stabilization in cv. Rubired grape juice was evaluated in the study by Ntuli et al. (2020), observing that there was no significant difference in the color maintenance of the juices when compared to the control.

Evolution of individual phenolic profile and antioxidant capacity of juices during storage

Table 4 presents the values obtained at 30, 90 and 150 days of storage in order to observe the influence of adding color stabilizing compounds on the phenolic profile and antioxidant capacity of grape juices during storage. There were generally no considerable differences for flavanols, flavonols, anthocyanins, flavanones, phenolic acids and trans-resveratrol in the juices that received the antioxidants, with the exception of the increase in the gallic acid content in the juice that received the enological tannin, in which a value of 9.7 mg/L in the control was increased to 28.7 mg/L. No major changes were observed regarding the antioxidant capacity in the juices that received the antioxidant treatments, with the exception of the encapsulated grape seed extract at dose 2 (1.0 g/L), which significantly increased (p < 0.05) the average ​​ FRAP values in the time of 30 days when compared to the control. However, all treatments evaluated similarly maintained the antioxidant capacity values during the evaluated period. The main significant losses of compounds were related to anthocyanins and color caused by the storage time factor, which according to Garrido and Borges (2013) is a natural phenomenon in wine aging due to the formation of conjugated anthocyanins. In addition to anthocyanins, one of the main compounds that decreased in juices during storage was procyanidin B2, which according to Garrido and Borges (2013) decreases its concentration during aging due to oxidation and/or precipitation.

It is also noteworthy that the phenolic content of BRS Magna juice in the present study was considered high (Table 4), with emphasis on petunidin 3-glucoside (727 mg/L) and malvidin 3,5-diglucoside (492 mg/L) anthocyanins, trans-caftaric acid (129 mg/L) and procyanidin B1 (78 mg/L). These results corroborate previous phenolic characterization studies of juices of this cultivar in Brazil (Lima et al. 2014; Dutra et al. 2018, 2021; Padilha et al. 2019).

A multivariate principal components analysis (PCA) was performed in order to study the behavior of the juices that received the antioxidant treatments during storage (30, 90 and 150 days) in relation to the chemical profile, as shown in Fig. 2. In this discussion we will consider separations by variables with component loads (component loadings) > 0.70. Components 1 and 2 (PC1 and PC2, respectively) explained 66.5% of the experiment’s variance. PC1 had the highest explained variance weight (51.5%) and grouped all the juices that received the antioxidant treatments and the control at 30 days, associated with greater color intensity, less hue, and greater individual anthocyanin values. PC2 (15% of variance) separated the juices that received sulphite and encapsulated grape seed extract at a dose of 0.5 g/L (time 150 days) in the positive part associated with the highest values of trans-caftaric acid, p-coumaric and quercetin 3-glucoside. The main differentiation cause of juices was due to the storage time factor (30 days in PC1); however, PC2 showed that sulphite and encapsulated grape seed extract at dose 1 were the color stabilization treatments which presented better results due to maintaining trans-caftaric acid. According to Ntuli et al. (2020), trans-caftaric acid is an important phenolic used as a parameter to study the oxidation of grape/wine juices, as it is easily oxidized during the elaboration and storage stages, and its maintenance is a positive factor in color preservation.

The PCA result corroborated those obtained in the study of color stability during storage, demonstrating that the use of sulphite and encapsulated grape seed extract had a benefit for the color stability of whole grape juice during the 150 days of storage. Based on this, the color stabilizing capacity of the grape juices by grape seed polyphenols is evidenced, and that Brazilian grapes, especially BRS Magna and BRS Violeta cultivars, are a raw material of high technological value.

Conclusion

The Brazilian grape seeds showed high phenolic content and antioxidant capacity for the preparation of juices, with emphasis on the BRS Magna and BRS Violeta cultivars due to the high catechin, epicatechin, and procyanidins B1 and procyanidin B2 contents. The encapsulated BRS Magna grape seed extract preserved the phenolic compounds and high antioxidant capacity of the seed. The use of the encapsulated extract in grape juice improved the color stability and maintained the main anthocyanins such as petunidin 3-glucoside and malvidin 3,5-diglucoside when compared to the control and the use of enological tannin during 150 days of storage. Sulphite was considered the best color stabilizer, but it has limitations in its use in 100% natural whole grape juice. In this context, the color stabilizing capacity of natural grape juices is evidenced by the use of encapsulated Brazilian grape seed extract, with emphasis on the BRS Magna and BRS Violeta cultivars, which constitute raw material of high technological value.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

HPLC

High performance liquid chromatography

DAD

Diode arrangement detector

DPPH

2,2-Diphenyl-1-picrylhydrazyl; di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium)

FRAP

Ferric reducing antioxidant power

TPTZ

2,4,6-Tris(2-pyridyl)-s-triazine

Trolox

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

AOX

Antioxidant capacity

Author contributions

Investigation, formal analysis, writing—original draft preparation, ETSN; formal analysis, writing—original draft preparation, MCPD; formal analysis, RGBS; visualization, AJBAC; conceptualization, methodology, writing—review and editing, supervision, project administration, MdSL.

Funding

None.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors have declared that they have no conflict of interest.

Ethics approval

Consent for publication

Consent to participate

Not applicable.

Consent for publication

Not applicable.