Chemical and Biological Properties of Peach Pomace Encapsulates: Chemometric Modeling

Abstract

Background: Bioactive compounds need to resist food processing, be released from the food matrix, and be bioaccessible in the gastrointestinal tract in order to provide health benefits. Bioactive compounds isolated from peach pomace (PP) were encapsulated using four different wall materials to improve their stability and to evaluate the effects of in vitro gastrointestinal digestion, as well as chemometric modeling among obtained encapsulates. Methods: Phenolics and carotenoids content, antioxidant, antihyperglycemic, anti-inflammatory, and cell growth activities were evaluated after gastric and intestinal digestion steps. Chemometrics classification analysis–principal component analysis and hierarchical cluster analysis revealed grouping among encapsulates. Results: The encapsulation of PP bioactive compounds showed a protective effect against pH changes and enzymatic activities along digestion, and thereby contributed to an increase in their bioaccessibility in gastric and intestinal fluids. Conclusions: The obtained results suggest protein and polysaccharide carriers and the freeze-drying technique, as an efficient method for the encapsulation of bioactives from PP, could find use in the food and pharmaceutical industry.

1. Introduction

Peach (Prunus persica L.) is one of the most popular stone fruits consumed worldwide with important economic value [1]. It is known as a fruit recommended by nutritionists, rich in vitamin A and potassium [2], which also contains a wide range of phenolics and carotenoids that certainly elevate its nutritional status [1]. Daily intake of this fruit has positive effects on the reduction in generating reactive oxygen species (ROS) in human blood plasma, which gives direct positive effects on the prevention of a number of chronic diseases. Peach production is constantly growing since it is used for various products, especially juice production, resulting in the generation of a large quantity of peach production waste. Peach waste does not only represent a wasted investment but also has a negative impact on the environment, due to greenhouse gas emissions. The utilization of the food industry’s by-products, which include peach waste as a valuable source of bioactive compounds, would have a positive impact on the economy and ecology.

Overwhelming scientific research has found that a fruit and vegetable-rich diet has a positive effect on the prevention of chronic diseases. The studies showed that phytochemicals, such as polyphenolics, act as antioxidants or compounds with other therapeutic properties: anti-inflammatory, antihyperlipidemic, or anticancer agents. Phenolic compounds could act as better antioxidants than vitamins E and C, which is based on two fundamental properties which participate in their antioxidant capacities: free radical scavenging and metal chelating properties towards potentially pro-oxidant metal ions such as Fe³⁺, Al³⁺, Cu²⁺, etc., or by direct trapping reactive oxygen species [3,4]. The most widespread fat-soluble group of yellow to red pigments in nature is carotenoids. These compounds have been associated with anti-aging, anti-inflammatory, and immune system enhancement, with an ability to prevent chronic diseases, such as cancer and cardiovascular disease, in preserving visual function and protecting against age-related macular degeneration. Autoxidation of carotenoids is one of the factors that significantly restricts the incorporation of carotenoids in processed products [5].

However, polyphenols and carotenoids extracted from peach waste are unstable and it makes their usage limited. A small portion administrated orally is absorbed, because of instability in the gastric system, low permeability, and low solubility in freeform [4]. With the aim of stabilizing these molecules, microencapsulation technologies are used. Different carriers are used to encapsulate bioactive compounds and protect them from water, oxygen, light, pH, and other degradable causes [6]. The wall materials used for microencapsulation in this study are maltodextrin (M), gum Arabic (A), whey protein isolate (W), and peas protein isolate (P). In order to evaluate the potential bioaccessibility of polyphenolics and carotenoids, in vitro digestions were performed. These results provide valuable information about appropriate sources, dosage, and encapsulation techniques [7]. Vulic et al. [8] designed a study to investigate the bioavailability and bioactivity of encapsulated phenolics and carotenoids, isolated from red pepper waste, during in vitro simulated gastrointestinal digestion, using a whey protein carrier. Moreover, in this study, after simulated gastric and intestinal digestions, the amounts of phenolics and carotenoids, antioxidant (DPPH•, ABTS•+, and O₂•⁻ free radical tests), anti-inflammatory, antihyperglycemic, and anticancer activities were determined. The total phenolic and carotenoid contents, antioxidant, anti-inflammatory, and antihyperglycemic activities were tested spectrophotometrically, individual polyphenolic compounds were quantified by the HPLC method, while anticancer potentials were tested on three different cancer cell lines using the sulforhodamine B (SRB) assay. However, up to now, studies of phenolics and carotenoids isolated from peach waste and encapsulated on different carriers after in vitro digestion have not been carried out. Nowadays classification methods are widely used in food chemistry for observing differences between samples of investigated confectionery products [9], lettuce [10], beetroot [11], kombucha fermented milk products [12], etc. Principal component analysis is based on data reduction when a certain correlation among the data is present. It is a powerful pattern recognition technique used for similarity and dissimilarity detection among the samples, as well as for the grouping of samples. Scores and loadings plots are used to present the results of PCA analysis. Scores represent the new coordinates of the projected objects and loadings reflect the direction with respect to the original variables. Hierarchical cluster analysis divides a group of objects into classes so that similar objects are grouped in the same cluster [13]. In this type of analysis, objects that are close together in the variable space are searched. The results of cluster analysis are displayed as a tree diagram called a dendrogram. The horizontal axis in a dendrogram represents the distance or dissimilarity between the clusters. Clustering is based on Ward’s linkage method and Euclidean distance [14]. Another aim of the study was to apply popular chemometric tools—principal component analysis (PCA) and hierarchical cluster analysis (HCA), in order to detect similarity and dissimilarity among the encapsulates tested before and after in vitro digestions and to notice the grouping of the encapsulates.

2. Materials and Methods

2.1. Chemicals and Instruments

Folin-Ciocalteau reagent, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), β-carotene, pancreatin, pepsin, Trolox and trichloroacetic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA), ferric chloride was obtained from J.T. Baker (Deventer, Holland), and sodium nitrite from LACH-NER (Brno, Czech Republic). Other chemicals and solvents were of the highest analytical grade. Whey protein isolate was purchased from Olimp Laboratories (Debica, Poland), pea protein (Beyond, Serbia), maltodextrin (Battery Nutrition Limited, London, UK), and gum arabica (Carlo Erba Reagents, Chau. du Vexin, Val-de-Reuil, France). Distilled water was produced using a water purification system DESA 0081 Water Still distiller (POBEL, Madrid, Spain). Absorbances in spectrophotometrical assays were measured on a Multiskan GO microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). For HPLC analysis, a Shimadzu Prominence chromatographic system was used, which consisted of an LC-20AT binary pump, CTO-20A thermostat and SIL-20A autosampler connected to the SPD-20AV UV/Vis detector (Shimadzu, Kyoto, Japan). Freeze dryer, model Christ Alpha 2–4 LSC, was from Martin Christ (Osterode am Harz, Germany).

2.2. Plant Material

Peach pomace (PP) was obtained as a by-product from the beverage industry after juice production in factory “Nectar” (Bačka Palanka, Serbia). Waste material was freeze-dried, ground, packed in vacuumed plastic bags, and stored at −20 °C until further analysis.

2.3. Extraction Procedure

Freeze-dried peach pomace (2.5 g) was extracted three times using acetone:ethanol mixture (36:64 v/v) in a solid to solvent ratio of 1:10 w/v, with the same volume of solvents. The extraction was performed using a laboratory shaker (Unimax 1010, Heidolph Instruments GmbH, Kelheim, Germany) at 300 rpm, under light protection, at room temperature, for 10 min. The obtained three extracts were filtered (Whatman paper No.1), combined, and stored in dark bottles at −20 °C until further analysis.

2.4. Encapsulation Process

Four carrier agents, including whey protein (W), pea protein (P), maltodextrin (M), and gum arabica (A) were used as wall materials for encapsulation of bioactive compounds from PP extract. Freeze-dried encapsulates were prepared following the method described by Šeregelj et al. [15] with some modifications. Each carrier agent (7 g) was dissolved in 10 mL of water at 60 °C and kept under stirring until the temperature reached 30 °C, except for pea protein which was dissolved in the same way in 30 mL of water. Separately, 50 mL of P extract was combined with sunflower oil (1.5 mL), concentrated under reduced pressure on a rotary evaporator set at 40 °C to remove the organic solvent, and immediately mixed with previously prepared carrier solution. The mixtures were homogenized at 11,000 rpm for 3 min at room temperature and iced overnight at −20 °C. All samples were freeze-dried at −40 °C for 48 h. Collected encapsulates (PPW, PPP, PPM, and PPA) were stored at −20 °C until further use.

2.5. HPLC Analysis of Encapsulated Phenolic Compounds

For HPLC analysis of phenolic compounds, two mobile phases, A (acetonitrile) and B (1% formic acid), were used at flow rates of 1 mL/min with the following gradient profile: 0–10 min from 10% to 25% A; 10–20 min linear rise up to 60% A, and from 20 min to 30 min linear rise up to 70% A, followed by 10 min reverse to initial 10% A with additional 5 min of equilibration time. Reference substances were dissolved in 50% methanol. Phenolic compounds were recorded using different wavelengths: 280 nm for hydroxybenzoic acids, 320 nm for hydroxycinnamic acids, and 360 nm for flavonoids [16].

2.6. In Vitro Simulated Gastrointestinal Digestion

In vitro digestion of encapsulates was determined by simulation of digestion in gastric fluid (SGF) and intestinal fluid (SIF) according to the method described by Vulic et al. [7]. The first step of digestion was treating encapsulates (2.5% water suspension) with pepsin at pH 2.0 and 37 °C for 1 h. After gastric digestion, the pH of the solution was adjusted to 7.5, pancreatin was added and the solution was stirred at the same conditions for 2 h. At the end of each step of digestion, the sample aliquots were concentrated on a rotary evaporator and dissolved in acetone:ethanol mixture (36:64 v/v) or hexane. In this way, lipophilic and hydrophilic fractions were obtained and analyzed for bioaccessibility and bioactivity of encapsulated bioactive compounds isolated from PP.

2.7. Bioaccessibility of Encapsulated Bioactive Compounds during In Vitro Simulated Gastrointestinal Digestion

Spectrophotometric analysis of total phenolics (TPh) in hydrophilic fractions was performed by the Folin-Ciocalteau method adapted to microscale. Results were expressed as gallic acid equivalents (GAE) per 100 g of encapsulates. Spectrophotometric analysis of total carotenoids (TCar) in lipophilic fractions was performed by the method of Nagata and Yamashita [17] and the results were expressed as mg of β-carotene equivalents per 100 g of encapsulates.

2.8. Bioactivity of Encapsulated Bioactive Compounds during In Vitro Simulated Gastrointestinal Digestion

The antioxidant activity, expressed as μmol Trolox equivalent (TE) per 100 g of encapsulates, was performed by three methods: 2,2-diphenyl-1-picrylhydrazyl method (DPPH) described by Girones-Vilaplana et al. [18], reducing power (RP) by Oyaizu [19], superoxide anion assay (SOA) by Girones-Vilaplana et al. [20], and β-carotene bleaching assay (BCB) by Al-Shaikan et al. [21]. The SA, RP, and SOA were measured with hydrophilic fractions, while BCB was performed with hexane fractions. Antihyperglycemic activity (AHgA) was determined by measuring α-glucosidase inhibitory potential following the Tumbas Šaponjac et al. [15] method. In vitro assessment of anti-inflammatory activity (AIA) was determined by protein denaturation bioassay, according to the method adopted by Ullah et al. [22]. Diclofenac sodium was used as a drug reference.

2.9. Antiproliferative Activity

Human cell lines: MCF7 (breast adenocarcinoma), MRC-5 (normal fetal lung fibroblasts), and HT-29 (colon adenocarcinoma) were used for the estimation of antiproliferative effects of four peach pomace encapsulates. Cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; PAA Laboratories GmbH, Pashing, Austria) with 4.5% glucose, supplemented with 10% heat-inactivated fetal calf serum (FCS; PAA Laboratories GmbH, Pashing, Austria), 100 IU mL⁻¹ of penicillin, and 100 µg mL⁻¹ of streptomycin (Galenika, Belgrade, Serbia). All investigated cell lines were grown attached to the surface. They were cultured in 25 cm³ flasks (Corning, New York, NY, USA) at 37 °C in an atmosphere of 5% CO₂, high humidity, and subcultured twice a week. A single-cell suspension was obtained using 0.1% trypsin (Serva, UK) with 0.04% EDTA.

2.9.1. Samples Used in the Antiproliferative Assay

For the analysis of antiproliferative effects, four PP encapsulates were dissolved and diluted in DMSO to obtain the required final concentrations. The final concentration of extracts was in the range of 32.5–1000 µg/mL, whereas the final concentration of DMSO in the samples was ≤0.05% (v/v).

2.9.2. Sulforhodamine B (SRB) Assay

Cell lines were harvested and plated into 96-well microtiter plates (Sarstedt, Newton, NC, USA) at a seeding density of 4 × 10³ cells per well, in a volume of 199 µL, and preincubated in complete medium supplemented with 5% FCS, at 37 °C for 24 h. Serial dilutions of extracts or solvent (1 µL per well) were added to the test and control wells, respectively. Microplates were then incubated at 37 °C for an additional 48 h. Cell growth was evaluated by the colorimetric SRB assay according to Skehan et al. (1990) [23]. Cells were fixed with 50% TCA (1 h, +4 °C), washed with distilled water (Wellwash 4; Labsystems; Helsinki, Finland), and stained with 0.4% SRB (30 min, room temperature). The plates were then washed with 1% acetic acid to remove the unbound dye. Protein-bound dye was extracted with 10 mM Tris base. Absorbance was measured on a microplate reader (Multiscan Ascent, Labsystems) at 540⁄620 nm. Antiproliferative activity, that is, the effect on cell growth, was expressed as a percent of the control and calculated as: % Control = (At⁄Ac)·100 [%], where At is the absorbance of the test sample and Acis the absorbance of the control.

2.10. Statistical Analysis

All experiments were run in triplicate. The results represented are the means ± standard deviation (±SD, n = 3). Statistical analyses were carried out using Origin 8.0 SRO software package and Microsoft Office Excel 2010 software. Significant differences were calculated by ANOVA (p < 0.05). Experimentally obtained data for four different encapsulates regarding their bioactivity before and after in vitro digestions were subjected to PCA and HCA analysis. Prior to the analysis, all experimental data were normalized using the min-max normalization method [24]. PCA and HCA were performed using Statistica v 10.0 software [25].

3. Results

3.1. HPLC Analysis of Individual Phenolic Contents in Peach Pomace Encapsulates

The results of individual phenolic contents determined by HPLC analysis are presented in

Table 1. Phenolics content in four peach encapsulates before digestion identified and quantified by HPLC.

Compound (mg/100 g)	PPW	PPP	PPM	PPA
Epicatechin	0.41 ± 0.01 c	0.18 ± 0.01 b	0.15 ± 0.01 a,b	0.86 ± 0.03 d
Catechin	10.26 ± 0.49 d	6.65 ± 0.31 c	1.99 ± 0.08 a	3.82 ± 0.17 b
Caffeic acid	0.51 ± 0.02 d	0.02 ± 0.00 a	0.19 ± 0.01 c	0.09 ± 0.00 b
p-coumaric acid	0.26 ± 0.01 b	0.48 ± 0.02 c	2.01 ± 0.01 d	0.13 ± 0.00 a
Chlorogenic acid	2.17 ± 0.09 d	0.63 ± 0.02 b	1.31 ± 0.06 c	0.11 ± 0.00 a
p-hydroxybenzoic acid	22.02 ± 1.00 d	4.52 ± 0.22 c	0.50 ± 0.02 a	0.87 ± 0.04 b
Rutin	0.15 ± 0.00 b	0.20 ± 0.00 d	0.08 ± 0.00 a	0.18 ± 0.01 c
Quercetin	0.23 ± 0.00 c	0.31 ± 0.01 d	0.06 ± 0.00 a	0.11 ± 0.00 b
Total	36.01 d	12.99 c	6.29 a	6.17 b

The results are presented as the mean value ± standard deviation (n = 3); Values sharing the same letter in the same raw are not significantly different at the 0.05 level; PPW: peach pomace whey protein; PPP: peach pomace pea protein; PPM: peach pomace maltodextrin; PPA: peach pomace gum arabica.

.

Data from the HPLC analysis revealed the presence of three phenolic acids (p-coumaric, caffeic, and chlorogenic acid), and four flavonoids (catechin, epicatechin, rutin, and quercetin) in all four peach pomace encapsulates. HPLC results showed the highest TPh in PPW.

Similar phenolic and flavonoid profiles of peach have also been reported in different studies. Stojanovic et al. [26] published a study where peach pulp and peel differed significantly in their phenolic profiles: the pulp contained mainly chlorogenic, neochlorogenic, and p-coumaric acids, whereas the peel possessed chlorogenic, neochlorogenic, and p-coumaric acids together with several flavonol glycosides in large amounts. In the study by Mokrani et al. [27], cinnamic acids were represented by two compounds: neochlorogenic and chlorogenic acids. Chlorogenic acid was the main compound of this class of phenolics in all cultivars. The maximum level of chlorogenic acid was found in the Spring Belle peach cultivar (13.1 mg/g extract). Six peach and six nectarine cultivars were evaluated for the phenolic content in their pulp and peel tissues in the paper by Andreotti et al. [28]. The authors of this paper detected chlorogenic acid, catechin, epicatechin, rutin, and cyanidin-3-glucoside as the main phenolic compounds of ripened peach and nectarine fruits. Moreover, our HPLC results showed the presence of the same phenolic compounds groups as in the available literature.

3.2. Total Phenolic (TPh) and Carotenoids Content (TCar)

Total phenolic and carotenoid contents results, before and after simulated in vitro gastrointestinal digestion, are presented in

Table 2. Spectrophotometric analysis of bioactive compounds content before and after in vitro gastrointestinal digestion.

Compounds	Before Digestion		SGF		SIF
	TPh A	Tcar B	TPh A	Tcar B	TPh A	Tcar B
PPW	543.05 ± 23.73 b	132.77 ± 9.98 a	3580.23 ± 44.30 d	32.08 ± 1.02 b	2242.99 ± 20.89 d	113.03 ± 1.27 c
PPP	818.62± 34.02 c	126.46 ± 2.72 a	2369.36± 22.98 c	27.23± 2.02 a	1846.02± 36.97 c	123.30 ± 2.30 d
PPM	251.13 ± 2.04 a	105.25 ± 2.21 a	1038.11± 62.44 b	33.02 ± 0.40 b	911.91 ± 21.12 b	103.80 ± 1.06 b
PPA	624.98 ± 94.20 b	128.34 ± 33.62 a	454.28 ±12.19 a	52.31 ± 1.13 c	431.54± 0.18 a	59.04 ± 2.37 a

Data present the mean value of three replicates ± standard deviation (n = 3); ^A expressed as mg GAE/100 g; ^B expressed as mg β-carotene/100 g; values for TPh or TCar sharing the same letter in the same raw are not significantly different at the 0.05 level; PPW: peach pomace whey protein; PPP: peach pomace pea protein; PPM: peach pomace maltodextrin; PPA: peach pomace gum arabica.

. Simulated gastrointestinal digestion was monitored in simulated gastric (SGF) and intestinal (SIF) conditions. After simulated in vitro digestion, contents of phenolics increased for all encapsulates, compared to samples before digestion, except for PPA. The results presented in Table 1 show that before in vitro digestion the highest TPh was observed for PPP, while the lowest value was determined in PPM. Namely, PPW, PPG, and PPM exhibited higher TPh values after simulated digestions, while for PPA, higher TPh was observed before digestion. After in vitro digestion, PPW_SGF had the highest TPh content (3580.23 mg GAE/100 g). However, TCar was higher for all samples before simulated gastrointestinal digestion. There was no statistical difference (p < 0.05) between TCar for all samples before digestion, where the highest value was observed for PPW (132.77 mg β-carotene/100 g). The highest value after in vitro digestion was observed for PPP_SIF (123.30 mg β-carotene/100 g).

3.3. Antioxidant Activity

Natural bioactive compounds from peach pomace are considered to exert health beneficial properties mainly through antioxidant activities. There is no single chemical assay that can accurately evaluate the contribution of hydrophilic and lipophilic compounds to the total antioxidant activity of the plants and the antioxidant activity of these compounds might be affected by the chemical transformations resulting during gastrointestinal digestion. The antioxidant activities of four peach waste encapsulates before and after simulated in vitro gastrointestinal digestion are presented in

Table 3. Antioxidant activity of encapsulates before and after in vitro gastrointestinal digestion.

Compounds	DPPH A	RC A	SOA A	BCB B
Before Digestion
PPW	1059.94 ± 29.38 c	1673.08 ± 26.08 c	274.32 ± 2.67 d	140.66 ± 2.86 a,b
PPP	761.58 ± 13.18 a	763.06 ± 4.21 a	32.95 ± 0.52 a	132.93 ± 2.88 a
PPM	1039.86 ± 11.45 b,c,d	1648.63 ± 41.42 c	218.69 ± 3.95 c	129.48 ± 2.83 a,b,c
PPA	1062.77 ± 25.42 d,c	1134.78 ± 17.09 b	56.31 ± 1.93 b	139.91 ± 4.88 a,c
SGF
PPWsgf	1233.17 ± 59.09 d	2607.42 ± 7.74 d	79.71 ± 2.34 a	135.21 ± 1.65 b
PPPsgf	1012.23 ± 85.23 c	1406.54 ± 95.27 c	102.88 ± 7.06 b	134.79 ± 4.88 b
PPMsgf	542.03 ± 18.42 b	536.51 ± 11.71 b	215.79 ± 1.96 d	139.43 ± 0.73 c,b
PPAsgf	369.24 ± 13.29 a	102.77 ± 1.38 a	104.38 ± 7.81 c	129.51 ± 0.01 a,b
SIF
PPWsif	661.44 ± 14.86 b	657.11 ± 6.48 d	250.88 ± 5.14 c	139.88 ± 2.37 a
PPPsif	717.27 ± 23.51 c	292.89 ± 17.28 c	254.21 ± 2.97 c	137.69 ± 0.01 a
PPMsif	348.87 ± 19.53 a	175.23 ± 5.85 b	224.88 ± 8.87 b	139.78 ± 0.57 a
PPAsif	347.69 ± 12.18 a	83.67 ± 3.42 a	209.21 ± 3.67 a	139.51 ± 0.38 a

Data present the mean value of three replicates ± standard deviation (n = 3).; ^A expressed as µmol Trolox equivalents (TE)/100 g encapsulate; ^B expressed as percent of inhibition relative to control; values for the same test and sample sharing the same letter in the same column are not significantly different at the 0.05 level; PPW: peach pomace whey protein; PPP: peach pomace pea protein; PPM: peach pomace maltodextrin; PPA: peach pomace gum arabica.

.

The samples were tested against DPPH radicals and the obtained results showed that DPPH• scavenging activity of SIF samples was higher for all samples compared to SGF and samples before digestion. Moreover, the results of the RC assay showed better results for SIF than SGF samples. The sample with the highest activity in both assays was PPW and the same sample showed the highest TPh content in both spectrophotometric and HPLC methods. In contrast, SOA and BCB assays showed that SIF samples had higher activities compared to SGF samples. The increases in SOA were higher for encapsulates with protein wall material than polysaccharide wall material. However, the best-obtained results in the SOA test were for the PPW sample, where the activity increased 68.2% after SGF. The BCB results correspond to slightly higher values for SIF than SGF samples. The PPA showed the highest BCB activity with a 7.2% increase.

Enzymatic reactions and different pH conditions, during simulated digestion, lead to decreasing molecule size [7]. This could explain the increase in SA after SGF and SIF in RC and BCB tests, as well as the higher phenolics amounts released for all samples, except for PPA.

3.4. Antihyperglycemic Activity (AHgA)

Hyperglycemia in type two diabetes is a consequence of insulin deficiency [29]. Enzyme inhibitors such as α-glucosidase and α-amylase play a role in the management of postprandial hyperglycemia for diabetics [30]. The antihyperglycemic activity (AHgA) was determined as the potential of peach encapsulates to inhibit the enzyme α-glucosidase. AHgA of peach encapsulates before and after simulated gastric and intestinal digestions are presented in Figure 1. It was shown that the SGF exhibited higher values compared to SIF for all encapsulates except PPM, where no significant difference was observed between SGF and SIF values. In our study, the highest AHgA values for SGF (76.34%) and SIF (47.87%) were observed for PPW.

3.5. Antiinflammatory Activity (AIA)

Antidenaturation of the egg albumin method was chosen to evaluate the anti-inflammatory activity of four different peach pomace encapsulates (Figure 2). Protein denaturation is a process in which proteins lose their tertiary structure and secondary structure by the application of external stresses or compounds such as strong acid or base inorganic salts, organic solvents, or heat. Namely, most biological proteins lose their biological function after denaturation [31]. The denaturation of proteins is a well-documented cause of inflammation. In our study, diclofenac sodium was used as a positive control, as a standard anti-inflammatory drug. Therefore, the search for natural sources of bioactive compounds with anti-inflammatory activity has greatly increased. In this study, maximum inhibition of 83.88% was observed after SGF digestion of PPP at a concentration of 30 mg/mL. However, the AIA of diclofenac sodium at a concentration of 20 mg/mL was 77.47%.

Since these encapsulates are designed for possible incorporation into food or pharmaceutical products, it is essential that release be controlled by the applied wall material during gastrointestinal digestion, to preserve their biological activities and protect them from degradation.

3.6. Antiproliferative Activity

The antiproliferative activity of four peach pomace encapsulates was investigated using three cancer cell lines (MCF7, HT-29, and MRC-5). PPW_SIF showed the strongest antiproliferative effects on the MCF7 (IC50MCF7 = 47.39 μg/mL) and HeLa (IC50HeLa = 34.89 μg/mL) cell lines (

Table 4. Antiproliferative activity of four peach encapsulates.

Sample	IC50 (μg/mL)
	MCF7	HT-29	MRC-5
PPASGF	>500	>500	>500
PPASIF	214.00 ± 35.21	185.25 ± 7.24	163.15 ± 3.17
PPMSGF	>1000	>1000	714.24 ± 96.11
PPMSIF	135.29 ± 30.36	109.21 ± 4.20	<62.5
PPWSGF	111.97 ± 3.12	101.85 ± 24.30	66.10 ± 0.86
PPWSIF	47.39 ± 3.07	34.89 ± 1.61	<62.5
PPPSGF	>1000	>1000	>1000
PPPSIF	91.15 ± 6.16	81.13 ± 2.53	<62.5

Data present the mean value of three replicates ± standard deviation (n = 3); investigated in 32.5–1000 μg/mL concentration range. PPW: peach pomace whey protein; PPP: peach pomace pea protein; PPM: peach pomace maltodextrin; PPA: peach pomace gum arabica.

). The highest antiproliferative effect on the MRC-5 cell line was obtained by PPM_SIF, PPW_SIF, and PPA_SIF. It could be observed that all peach encapsulates showed better antiproliferative results after intestinal, compared to gastric in vitro digestion.

4. Discussion

4.1. Bioactive Compounds Contents and Antioxidant Activity

Since peach waste encapsulates were prepared with different wall materials, and designed for possible incorporation into food products, it is essential that present bioactive compounds (phenolics and carotenoids) during gastrointestinal digestion preserve their biological activities and be protected from degradation. It is known that the biological activity of carotenoids may be reduced due to their relatively low chemical stability and bioaccessibility in the gastrointestinal tract. Fu et al. [32] showed that polysaccharide coating improved emulsion aggregation stability in the gastric phase, which further led to a larger surface area for the pancreatic lipase molecules to attach to. The results of our study are in accordance with the previously mentioned theory, where TCarSIF increased by 11.4% compared to TCarSGF for PA encapsulates, while the highest increase in TCarSGF had PP with an increase of 77.9%. This increase might be due to heat-induced protein–protein interactions and aggregation on proteins at low pH in the gastric environment.

The changes of bioactive compound contents were affected by the digestion processes. These results are in accordance with the literature, which also found differences in the release of the bioactive component from encapsulates exposed to simulated gastrointestinal fluids [33]. The used wall material has an important role in bioactive compounds released during gastrointestinal digestion. The spectrophotometric analysis results presented in Table 1 revealed that after intestinal digestion, PWSIF, PPSIF, and PMSIF had TPh values higher than TPh before digestion, while PASIF showed a decrease in TPh. Ćujić Nikolić et al. [34] showed that gastrointestinal digestion caused a decrease in the content of phenolics chokeberries extract encapsulated with gum Arabica as wall material after simulated intestinal digestion. In the same study, TPh after intestinal digestion was 6145 mgGAE/100 g, while in our study, TPh in PASIF was 431.54 mg GAE/100 g. In contrast, Işık et al. [35] studied the impact of wall material containing carbohydrates, such as gum Arabica, and demonstrated a high and immediate release rate of encapsulated phenols after simply adding the gastric enzyme pepsin with the relatively high water solubility of these materials. Possibly, contact with water may destroy the structure of the particles. Moreover, these results are in agreement with the study on the various phenolics, anthocyanidins, and antioxidant effects in strawberry grape after simulated gastrointestinal digestion.

The DPPH radical scavenging assay is a widely used assay to determine the antioxidant activity of phenolic compounds. It is based on the capacity of stable DPPH free radicals to react with hydrogen donors. Pazinatto et al. [36] reported digestive enzymes were able to change the chemical structure of soluble and insoluble components leading to an increase in their availability on the matrix surface, thus giving their reducing properties to DPPH radicals. Phenolics have been reported to be responsible for the antioxidant activities of plant extracts. These compounds are sensitive to pH in the small intestine conditions, which leads to their transformation into compounds with different structural forms and chemical properties [37]. This could explain a decrease in DPPH radical scavenging after gastric digestion and it is also correlated to a decrease in TPhSIF content compared to SGF content for all samples, except for PPA. However, PPW showed a higher decrease in DPPH radical scavenging activity during intestinal digestion (46.4%). Balhadj et al. [38] reported significant DPPH radical scavenging activity of peach extract (591.94 TE/gFW).

The principle of the reducing power assay is based on the reduction potential for a compound to react with potassium ferricyanide (Fe³⁺) to form potassium ferrocyanide (Fe²⁺), which further reacts with ferric chloride to form a ferric–ferrous complex with an absorption maximum at 700 nm. In our study, peach pomace encapsulates with different wall materials showed a greater decrease in reduction potential in SIF compared to SGF samples. The highest RP was observed in PPW_SGF (2607.42 ± 7.74 TE/100 g encapsulate) with a total decrease of 77.1%. However, these assays decreased values for SGF and SIF samples which are in accordance with the presented results in Table 2 for TPh content and Table 3 for DPPH activity.

When the superoxide anion scavenger capacity was evaluated, all SIF samples had higher values than SGF samples. The most reactive oxygen species is the hydroxyl radical, in which its formation can be accelerated by the superoxide radical, which cannot directly initiate oxidative reaction [39]. Therefore, the good superoxide scavenging ability of PPSIF might contribute to its antioxidant potential. Cardounel et al. [40] demonstrated direct scavenging of the superoxide anion by carotenoid derivatives. The PPSIF showed the best SOA (254.21 μmolTE/100 g) which could be correlated to released carotenoids in this phase (Table 2). Previous studies with peptides that contained histidine showed that these peptides potentially have active-oxygen quencher, hydroxy radical scavenger, and metal-ion chelating activity. The nature and composition of the different peptide fractions produced during the digestion process dictate the potential ability of protein hydrolysates to inhibit some changes caused by lipid oxidation [41].

The antioxidant activity of carotenoids is based on the radical adducts of carotenoids with free radicals from linoleic acid. The highly unsaturated β-carotene models have been attacked by linoleic acid-free radicals. The presence of different antioxidants can hinder the extent of β-carotene bleaching by neutralizing the linoleate-free radical and other free radicals formed in the system [42]. The antioxidant activity of tested samples before and after in vitro digestion could be directly related to the released carotenoids during the digestive process. All SIF samples showed higher BCB compared to SGF, with no significant differences (p < 0.05). The PPW and PPA exhibited the highest activity among all the tested samples, while the PpG sample showed the highest increase in BCB (7.2%), followed by PPW with a 3.3% increase in β-carotene bleaching activity.

4.2. Antihyperglycemic, Anti-Inflammatory and Antiproliferative Activity

Insulin deficiency causes exaggerated glucose production from the liver, reduced insulin-mediated glucose uptake from muscle, and increased free fatty acid mobilization from adipose tissue [43]. The dietary recommendations for the prevention of obesity, hepatic steatosis, and insulin resistance include the consumption of dietary fiber and polyphenol-rich sources [44,45]. The novel approach to overcome diabetes mellitus complications is aimed at avoiding synthetic drugs due to their high price and considerable clinical side effects. It is reported that plants have great potential to retard the absorption of glucose by inhibiting saccharide-hydrolyzing enzymes; therefore, in recent years, their screening and isolation have grown (Husain et al., 2018). According to Olocoba et al. [46], there is a direct relationship between phenolic compounds, flavonoids, and condensed tannin in plant extracts and their ability to inhibit α-glucosidase activity. Noratto, Martino, Simbo, Byrne, and Mertens-Talcott [47] reported that peach juice rich in phenolic acids improved glucose and insulin levels in the plasma of Zucker rats.

Inflammatory diseases include different types of rheumatic diseases characterized by pain, swelling, and disturbed physiological functions. The greatest disadvantage in today’s used synthetic anti-inflammatory drugs lies in their gastric irritation leading to the formation of gastric ulcers, toxicity, and the reappearance of symptoms after discontinuation [31]. All tested peach samples exhibited significant anti-inflammatory activity, whereas encapsulates with protein wall materials showed slightly better results.

All peach encapsulates showed better antiproliferative results on three human cell lines: MCF7 (breast adenocarcinoma), MRC-5 (normal fetal lung fibroblasts), and HT-29 (colon adenocarcinoma) after intestinal, compared to gastric in vitro digestion. In the study by Sun et al. [48], antiproliferative activities of fruit extracts on the growth of HepG2 human liver cancer cells in vitro were examined. Among the 11 selected common fruits, phytochemical extracts of peach showed a weak antiproliferative activity at higher doses with an EC50 of 156.3 mg/mL. The cancer suppression activity of extracts from a commercial variety of yellow-fleshed peach Rich Lady effectively inhibited the proliferation of the estrogen-independent MDA-MB-435 breast cancer cell line. The obtained IC50 value was 42 mg/L for this cell line compared to an IC50 of 130 and 515 mg/L for the noncancerous breast cell line MCF-10A and the estrogen-dependent breast cancer cell line MCF-7, respectively [49]. Rodríguez-González et al. [50] published a study where obtained results suggested that peach (Prunus persica L.) juice by-product exerts a greater beneficial effect on obesity-related complications than dietary fiber. The authors concluded that those effects were associated with caffeoylquinic, p-coumaroylquinic, and 4-feruloylquinic acids, and kaempferol derivatives.

Today, the major goal in food waste management is to establish new technologies to reduce and reuse generated waste material as a source of bioactive compounds with health-promoting properties. Encapsulated peach pomace, a food industry by-product, investigated on total and individual phenolics and carotenoids, antioxidant, antihyperglycemic, anti-inflammatory, and antitumor activities, showed good potential for further utilization in functional food systems. Additionally, the good choice of used wall materials may represent an additional improvement in the nutritional value of the final product. These results contribute to the development of new food health ingredients with possibly improved bioaccessibility, increased stability, and controlled release.

Fourier transform infrared (FTIR) analysis is a fundamental method in identifying the molecular structure and observing functional groups’ shifting and/or stretching. Azad et al. [51] showed the cross-linking between compounds and biopolymers or structural modification in the functional groups in purple potato. The obtained peaks were assigned to the functional group of phenolic compounds, confirming a good cross-linking between bioactive compounds and the carrier. The surface characteristics of the same formulation have been observed by SEM (scanning electron microscopy analysis). Non-crystalline, super-porous, and multiple water channels were noticed in the formulation of purple potato, lecithin, and ascorbic acid, indicating the good miscibility of used biopolymers. Such super-porous structures significantly increase the penetration and permeation of water into the core matrix, resulting in a high swelling rate and faster dissolution [52]. In the study by Adnan et al. [52], kenaf seed formulation SEM results showed the formation of multiple pores, etching, and perforations. This observation suggested that after used encapsulation more water channels were produced, leading to swelling and high water absorption by the rapid permeation and penetration of water into the core matrix. In the same study, FTIR recorded many functional groups in kenaf seed, showing the presence of various vital metabolites. Moreover, no degradation of phenolic or bioactive compounds and only physical crosslinks were involved between kenaf seed and utilized carriers.

4.3. Chemometric Modeling

The PCA analysis resulted in a two-component model that covered 72.89% of the total variance—PC1 described 52.27% and PC2 20.62%. The obtained results are considered through the loadings (Figure 3a) and scores (Figure 3b) plots. On the basis of the loadings plot (Figure 3a), it can be noticed that the distribution of the encapsulates along the dominant PC1 axis is achieved based on the AHgA, DPPH, and RC values that all have a very similar influence on the position of the investigated encapsulates on the scores plot. All three bioactivity values have a negative coefficient of latent variables regarding the PC1 axis. Considering the scores plot (Figure 3b), the encapsulates placed closer to the negative end of the PC1 axis (group A—PPASGF, PPPSGF, PPWSGF, PPA, PPP, PPM, and PPW) generally have higher values of AHgA, DPPH, and RC, while encapsulates placed closer to the positive end of the PC1 axis (group B—PPASIF, PPMSIF, PPPSIF, PPWSIF, and PPMSIF) generally have lower values of AHgA, DPPH, and RC. Moving along the PC2 axis, from the loadings plot (Figure 3a) it can be noticed that the distribution of the encapsulates is conditioned based on the BCB and SOA values that have a similar influence on the position of the investigated encapsulates on the scores plot. Both of these values have a positive coefficient of latent variables regarding the PC2 axis. From the scores plot (Figure 3b), it can be observed that the encapsulates placed closer to the negative end of the PC2 axis generally have lower values of BCB and SOA, while encapsulates placed closer to the positive end of the PC2 axis generally have higher values of BCB and SOA.

The HCA analysis confirmed the results obtained with PCA analysis regarding the dominant PC1 axis. The obtained clustering is shown as a dendrogram (Figure 4) that presents two main clusters (A and B) with subcluster (A1 and A2). Cluster A contains seven encapsulates (three in A1 subcluster and four in A2 subcluster), while cluster B consists of five encapsulates. These two clusters are very well separated, indicating that there is a significant difference between the separated groups of encapsulates. On the basis of the results, it can be concluded that encapsulates with generally higher values of DPPH, RC, and AHgA and lower values of SOA, BCB, and AIA can be found in cluster A. In this cluster, there are two subclusters: A1 containing PPASGF, PPPSGF, and PPWSGF and A2 containing PPA, PPP, PPM, and PPW encapsulates. Therefore, generally speaking, the majority of the analyzed encapsulates that were subjected to the in vitro gastric digestion and encapsulates tested before digestion have high values of DPPH, RC, and AHgA (as desired bioactivity profiles) in comparison with encapsulates that were subjected to the in vitro intestinal digestion positioned in cluster B (PPASIF, PPMSGF, PPPSIF, PPWSIF, and PPMSIF).

Applied classification methods PCA and HCA contributed to the grouping and emphasized some differences between the examined encapsulates tested before and after in vitro digestions.

5. Conclusions

In this study, valuable bioactive compounds were isolated from PP and encapsulated using four different wall materials to improve their stability, and to evaluate the effects of in vitro gastrointestinal digestion. Antioxidant, antihyperglycemic, anti-inflammatory, and cell growth activities of peach waste were evaluated after each digestion step. Moreover, chemometrics classification analysis among encapsulates was examined before and after in vitro digestions.

Overall, it could be concluded that PP, a rich source of phenolics and carotenoids, encapsulated using protein and polysaccharide carriers, as well as the freeze-drying technique, could find a possible use in the food and pharmaceutical industry, as a value-added additive or as a dietary supplement. In addition, the encapsulation of bioactive compounds present in PP could prolong the shelf-life and storage period of enriched products, better appearance, health benefits, and generally better acceptability among consumers.