**Colon Bioaccessibility under In Vitro Gastrointestinal Digestion of a Red Cabbage Extract Chemically Profiled through UHPLC-Q-Orbitrap HRMS**

**Luana Izzo 1,\*, Yelko Rodríguez-Carrasco 2, Severina Pacifico 3, Luigi Castaldo 1, Alfonso Narváez <sup>1</sup> and Alberto Ritieni 1,4**


Received: 4 September 2020; Accepted: 2 October 2020; Published: 6 October 2020

**Abstract:** Red cabbage is a native vegetable of the Mediterranean region that represents one of the major sources of anthocyanins. The aim of this research is to evaluate the antioxidant capability and total polyphenol content (TPC) of a red cabbage extract and to compare acquired data with those from the same extract encapsulated in an acid-resistant capsule. The extract, which was qualitatively and quantitatively profiled by UHPLC-Q-Orbitrap HRMS analysis, contained a high content of anthocyanins and phenolic acids, whereas non-anthocyanin flavonoids were the less abundant compounds. An in vitro gastrointestinal digestion system was utilized to follow the extract's metabolism in humans and to evaluate its colon bioaccessibility. Data obtained showed that during gastrointestinal digestion, the total polyphenol content of the extract digested in the acid-resistant capsule in the Pronase E stage resulted in a higher concentration value compared to the extract digested without the capsule. Reasonably, these results could be attributed to the metabolization process by human colonic microflora and to the genesis of metabolites with greater bioactivity and more beneficial effects. The use of red cabbage extract encapsulated in an acid-resistant capsule could improve the polyphenols' bioaccessibility and be proposed as a red cabbage-based nutraceutical formulation for counteracting stress oxidative diseases.

**Keywords:** red cabbage; in vitro gastrointestinal digestion; antioxidants; acid-resistant capsule; bioaccessibility; UHPLC-Q-Orbitrap HRMS

#### **1. Introduction**

Red cabbage (*Purple Brassica oleracea* L. var. *capitata F. rubra*) is a native vegetable of the Mediterranean region that originated in Europe in the 16th century and nowadays is largely consumed worldwide. Among different vegetables in the human diet, it represents one of the major sources of polyphenolic compounds, especially anthocyanins [1]. Anthocyanins are natural glycoside compounds belonging to the flavonoids group, and they are mainly responsible for the colors of fruits, vegetables, and flowers. The flavylium (2-phenylchromenylium) ion represents the basic molecular skeleton of anthocyanins, whose sugar-free components are anthocyanidin aglycones. Among naturally anthocyanidins known until now, merely six principal types are common in fruit and vegetables, which mainly differ in the oxygenation degree of the flavonoid B-ring (e.g., hydroxyl and/or methoxyl

groups). Thus, anthocyanins variability in plants is mainly due to the sugar moieties identity and number as well as to the diversity in acylated substituents [both aromatic (largely hydroxycinnamic acids, and/or simple C6C1 acids) or aliphatic acids (e.g., malonic acid)], which could be linked to the anthocyanin core or directly to the anthocyanidin nucleus. Particularly, the red cabbage matrix contains cyanidin 3-diglucoside-5-glucoside derivatives highly conjugated with sugars such as glucose and xylose and acyl groups including caffeoyl, *p*-coumaroyl, feruloyl, *p*-hydroxybenzoyl, sinapoyl, and oxaloyl [2,3].

The overproduction of reactive oxygen and nitrogen species (ROS/RNS) could occur in living organisms at an uncontrolled rate, defining oxidative stress condition onset, which is correlated with various forms of health damage including chronic age-related diseases, atherosclerosis, carcinogenesis, and neurodegenerative disorders. A healthy nutrition, mainly based on fruits and vegetables, is suggested to delay or positively modulate the dynamic balance between oxidants and antioxidants, thanks to plant foods diversity in bioactive compounds, such as polyphenols. Polyphenols are exogenous antioxidant compounds that are able to prevent and/or inhibit the genesis of pathophysiological perturbations in redox circuitry. According to Sies et al. [4], the oxidative stress represents "a disturbance in the pro-oxidant–antioxidant balance in favor of the former, leading to potential damage". Anthocyanins are known to have a wide range of health-promoting properties for human health including cytoprotective activity, which might be due to the ability of anthocyanins to decrease cell death, lactate dehydrogenase (LDH) release, caspase 3 activation, and DNA damages [5]. Moreover, scientific reports support an increase in the cytoprotective effect as a result of the anthocyanin-rich diet [6].

To fully preserve or augment polyphenols bioactivity and achieve an efficient therapeutic activity, these compounds often need to be formulated into bioavailable dosage nutraceutical forms [7,8]. Indeed, new formulations by using polyphenols from dietary plants are continuously investigated also for safeguarding polyphenol chemical features that could be compromised during their metabolism fates in humans. This is particularly true for anthocyanins and anthocyanidin compounds, which are highly instable and very sensitive to degradation by oxygen, temperature, light, enzymes, and pH, and whose availability after gastrointestinal digestion could limit their beneficial effect on health.

The correlation of the anthocyanins' consumption and beneficial effects on human health has been reported by many scientific studies and includes sundry protective effects on human health such as antioxidant, anti-inflammatory, anticancer activity, and effects on the cardiovascular, neurodegenerative, and metabolic systems [9–11]. To confer beneficial health effects, bioactive compounds need to be bioavailable and reach, after gastrointestinal (GI) digestion, target tissues in the human body. Although fruits and vegetables are an abundant source of polyphenols and other bioactive compounds, the available amount of these substances after small intestine digestion is significantly reduced [12].

Anthocyanins are stable in the acid conditions existing in the stomach, whereas their stability decreases with the increase in the pH value in the small intestine. Therefore, the bioavailability of these compounds is affected [13]. In particular, it was observed that at pH > 7, anthocyanidins undergo degradation, and that the presence of sugar moieties in anthocyanins favors an increase in terms of stability at neutral pH in respect to their aglycones [14]. Thus, diglycosides are more stable than monoglycosides, which is explained by the hindrance from sugar parts that prevent the degradation into phenolic acids and aldehydes [2]. Accordingly, it is important to define the effect of such polyphenols and their stability during the digestion process and consequentially, their bioaccessibility and their possible beneficial effects. Until now, limited information describing the in vivo effects of the GI process on dietary polyphenols has been reported [15–17].

Thus, the aim of this scientific research is as follows: (i) to prepare red-cabbage extracts based on different extracting mixtures, evaluated for their total polyphenol content (TPC) and the antiradical and reducing properties; (ii) to compare the antioxidant activity of red cabbage extract showing the highest TPC value with data from the same extract encapsulated in an acid-resistant capsule, and (iii) to assess the bioaccessibility of the extract, such as it is, and its encapsulated formula through an in vitro gastrointestinal digestion, with a view to propose the use of the red-cabbage nutraceutical formulation in slowing down or delaying oxidative stress onset.

The polyphenolic profile of the prepared red cabbage extract was ascertained through ultra-high-performance liquid chromatography coupled to a high-resolution Orbitrap mass spectrometry.

#### **2. Materials and Methods**

#### *2.1. Reagents and Materials*

Methanol (MeOH), ethanol (EtOH), acetic acid (AcOH), formic acid (FA), and acetonitrile (AcN) HPLC grade were purchased from Merck (Darmstadt, Germany). Deionized water (<18 MΩ x cm resistivity) was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Potassium chloride (KCl), potassium thiocyanate (KSCN), monosodium phosphate (NaH2PO4), sodium sulfate (Na2SO4), potassium persulphate (K2S2O8), sodium chloride (NaCl), sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), hydrochloric acid (HCl), pepsin (250 U/mg solid) from porcine gastric mucosa, pancreatin (4 USP) from porcine pancreas, protease from *Streptomyces griseus*, also named Pronase E (3.5 U/mg solid), and Viscozyme L were purchased from Sigma Aldrich (Milan, Italy).

The compound of 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), ferrous chloride (FeCl2), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ), Folin–Ciocalteu reagent, (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid commonly called Trolox (C14H18O4), and gallic acid (C7H6O5) were acquired from Sigma Aldrich (Milan, Italy). All other chemicals and reagents were of analytical grade.

#### *2.2. Sampling*

Red cabbage (*Brassica oleracea L*. var. *capitata F. rubra*) plants were grown in different fields located in Campania, South of Italy. All bulbs (*n* = 10) were harvested in September 2019. After the samples arrived in the laboratory, the cabbage samples with mechanical damage and visible spoilage were separated. Red cabbage samples were rapidly washed under running tap water and chopped into small pieces before being frozen and freeze-dried (Lyovapor™ L-200, Buchi srl, Milan, Italy). After lyophilization, the dry weight of the samples obtained was recorded; then, they were milled into powder (particle size 200 μm) using a laboratory mill. The powders were stored at −80 ◦C until analysis. All the analyses were performed in triplicate, the replicates were independents and results expressed as mean ± SD. Dry matter content of red cabbage corresponded to 12%.

#### *2.3. Red Cabbage Polyphenolic Extraction*

Polyphenols were extracted according to the procedure reported by Grace et al. [18] with some modifications. Briefly, 2.5 g of freeze-dried red cabbage was introduced into a 50-mL Falcon tube (Conical Polypropylene Centrifuge Tube; Thermo Fisher Scientific, Milan, Italy) and extracted with 30 mL of five different mixtures: (1) MeOH:H2O (60:40) 0.1% FA; (2) MeOH:H2O (70:30) 0.1% AcOH; (3) MeOH:H2O (80:20) 0.1% FA; (4) H2O 0.1% FA; (5) EtOH:H2O (70:30) 0.1% AcOH. The samples were vortexed (ZX3; VEPL Scientific, Usmate, Italy) for 2 min and sonicated (LBS 1; Zetalab srl, Padua, Italy) for 30 min (vortexed at 10-min intervals). Then, the mixture was centrifuged for 10 min at 4000 rpm at 20 ◦C. The supernatant was collected and filtrated through a 0.22 μm filter. A portion of the extracts was kept in refrigeration conditions until further analysis.

Moreover, another part of the red cabbage polyphenol extract obtained with mixture 1 after lyophilization was employed for the capsules' formulation. In particular, the capsules contained 1000 mg of red cabbage polyphenolic extract. The capsules used were acid-resistant (hydroxypropyl cellulose E464, gellan gum E418, titanium dioxide E171).

#### *2.4. Determination of Total Phenolic Content (TPC)*

The Folin–Ciocalteu method was used for determining the total phenolic content in accordance with the procedure reported by Izzo et al. [19]. Briefly, 500 μL of deionized water and 125 μL of the Folin–Ciocalteu reagent 2 N were added to 125 μL of red cabbage extract. The tube was mixed and incubated for 6 min in dark conditions. Then, 1.25 mL of 7.5% of sodium carbonate solution and 1 mL of deionized water were added. The reaction mixture was maintained in dark for 90 min. Finally, the absorbance at 760 nm was measured through a spectrophotometer system. Results were expressed as mg of gallic acid equivalents (GAE)/g of dry weight sample.

#### *2.5. UHPLC-Q-Orbitrap HRMS Analysis*

The qualitative and quantitative profile of bioactive compounds were performed by Ultra High-Pressure Liquid Chromatograph (UHPLC, Dionex UltiMate 3000, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a degassing system, a Quaternary UHPLC pump working at 1250 bar, and an autosampler device. Chromatographic separation was carried out with a thermostated (T = 25 ◦C) Kinetex 1.7 μm F5 (50 × 2.1 mm, Phenomenex, Torrance, CA, USA) column. The mobile phase consisted of 0.1% FA in water (A) and 0.1% FA in methanol (B). The injection volume was 1 μL. The gradient elution program was as follows: an initial 0% B, increased to 40% B in 1 min, to 80% B in 1 min, and to 100% B in 3 min. The gradient was held for 4 min at 100% B, reduced to 0% B in 2 min, and another 2 min for column re-equilibration at 0%. The total run time was 13 min, and the flow rate was 0.5 mL/min.

The mass spectrometer was operated in both negative and positive ion mode by setting 2 scan events: full ion MS and all ion fragmentation (AIF). The following settings were used in full MS mode: resolution power of 70,000 Full Width at Half Maximum (FWHM) (defined for *m*/*z* 200), scan range 80–1200 *<sup>m</sup>*/*z*, automatic gain control (AGC) target 1 <sup>×</sup> 106, injection time set to 200 ms and scan rate set at 2 scan/s. The ion source parameters were as follows: spray voltage 3.5 kV; capillary temperature 320 ◦C; S-lens RF level 60, sheath gas pressure 18, auxiliary gas 3, and auxiliary gas heater temperature 350 ◦C.

For the scan event of AIF, the parameters in the positive and negative mode were set as follows: mass resolving power = 17,500 FWHM; maximum injection time = 200 ms; scan time = 0.10 s; ACG target = 1 <sup>×</sup> 105; scan range = 80–120 *m*/*z*; isolation window to 5.0 *m*/*z*; and retention time to 30 s. The collision energy was varied in the range of 10 to 60 eV to obtain representative product ion spectra.

For accurate mass measurement, identification and confirmation were performed at a mass tolerance of 5 ppm for the molecular ion and for both fragments. Data analysis and processing were performed using Xcalibur software, v. 3.1.66.10 (Xcalibur, Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.6. Antioxidant Activity*

#### 2.6.1. ABTS Radical Cation Scavenging Assay

Free radical-scavenging activity was measured by using the method reported by Luz et al., [20]. Briefly, 9.6 mg of 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt was solubilized in 2.5 mL of deionized water (7 mM) and 44 μL of solution of potassium persulfate (K2S2O8; 2.45 mM) were added. The solution was kept in dark conditions at room temperature for 16 h prior to use. Afterward, ABTS•<sup>+</sup> solution was diluted with ethanol to reach an absorbance value of 0.70 (±0.02) at 734 nm. Then, to 1 mL of ABTS•<sup>+</sup> solution with an absorbance of 0.700 <sup>±</sup> 0.050, 0.1 mL of opportunely diluted sample was added. After 2.5 min wait, the absorbance was immediately measured at 734 nm. Results were expressed as millimoles of Trolox Equivalents (TE)/kg of dry weight sample.

#### 2.6.2. DPPH Free Radical-Scavenging Assay

The total free radical-scavenging activity of the analyzed samples was determined using the method suggested by Brand-Williams et al. [21] with modifications. Briefly, 1,1-diphenyl-2-picrylhydrazyl (4.0 mg) was solubilized in 10 mL of methanol and then diluted to reach a value of 0.90 (±0.02) at 517 nm. This solution was used to perform the assay, and 200 μL of sample extract was added to 1 mL of working solution. Results were expressed as mmol Trolox Equivalents (TE)/kg of dry weight sample.

#### 2.6.3. Ferric Reducing Antioxidant Power

The antioxidant capacity of red cabbage samples was estimated spectrophotometrically following the procedure of Benzie and Strain [22]. The ferric reducing/antioxidant power (FRAP) reagent was prepared by mixing acetate buffer (0.3 M; pH 3.6), TPTZ solution (10 mM), and ferric chloride solution (20 mM) in the proportion of 10:1:1 (*v*/*v*/*v*). Freshly prepared working FRAP reagent was used to perform the assay. Briefly, 0.150 mL of the appropriately diluted sample was added to 2850 mL of FRAP reagent. The value of absorbance was recorded after 4 min at 593 nm.

The method is based on the reduction of Fe3<sup>+</sup> TPTZ complex (colorless complex) to Fe2+-tripyridyltriazine (intense blue color complex) formed by the action of electron-donating antioxidants at low pH. Results were expressed as mmol Trolox Equivalents (TE)/kg of dry weight sample. All the determinations were performed in triplicate.

#### *2.7. In Vitro Simulated Gastrointestinal Digestion*

The in vitro gastrointestinal digestion, composed by oral, gastric, and intestinal phases (both duodenal and colon phases), was performed according to the standardized in vitro digestion model (INFOGEST method) [23] with some modifications (Figure 1). The simulated salivary, gastric, and intestinal fluid was prepared in accordance with the proportion salts reported by Castaldo et al. in Table 8 of the materials and methods section.

In the oral phase, 1 g of extract and one capsule contained 1 g of extract were mixed with 3.5 mL of warmed simulated salivary fluid (SSF). Then, 0.5 mL of α-amylase enzyme (50 mg of 250 U/mg solid), 25 μL of 0.3 M CaCl2 (H2O)2, and 975 μL of water were added and thoroughly mixed. Afterward, the pH of the mixture was adjusted to 7 with HCl 1 M, and the sample was incubated for 2 min at 37 ◦C at 150 rpm in an orbital shaker (KS130 Basic IKA, Argo Lab, Milan, Italy).

In order to simulate the gastric phase, 7.5 mL of simulated gastric fluid (SGF), 1.6 mL of pepsin (59.2 mg of 4 USP), 5 μL of 0.3 M CaCl2 (H2O)2, and 695 μL of water were added and thoroughly mixed. The pH value was adjusted to 3 using HCl 6 M. The sample was incubated for 120 min at 37 ◦C at 150 rpm in an orbital shaker (KS130 Basic IKA, Argo Lab, Milan, Italy).

In the intestinal phase, 11 mL of simulated intestinal fluid (SIF), 5 mL of pancreatin (20 mg of 4 USP), 2.5 mL of bile salts (150 mg), 40 μL of 0.3 M CaCl2 (H2O)2, and 1300 μL of water were added and thoroughly mixed. The pH value was adjusted to 7 using NaOH 6 M. The sample was incubated for 120 min at 37 ◦C at 150 rpm in an orbital shaker (KS130 Basic IKA, Argo Lab, Milan, Italy).

To the end of the intestinal phase, the tube was centrifuged at 5000 rpm for 10 min. To simulate the colon digestion process, the supernatant was collected, while 5 mL of Pronase E (1 mg/mL water solution) was added to the pellet. In this step, the pH value was adjusted to 8 using NaOH 1M and incubated for 60 min at 37 ◦C. Afterward, the supernatant was collected, lyophilized, and stored. The residue pellet was mixed with 150 μL of Viscozyme L, and the pH value was adjusted to 4 and incubated for 16 h at 37 ◦C. Finally, the supernatant was collected, stored, and lyophilized whereas the pellet was eliminated. All the supernatants collected during the different in vitro digestion phases were freeze-dried and then dissolved in MeOH:H2O (6:4, *v*/*v*) containing 0.1% FA for the evaluation of antioxidant activity and total polyphenols content.

**Figure 1.** Overview and flow diagram of a simulated in vitro digestion method. SSF: simulated salivary fluid; SGF: simulated gastric fluid; SIF: simulated intestinal fluid.

#### *2.8. Statistical Analysis*

Statistical analysis of data was performed by two-way ANOVA analysis (SPSS 13.0) followed by the Tukey–Kramer multiple comparison test to evaluate significant differences; *p*-values ≤ 0.05 were considered as significant. All the determinations were performed in triplicate, and results were expressed as mean ± standard deviation (SD).

#### **3. Results and Discussion**

In this context, considering the diversity in polyphenol compounds of red cabbage, herein, the total polyphenol content and the antioxidant capability of red cabbage extracts obtained by using five different extracting mixtures were firstly evaluated. The red cabbage extract from the hydroalcoholic solution MeOH:H2O (6:4, *v*/*v*), acidified with 0.1% FA, appeared to be the most active. Thus, this extract, which was chemically profiled by UHPLC-Q-Orbitrap HRMS, underwent an in vitro gastrointestinal digestion simulation, together with its encapsulated form. Accordingly, the effects of the GI process on the extract as it is and that formulated in an acid-resistant capsule were compared.

#### *3.1. Red Cabbage Extract Bioactivity*

In the first approach, efficient extraction mixtures to maximize polyphenol recovery, mainly in its anthocyanin component, were explored. The choice of the solvent represents an important step in the extraction process because of its impact on the yield of bioactive compounds and consequently on human system effects. As reported by Lapornik et al. [24], who studied the solvent effect on the extraction of anthocyanins and other polyphenols from grape and red currant, ethanol and methanol

extracts resulted in a major amount of bioactive compounds than water extracts. Methanol exhibits slightly better characteristics than ethanol as an extracting solvent. Since methanol and ethanol are less polar than water, they are more effective in degrading cell walls (due to their apolar feature), favoring anthocyanins and polyphenols releasing from cells. However, it must be considered that ethanol is more suitable than methanol for a safe application in the food sector [25,26]. Taking into account these previous observations, in the current scientific research, total polyphenol content (TPC) and antioxidant activity of red cabbage extracts obtained by using five different mixtures of extraction solvents are evaluated and reported in Table 1. TPC data ranged from 15.798 to 19.986 mg GAE/g. Acidified water is less suitable for the extraction of phenolic compounds from red cabbage, followed by acidified ethanol/water mixture, which showed a 2.95% increase in extraction efficiency in respect to water extract. Hydroalcoholic solution based on methanol as an alcoholic component also differed in their TPC content, and the MeOH:H2O (6:4) solution acidified with 0.1% formic acid appeared to elicit the best extracting properties. Indeed, beyond the ratio of alcohol to water, the type of acid component also could affect extraction overall. In fact, considering a variation of the MeOH/water ratio from 7:3 (*v*/*v*) to 8:2 (*v*/*v*) leads to a comparable TPC yield where acetic acid is used instead of formic acid. Moreover, the phenol recovery was estimated, taking account of the relative TPC value, and it showed a percentage increase of 26.5% in MeOH:H2O (6:4) plus 0.1% FA with respect to acidified water extract. A similar trend was observed also assessing antioxidant activity through ABTS and DPPH antiradical tests, as well as by the ferric reducing/antioxidant power assay. Data acquired are in the range of 45.128–50.849, 23.498–36.242, and 67.759–87.095 mmol Trolox®/kg for ABTS, DPPH, and FRAP, respectively. It appears clear that the potential antioxidant capacity of red cabbage extract could be affected by the typology and polarity of the extraction mixture used or methodology applied [26], and a great variability could be verified when the total phenolics content from previous scientific studies in the literature was consulted.


**Table 1.** Total polyphenol content and antioxidant data of red cabbage extracts obtained using five different extractants. Values are reported as mean ± SD of independent experiments performed in triplicate. Statistic significance was calculated with two-way ANOVA analysis.

\* results are referred to dry weight (dw). a–h Mean values with different superscript letters are significantly. different by Tukey–Kramer multiple comparison test.

Antioxidant activity data measured through the three spectrophotometrical assays (ABTS, DPPH, and FRAP test) are in line with those from other studies [27,28]. The Relative Antioxidant Capacity Index (RACI) was determined in accordance with the method previously reported by Pacifico et al. [29]. The standard score was calculated as the average of the standard scores obtained from the raw data for the various antioxidant methods. The TPC value highlights that MeOH:H2O (6:4, *v*/*v*) 0.1% FA extract was more active than the others (Figure 2) and that an increase in methanol amount corresponds to a gradual decrease in antioxidant capacity. As shown in Table 2, a wide variability in the TPC of different red cabbage extracts was found in the literature. The concentration ranged between 0.10 and 116.00 mg/kg dry weight (dw). Presumably, the mixture MeOH:H2O (6:4, *v*/*v*) results were a good compromise applicable to the extraction of red cabbage bioactive compounds. By increasing the percentage of methanol, the TPC content decreases. This effect could be due to the major solubility of

anthocyanins, which are contained in high quantity in red cabbage, in a high percentage of acidified water, whereas polyphenols are more soluble in methanol.

**Figure 2.** Relative Antioxidant Capacity Index (RACI) was used to integrate the antioxidant capacity values generated from the different applied methods.

**Table 2.** Recent surveys reporting the total phenolic content (mg GAE/g) in red cabbage extract. GAE: gallic acid equivalents.


\* results are adjusted and expressed in the same measurement unit as mg GAE/g dry weight.

Cruz et al. [40] reported higher TPC content in red cabbage extract (up to 116.00 mg/g) obtained with a hydromethanolic mixture (7:3, MeOH:H2O, *v:v*), whereas in the here analyzed samples, all extracts were obtained by different mixtures acidified with AcOH or FA.

Murador et al. [27] evaluated the effects of different home cooking techniques on kale and red cabbage and demonstrated that these procedures seemed to have no significant effect on TPC in red cabbage. On the contrary, the cooking process facilitated the extraction of bioactive compounds due to the capability to soften the vegetable tissues, ameliorating the activity. Moreover, Wiczkowski et al. [42] determined the antioxidant activity of red cabbage varieties (Langedijker Dauer 2, Kissendrup, Koda, Kalibos, and Langedijker Polona) in two diversified lengths of the vegetation period (year 2008, 2009). The result of antioxidant activity measured through ABTS assay ranged between 87.99 and 168.76 and from 101.06 to 169.46 mmol Trolox/kg dry weight (dw) for two diversified lengths of the vegetation period, respectively. The varieties of red cabbage obtained in 2009 were characterized by a higher ability to radical scavenge than that grown in 2008, indicating that the differences in antioxidant capacity of red cabbage occurred in a variety-dependent manner. Nevertheless, amongst *Brassica* vegetables, red cabbage, brussels sprouts, and broccoli possess the highest antioxidant capacity and the contribution to health improvement can be related to their capacity [43].

#### *3.2. In Vitro Bioaccessibility of Red Cabbage Polyphenols*

The bioaccessibility of total phenolic compounds, which was calculated using the Folin–Ciocalteu method, and antioxidant activity determined by DPPH, ABTS, and FRAP tests during the in vitro gastrointestinal digestion are presented in Tables 3 and 4. The in vitro gastrointestinal digestion was evaluated according to the INFOGEST procedure until the duodenal stage. Subsequently, to reproduce the microbiota occurring in the colon phase, the combined action of Pronase E and Viscozyme L. was utilized [44,45].

**Table 3.** Total polyphenol content of red cabbage extract compared to that of the same extract encapsulated in an acid-resistant capsule. Values are reported as mean ± SD of independent experiments performed in triplicate. Statistic significance was calculated with two-way ANOVA analysis.


\* results are referred to dry weight (dw) of extract; red cabbage extract was encapsulated in polyethylene capsule and used in in vitro digestion process. a–e Mean values with different superscript letters are significantly different by Tukey–Kramer multiple comparison test.

**Table 4.** Antioxidant data by means of 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and ferric reducing/antioxidant power (FRAP) methods of red cabbage extract compared to that of the same extract encapsulated in an acid-resistant capsule. Values are reported as mean ± SD of independent experiments performed in triplicate.


\* Results are referred to dry weight (dw) of extract; a–m Mean values with different superscript letters are significantly different by Tukey–Kramer multiple comparison test.

An improvement of bioactivity was observed for the extract digested in an acid-resistant capsule after the colonic stage compared to the extract digested without capsule. In fact, the total polyphenols content of extract digested in an acid-resistant capsule in the Pronase E resulted in a significantly higher concentration than the extract digested without capsule: 4.434 and 0.124 mg GAE/g, respectively. A similar trend was also observed by evaluating the antioxidant activity (Table 4); encapsulation appeared to favor an increase in antiradical and reducing capability mainly at the Viscozyme L stage. In GI digestion, polyphenols may interact with food constituents and be subjected to further degradation or metabolization that could affect their uptake. The presence of dietary fiber in fruits and vegetables is able to influence and modulate the phytochemicals bioaccessibility [46]. In this context, Podse¸dek et al. [47] have evaluated the stability of red cabbage antioxidant compounds, principally anthocyanins, during GI, concluding that the latter are affected by cabbage composition and vegetable constituents, including dietary fiber. During intestinal digestion, anthocyanins' stability is hard dependent on food matrices. Notwithstanding, compared to other matrices, red cabbage has demonstrated a greater stability [43].

Some studies showed that anthocyanins have low absorption and high metabolism, and their bioavailability is lower compared with other subclasses of polyphenols [48,49]. Despite the reduced bioavailability, Sodagari et al. [50] reported that up to 70% of anthocyanins derived by foods could reach the colon. Hence, a regular intake of foods rich in anthocyanins could result in beneficial effects on human health.

Chemical modifications occurring during gastrointestinal digestion including the activity of gut microbiota lead to a releasing of smaller compounds, the principal responsible for the increased antioxidant activity. Although polyphenols were poorly absorbed in the duodenum, they can exert their antioxidant activities in the lower gut, which is able to metabolize these compounds, generating metabolites with greater activity [43]. It is speculated that bioactive compounds could be metabolized by human colonic microflora, generating metabolites with greater bioactivity and more beneficial effects. Specifically, anthocyanins, forming the majority of the polyphenols of red cabbage, are metabolized by glycosidase from gut microflora through cleavage of the C-ring to produce easily absorbed phenolic acids. Glycosidase, in the ileum, supports metabolism and the absorption of glycosides [51]. Moreover, Chen et al. [52] demonstrated that mulberry's anthocyanins were metabolized to phenolic acids such as chlorogenic, protocatechuic, caffeic, and ferulic acids by the action of intestinal microflora in a percentage of 46.17%.

Several studies have also demonstrated that polyphenols from foods could have dissimilar bioaccessibilities. Most of the polyphenols are stable in the acidic gastric environment and degraded due to the neutral conditions encountered in the small intestine, which contribute to a reduced uptake into blood [12]. The in vitro GI digestion represents a valid tool to understand the behavior of compounds and the amounts that are effectively subjected to mucosal absorption. By using oral administration, macromolecules derived by food need firstly to be digestive and reduced in small bioactive compounds, and after withstanding to the pH in the GI tract, they could reach the absorption site in the small intestine [53]. Polyphenols metabolized by the combined action of Viscozyme and Pronase lead to the release of smaller compounds, the principal being responsible for the increased antioxidant activity [45]. Foods, including fruits and vegetables, represent an important source of bioactive compounds having numerous beneficial health effects such as antioxidant capacity [54]. As proved by scientific studies, GI digestion plays an important role in the antioxidant capacity, because the availability of bioactive compounds is influenced by the digestion process [55]. It seems that digestion could modify the antioxidant properties of foods, but there are contrasting opinions [56,57]. The antioxidant activity of dietary supplements commonly ingested as a source of antioxidant polyphenols was investigated by Henning et al. [58]. In addition, in this latest case, the stability was evaluated by determining the total phenolic content by Folin–Ciocalteu assay, and the antioxidant capacity was assessed by using DPPH, FRAP, and ABTS tests. Although polyphenols provide the major antioxidant potency, their results highlight that digestion may alter antioxidant properties depending on polyphenol content.

Naturally, the behavior of different classes of molecules during digestion is no overall standard. This finding was broadly demonstrated by Chen et al. [54], who evaluated the total phenolic content (TPC) and antioxidant activity before and after the in vitro digestion of thirty-three fruits, deriving large variations in the results. A significant improvement in TPC after gastric step was observed for eight fruits, but for the other twenty-five, it resulted in an increase after the duodenal phase. The same trend was observed by the antioxidant activity performed through DPPH, ABTS, and FRAP tests; the values significantly increased for some fruits but decreased for others.

It is demonstrated that some classes of polyphenols are able to increase their concentration after the gastric phase, justifying their high sensitivity to alkaline conditions. In fact, the majority of antioxidant compounds are degraded by alkaline pH, causing a substantial loss in the activity after the intestinal stage [57].

Unfortunately, polyphenols had not demonstrated high long-term stability and are characterized by sensitivity to heat and light. Moreover, polyphenols present a poor bioavailability because of low water solubility [59]. Finally, some of these compounds possess a bitter and astringent taste that limits their use in food or in oral medications. To circumvent these drawbacks, some processes that are able to enhance polyphenol bioavailability have been reported [60–62]. The use of an acid-resistant capsule in the digestion process acted as a protective agent and allowed us to avoid the consequence of gastric ambient on the bioactive molecules. In particular, acid-resistant capsules protect bioactive compounds from degradation or alteration of their chemical structure caused by changes in pH or the action of digestive enzymes. The acid-resistant capsules represent a useful strategy to conduct bioactive molecules until the intestinal district, where the bioactive compounds are favorably absorbed and exert their activity.

#### *3.3. Identification and Quantification of Red Cabbage Bioactive Compounds Analyzed through UHPLC-Q-Orbitrap HRMS*

Bioactive compounds of red cabbage MeOH:H2O (6:4, *v*/*v*) extract were profiled through UHPLC-Q-Orbitrap HRMS. A total of 40 different polyphenolic compounds including phenolic acids, flavonoids and anthocyanins were tentatively identified by combining MS and MS/MS spectra (Tables S1 and S2 in Supplementary Material). Experiments were carried out both in ESI- and ESI<sup>+</sup> mode. Phenolic acids and flavonoids exhibited better fragmentation patterns producing the deprotonated molecular ion [M-H]- , whereas anthocyanins were investigated in positive ESI mode. Unambiguous identification of some compounds is carried out by comparison to their relative reference pure standards. The quantitative determination of target analytes was carried out using calibration curves at eight concentration levels. Each calibration curve was prepared in triplicate. Regression coefficients >0.990 were obtained. The quantification of compounds that had no standard to generate a curve was based on a representative standard of the same group. This is the case of cyanidin derivatives, which are quantified by using the calibration curve of cyanidin 3,5-diglucoside.

Screening in full scan mass chromatograms enables the identification of untargeted compounds and retrospective data analysis. The confirmation of the structural characterization of untargeted analytes was based on the accurate mass measurement, elemental composition assignment, retention time, and MS/MS fragmentation. Sensitivity was assessed by the limit of detection (LOD) and limit of quantification (LOQ). LOD is defined as the min imum concentration that enables the analyte identification with a mass error below 5 ppm. LOQ is the lowest analyte concentration that allows the analyte quantitated at defined levels for imprecision and accuracy <20%.

Red cabbage has its own characteristic anthocyanin pattern that includes acylated anthocyanins, which affect the antioxidant activity. In particular, anthocyanins with cyanidin-3-diglucoside-5-glucoside core, non-acylated, mono-acylated, or diacylated with *p*-coumaric, caffeic, ferulic, and sinapic acids were found to be the predominant compounds, which was in accordance with data reported by several scientific studies [63–65]. Indeed, according to Charron et al. [66], the dominant form of anthocyanins in the investigated red

cabbage extract was cyanidin-3-diglucoside-5-glucoside, which appeared as the first main peak of the investigated extract. The compound showed the [M]<sup>+</sup> ion at *m*/*z* 773.21057 and the main MS<sup>2</sup> fragment ions at *m*/*z* 611.06012 [M-Glc]+, 449.16882 [M-2Glc]+, and 287.05219 [M-3Glc]<sup>+</sup> (Table S1). It constituted 39.5% of the anthocyanin fraction, which represented 26% of the investigated extract. Three abundant sinapoyl derivatives of the previous compound were also tentatively identified (Table 5). In particular, cyanidin-3-(sin)-diglucoside-5-glucoside and cyanidin-3-(sin)sophoroside-5-glucoside shared the molecular formula C44H51O25 and the neutral loss of a sinapic acid-H2O moiety, whereas cyanidin-3(sin)(sin)sophoroside-5-glucoside showed the [M]<sup>+</sup> ion at *m*/*z* 1185.32959 (Table S1). Cyanidin-3-(caf)(sin)soph-5-glucoside showed the [M]<sup>+</sup> ion at *m*/*z* 1141.30261, and cyanidin 3(sin)triglucoside-5-glucoside ([M]<sup>+</sup> ion at *m*/*z* 1141.32544) was also identified. The latter compound and cyanidin 3(caf)(fer)sophorosyl-5-glucoside, along with seven other acylated anthocyanidins (cyan-3(caf)(sin)soph-5-glu; cyan-3(*p*-coum)triglu-5-glu; cyan-3(*p*-coum)soph-5-(suc)glu; cyan-3(glucop-sin)(*p*-coum)soph-5-glu; cyan-3(glucop-sin)(fer)soph-5-glu; cyan-3(glucop-sin)(sin)soph-5-glu; cyan-3(fer)soph-5-(sin)glu)) were identified for the first time by Arapitsas et al. [64], who analyzed the red cabbage anthocyanin profile using HPLC/DAD-ESI/Qtrap MS.

Mizgier et al. [67] analyzed the phenolic compounds and antioxidant capacity of red cabbage extract, confirming that more than 80% was constituted from acylated compounds. The total anthocyanin content of the red cabbage extracts was reported to be equal to 175.1 mg/g dry weight, which was expressed as cyanidin-3-glucoside equivalents.

Wiczkowski et al. [63] studied the red cabbage anthocyanins profile and analyzed the correlation of antioxidant activity and acylated compounds, demonstrating that cyanidin 3-diglucoside-5-glucoside diacylated with sinapic acid had the highest radical-scavenging activities. Acylated cyanidin glycosides showed higher antioxidant capacity than the non-acylated form of cyanidin glycosides. In this case, the total content of anthocyanins in red cabbage was 2.32 mg/g on dry weight, which was calculated based on cyanidin equivalents [63]. Dominant forms of anthocyanins in this red cabbage were non-acylated compounds, which comprised 27.6% of the total red cabbage anthocyanins, whereas mono-acylated and diacylated anthocyanins covered 38.4% and 34.1%, respectively.

Other reports evidenced that red cabbage is an excellent vegetable containing a high content of anthocyanins, which were in the range from 40 to 188 mg Cy 3-glcE/100 g fresh weight [68,69].

Vo´ca et al. [70], who analyzed the difference in chemical composition between cabbage cultivars, reported the highest content of anthocyanins in red cabbage extract (until 750.71 mg/kg fresh weight). In red cabbage cultivars, even 3.9 times higher antioxidant capacity was reported compared to the other cultivars. Another report estimated the total anthocyanins content of the red cabbage extract equal to 4984.13 ± 101.62 μg/g dry weight [71]. In addition, the flavonoids composition was analyzed by Vo´ca et al. as well [70]; red cabbage contains, besides anthocyanins, other phenolic compounds also. In particular, the most abundant part of the extract (73.7%) consisted in C6C3 phenolic acids as sinapic acid (6325.025 ± 3.568 μg/g dry weight), ferulic acid (2768.48 ± 29.18 μg/g dry weight), and *p*-coumaric acid (4518.52 ± 15.83 μg/g dry weight), beyond C6C1 phenolic acids such as vanillic acid (5961.13 ± 29.08 μg/g dry weight) and protocatechuic acid (2881.17 ± 15.59 μg/g dry weight). Thus, sinapic acid covered 37.5% of phenolic acids.

Finally, non-anthocyanin flavonoids were found in a negligible portion, as they constituted only 0.38% of the extract. In particular, among flavonols, kaempferol appeared to mostly contain 17.371 ± 0.23 μg/g dry weight, followed by rutin and its aglycone quercetin, whose relative contents were estimated to be equal to 15.302 ± 0.96 and 4.359 ± 0.33 μg/g dry weight. The flavanol epigallocatechin constituted 23.965 ± 0.16 μg per g of dry extract.



\* Results are referred to dry weight (dw) of extract; sin = sinapoyl; soph = sophoroside; fer = feruloyl; *p*-coum = *p*-coumaroyl; caf = caffeoyl; glucofer = glucoferoyl.

#### **4. Conclusions**

Red cabbage is a rich source of phenolic acids and flavonoids. Among the latter, anthocyanins represent the most abundant class. Indeed, the full exploitation of the beneficial antioxidant efficacy of these compounds requires their extraction optimization. In this context, extractive procedures on red cabbage provided an extract rich in cyanidin-3-diglucoside-5-glucoside and its acylated compounds. In particular, UHPLC-Q-Orbitrap HRMS analysis highlighted the diversity in sinapoyl derivatives, and sinapic acid was also the most representative phenolic acid in the extract. The chemically profiled extract was screened for its antiradical and reducing capabilities, whereas its stability and bioaccesibility were proved to be preserved in an acid-resistant capsule. Indeed, based on the data acquired, improvements for all the evaluated bioactivities (TPC and antioxidant activity) were observed during the Pronase E and/or Viscozyme L phases of the in vitro gastrointestinal digestion of the red cabbage extract in an acid-resistant capsule. An increase in the colonic phase in TPC was also observed,

as well as a similar trend for the other evaluated antioxidant activities. During gastrointestinal digestion, bioactive compounds could be metabolized by human colonic microflora, generating metabolites with greater bioactivity and more beneficial effects. Thus, the use of an acid-resistant capsule in the digestion process could protect bioactive compounds, ameliorating their bioaccessibility. In this context, red cabbage extract encapsulated in an acid-resistant capsule could be a valid alternative to produce a new nutraceutical formulation useful for preventing or slowing down oxidative stress-related diseases onset.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/10/955/s1, Table S1: Chromatographic and spectrometric parameters including retention time, adduct ion, theoretical and measured mass (*m*/*z*), accuracy and sensibility for anthocyanins (n = 20) in the investigated red cabbage extract. RT = Retention Time, Table S2: Chromatographic and spectrometric parameters including retention time, adduct ion, theoretical and measured mass (*m*/*z*), accuracy and sensibility for phenolic acids and flavonoids (n = 20) in the investigated red cabbage extract. RT = Retention Time.

**Author Contributions:** Conceptualization, L.I. and A.R.; methodology, L.I.; formal analysis, L.I.; investigation, L.I., L.C. and A.N.; resources, A.R.; writing, original draft preparation, L.I.; writing, review and editing, Y.R.-C., S.P.; supervision, A.R and S.P.; project administration, A.R.; funding acquisition, A.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Improvement of Health-Promoting Functionality of Rye Bread by Fortification with Free and Microencapsulated Powders from** *Amelanchier alnifolia* **Nutt**

### **Sabina Lachowicz 1,\*, Michał Swieca ´ <sup>2</sup> and Ewa Pejcz <sup>1</sup>**


Received: 10 June 2020; Accepted: 10 July 2020; Published: 13 July 2020

**Abstract:** This study established the appropriate amounts of a functional Saskatoon berry fruit powder in fortified rye bread acceptable to consumers and determined the potential relative bioaccesibility of bioactive compounds exhibiting antioxidant activity, and enzymatic in vitro inhibitory activity against lipoxygenase, cyclooxigenase-1, cyclooxigenase-2, acetylcholinesterase, pancreatic lipase α-glucosidase, and α-amylase, as well as the relative digestibility of nutrients. The content of polyphenolic compounds and antioxidant capability were strongly, positively correlated with the content of the functional additive. The highest phenolics content and antioxidant activity were determined in the products enriched with the powders microencapsulated with maltodextrin (an increase by 91% and 53%, respectively, compared with the control). The highest overall acceptability was shown for the products with 3% addition of the functional additive, regardless of its type. The simulated in vitro digestion released phenols (with the highest bioaccessibility shown for anthocyanins) and enhanced the antioxidant activity of rye bread. In turn, the microencapsulation contributed to the improvement in the relative bioaccesibility of antioxidant compounds. Bread fortification led to an increased inhibitory activity against α-amylase, α-glucosidase, and lipoxygenase. Furthermore, the additive microencapsulated with maltodextrin and inulin improved the capacity to inhibit the activities of pancreatic lipase and cyclooxigenase-2. The results presented allowed concluding that the powders from Saskatoon berry fruits, especially microencapsulated ones, may be a promising functional additive dedicated for the enrichment of rye bread.

**Keywords:** rye bread; microencapsulation; phenolics; in vitro relative bioaccessibility; lipoxygenase; cyclooxygenase; acetylcholinesterase; biological activity

#### **1. Introduction**

Rye bread is an integral element of a man's diet as a good source of nutrients, including compounds that exhibit a broad spectrum of antioxidant activity. These properties are mainly ascribed to the phenolic compounds of cereal grains [1], which also exhibit anti-carcinogenic and anti-inflammatory activities and are recommended in the prevention of many degenerative diseases [1–3]. The consumption of rye bread has increased over the last years also owing to the nutritive value and higher bioaccesibility of its grain components, as well as a lower gluten content [2,3].

The production process of rye bread involves fermentation with sourdough, which positively affects the finished products by acidifying the dough, inhibiting α-amylase activity, developing flavor values, increasing the solubility of pentosans, and also by increasing their nutritive value and

antioxidant potential [1,2]. In addition, this technology extends the shelf life of bread by preventing crumb sticking and mold appearance [2].

Worthy of attention is the recent development of the sector of functional food products, which offer increased nutritive and health values that may help prevent many diseases [2,4]. Considering their high intake, food products such as bread can serve as an excellent carrier of functional ingredients that can be introduced at any stage of the technological process. However, when designing fortified/supplemented foods, attention should be paid to their biological value as well as to their sensory profile, including in particular their acceptability by consumers [5]. Investigations conducted so far have demonstrated the fortification of rye bread with grape and tomato pomace to have a positive effect on its nutritive value and its flavor values [4,6]. Considering the properties and composition of Saskatoon berry, a worthwhile alternative to common bread types can be offered by rye bread with the addition of powder from its fruits. Saskatoon berry fruits exhibit a high antioxidant activity that is strongly correlated with the contents of phenolic compounds, vitamins, minerals, free and bound amino acids, and organic acids [7–9]. Furthermore, Saskatoon berry fruits exhibit strong antibacterial activity against *Escherichia coli* (VTT E-94564), *Enterococcus hirae* (ATCC 10542), and *Staphylococcus aureus* (VTT E-70045). Their components also exhibit inhibiting activities of α-amylase and α-glucosidase [9–11]. The study conducted by Zhao et al. [12] demonstrated that powders from *Amelanchier alnifolia* Nutt. could alleviate hyperlipidemia, hyperglycemia, and blood vessel inflammation induced by high-saccharose and high-fat diet. According to Juricova et al. [7], the Saskatoon berry fruit is additionally responsible for the anti-inflammatory and chemo-protective potency. The sweet taste of the Saskatoon berry fruit can also contribute to the improvement of taste values of sourdough bread by masking its sour taste. On the other hand, establishing the optimal amount of the additive is important as well, because a too high amount of fruit powder can decrease the taste value and cause the appearance of bitterness resulting from a high content of polymerized procyanidins [9].

Another critical issue is the protection of thermolabile bioactive compounds during bread baking at high temperatures. A solution to this problem can be offered by entrapping functional additives in carriers in the microencapsulation process, which allows minimizing losses of health-promoting compounds valuable for a human body. This solution will be worth considering while designing food products with functional characteristics, where not only their production technology, but also their nutritional aspects need to be strongly considered. The effectiveness of microencapsulation has been confirmed in studies into the use of curcumin or Garcinia fruits as functional additives to wheat bread, which demonstrated a higher content of polyphenolic compounds in final products compared with the crude additives [13,14]. The cited authors demonstrated the microencapsulated additive not to affect the complexity of the production process or quality traits of bread, but to contribute to the greater stability of bioactive compounds. According to recent literature, the protection of compounds ensured by the carriers can also contribute to the mitigation of the adverse effects of polyphenolic compounds on sensory properties of the finished products [15] including, for example, the appearance of a bitter and an astringent taste [13]. What is more, the use of microencapsulated food additives has been reported to positively affect the quality traits of bakery products, including bread, by improving their color, dough yield, or even texture [16].

Other important aspects that need to be considered while designing fortified food products include the potential in vitro bioaccesibility of compounds identified in functional additives that are often neglected in the assessment of the real value of such products, as well as the effect their additive has on the nutritive value. As reported by Gawlik-Dziki et al. [17], the quality and quantity of extracted nutrients and their potential health-promoting value determined after simulated in vitro digestion can differ from those obtained for the chemical extracts only. What is more, the bioaccesibility of components of the functional additive will also depend on its interactions (antagonistic or synergistic) with food components or on of its behavior in the digestive system [14]. Therefore, the determination of the potential relative bioaccesibility and biological value of functional food products is essential for the assessment of the effectiveness and safety of the fortification process.

This study examined the effectiveness of rye bread fortification with pure and microencapsulated powders from Saskatoon berry fruits. The study of quality and health-promoting properties was extended with the analysis of potential relative bioaccesibility that indicates the real value of fortified functional food. Furthermore, it aimed to establish the appropriate amounts of a functional additive from Saskatoon berry fruits acceptable by consumers and to determine the antioxidant activity and enzymatic in vitro inhibitory activity against lipoxygenase, cyclooxigenase-1, cyclooxigenase-2, acetylcholinesterase, pancreatic lipase, α-glucosidase, and α-amylase, as well as the relative digestibility of nutrients of fortified rye bread.

#### **2. Materials and Methods**

#### *2.1. Reagents*

Acetonitrile, formic acid, methanol, ABTS (2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH), methanol, acetic acid, phosphate buffered saline (pH 7.4), LOX Activity Assay kit, AChE Assay kit, α-amylase from porcine pancreas, α-glucoamylase from Rhizopus sp., lipase from porcine pancreas, trini-trobenzenesulfonic acid, NaH2PO4, and 3,5-dini-trosalicylic acid were purchased from Sigma-Aldrich (Steinheim, Germany). COX Inhibitor Screening Assay Kit was purchased from Cayman (No. 560131; Ann Arbor, MI, USA). (−)-Epicatechin, (+)-catechin, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, dicaffeic acid, procyanidin A2, procyanidin B2, *p*-coumaric acid, caffeic acid, 4-caffeoylquinic, kampferol-3-*O*-galactoside, quercetin-3-*O*-rutinoside, quercetin-3-*O*-galactoside, quercetin-3-*O*-glucoside, cyanidin-3-*O*-arabinoside, cyanidin-3-*O*-xyloside, cyanidin-3-*O*-galactoside, and cyanidin-3-*O*-glucoside were purchased from Extrasynthese (Lyon, France). Acetonitrile for ultra-performance liquid chromatography (UPLC; gradient grade) and ascorbic acid were from Merck (Darmstadt, Germany). The carriers (30%) applied to produce powders were maltodextrin DE (20–40) and inulin (Beneo-Orafti, Belgium).

#### *2.2. Materials*

Fruits—"Smoky" cultivar of Saskatoon berry and rye flour type 720—were used in this work. Saskatoon berry was collected in the year 2019 from BIOGRIM company (Wojciechow, Lublin, Poland), and the rye flour was from Good Mills Poland company (Stradunia, Poland). The freeze drying pure fruit powder (FP) as additives was prepared as described previously by Lachowicz et al. [18]. Meanwhile, the freeze drying encapsulated fruit powder with maltodextrin (FPM) and inulin (FPI) as additives was prepared as described previously by Lachowicz et al. [19]. The resulting Saskatoon berry fruit was ground in Thermomix (Wuppertal, Vorkwek, Germany) at 40 ◦C for 10 min. For encapsulation fruit, 70% of grounded fruit (*w*/*w*) and 30% of carrier (maltodextrin or inulin) were mixed. Furth, grounded fruit and encapsulated fruit were frozen at −80 ◦C. Next, the freeze drying method of FP and FPM as well as FPI (≈200 g of each sample) was carried out in a freeze dryer (Christ Alpha 1–4 LSC; Germany) for 24 h at a reduced pressure of 65 Pa. The temperature in the drying chamber was −60 ◦C, while the heating plate reached 30 ◦C.

#### *2.3. Rye Bread Preparation*

The rye doughs were made according to a previously description by Pejcz et al. [3]. Dough samples were made using double-phase type. Yeasts (1%), salt (0.5%), and sourdough (50% of rye flour) were used to make rye flour. The rye dough was fermented by LV2 starter cultures. The next step was placing the dough in a baking tin (at 30 ◦C for last fermentation). Different rye breads were using different content of fruit powder: 1, 2, 3, 4, 5, and 6%. Breads were baked in a laboratory oven (Brabender, Duisburg, Germany) for 35 min/230 ◦C. Whole baking experiments were performed twice. Each sample of bread after 24 h was freeze-dried and milled for analysis. The rye bread sample without

addition was considered to be the control sample. Symbols of samples: rye bread without additives (BRC) and with fruit powder from 1% (BR1) to 6% (BR6); rye bread with fruit powder encapsulated with maltodextrin from 1% (BRM1) to 6% (BRM6); and rye bread with fruit powder encapsulated with inulin from 1% (BRI1) to 6% (BRI6). The amount of encapsulated fruit powders with maltodextrin (FPM) and encapsulated fruit powders with inulin (FPI) added to bread was calculated per the amount of crude fruit powder (FP).

#### *2.4. Sensory Attributes and Colour Parameters*

The sensory properties of the obtained rye bread with supplementation were determined using a ten-degree hedonic scale: 1—"I do not like it extremely" to 10—"I like it extremely". The assessment included the following quality attributes such as aroma, taste, crumb, crust colour, texture, and consistency. It was conducted by a group of 22 panelists (11 men and 11 women in the age group of 20–70). Coded samples were provided to the panelists for the evaluation at 20 ◦C in a plastic plate.

Colour properties such as L\*, a\*, and b\* of control and enriched rye bread were determined with a Konika Minolta CR-400 colorimeter (Wroclaw Poland). Bread samples were analysed against a white ceramic reference plate (L\* = 93.92; a\* = 1.03; b\* = 0.52), and colour parameters L\* (lightness or brightness: black = 0, white = 100), a\* (greenness = −a\*, redness = +a\*), and b\* (blueness = −b\*, yellowness = +b\*) values were recorded. Total change in colour of rye bread samples (ΔE\*) was calculated as follows:

$$
\Delta E \ast = \sqrt{(L\_0^\star - L^\star)^2 + (a\_0^\star - a^\star)^2 + \left(b\_0^\star - b^\star\right)^2}
$$

#### *2.5. Extraction Procedures*

#### 2.5.1. Digestion In Vitro

In vitro simulated digestion was made on the basis of the method described by Minekus et al. [20]. For gastrointestinal digestion, the freeze-dried rye bread (250 mg) was mixed with PBS buffer (phosphate-buffered saline) (0.5 mL; pH 7.4) and simulated salivary fluid (1 mL). After that, the sample was shaken (10 min/37 ◦C). Next, the pH of the mixture was changed to pH 3 using 6 M HCl, and the samples were shaken for 120 min at 37 ◦C. After that, pH of digests was changed to 7 using 1 M NaOH. The samples underwent intestinal digestion in vitro for 120 min. After that, the activity of enzymes was stopped using methanol (1:1 ratio). The samples were centrifuged (at 19,000× *g*/10 min) and used for the test.

#### 2.5.2. Chemical Extraction

Freeze-dried rye bread samples (1 g) were mixed with 30% of UPLC-grade methanol (10 mL) with 1% of acetic acid. After that, the extract was sonicated for 20 min (Sonic 6D, Polsonic, Warsaw, Poland) and centrifuged (at 19,000× *g*/10 min). Finally, the extract was filtered by hydrophilic PTFE (politetrafluoroetylen) 0.20 μm membrane (Millex Samplicity Filter, Darmstadt, Germany) and used for testing—chemical extract (CE)

#### 2.5.3. Buffer Extraction

Freeze-dried rye bread (~1 g) was mixed with 20 mL of PBS buffer (pH 7.4), and extracted for 1 h. After the extraction, the extracts were centrifuged (6900× *g*/15 min) and the samples were used for analysis—buffer extracts (BE).

#### *2.6. Identyfication and Quantyfication of Polyphenolic Compounds*

Determination of polyphenolic compounds of freeze-dried rye bread sample was carried out using an ACQUITY ultra performance liquid chromatography system equipped with a photodiode array detector with a binary solvent manager (Waters Corporation, Milford, MA, USA) series with a mass detector G2 Q/Tof Micro mass spectrometer (Waters, Manchester, U.K.) equipped with an electrospray ionization (ESI) source operating in negative and positive modes [21]. Separations of polyphenolic compounds were carried out using a UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters Corporation, Milford, MA, USA) at 30 ◦C. The extracts (10 μL) were injected, and the elution was completed in 15 min with a sequence of linear gradients and isocratic flow rates of 0.45 mL min<sup>−</sup>1. The mobile phase consisted of solvent A (2.0% formic acid, *v*/*v*) and solvent B (100% acetonitrile). The program began with isocratic elution with 99% solvent A (0–1 min), and then a linear gradient was used until 12 min, lowering solvent A to 0%; from 12.5 to 13.5 min, the gradient returned to the initial composition (99% A), and then it was held constant to re-equilibrate the column. The analysis was carried out using full-scan, data-dependent MS scanning from *m*/*z* 100 to 1500. Leucine enkephalin was used as the reference compound at a concentration of 500 pg/μL, at a flow rate of 2 μL/min, and the [M – H]− ion at 554.2615 Da was detected. The [M – H]− ion was detected during 15 min analysis performed within ESI–MS accurate mass experiments, which were permanently introduced via the LockSpray channel using a Hamilton pump. The lock mass correction was ±1.000 for the mass window. The mass spectrometer was operated in negative- and positive-ion mode, set to the base peak intensity (BPI) chromatograms, and scaled to 12,400 counts per second (cps) (100%). The optimized MS conditions were as follows: capillary voltage of 2500 V, cone voltage of 30 V, source temperature of 100 ◦C, desolvation temperature of 300 ◦C, and desolvation gas (nitrogen) flow rate of 300 L/h. Collision-induced fragmentation experiments were performed using argon as the collision gas, with voltage ramping cycles from 0.3 to 2 V. Characterization of the single components was carried out via the retention time and the accurate molecular masses. Each compound was optimized to its estimated molecular mass [M – H]−/[M + H]<sup>+</sup> in the negative and positive mode before and after fragmentation. The data obtained from UPLC–MS were subsequently entered into the MassLynx 4.0 ChromaLynx Application Manager software (Waters Corporation, Milford, MA, USA). On the basis of these data, the software is able to scan different samples for the characterized substances. The runs were monitored at the following wavelength: flavonol glycosides at 360 nm. The PDA spectra were measured over the wavelength range of 200–800 nm in steps of 2 nm [21]. The results were as mg per 100 g of dry substances (d.s.).

#### *2.7. Health-Promoting Properties*

#### 2.7.1. Antiradical Capacity

Rye bread (1 g) was mixed with 80% of methanol and water (10 mL) + 1% hydrochloric acid, and incubated for 20 min under sonication (Sonic 6D, Polsonic, Warsaw, Poland). Next, the slurry was centrifuged at 19,000× *g* for 10 min, and the supernatant was filtered through a hydrophilic PTFE 0.20 μm membrane (Merck, Darmstadt, Germany) and used for analysis.

The ABTS method was performed according to the method described by Re et al. [22]. ABTS•<sup>+</sup> was generated by oxidation of ABTS with potassiumpersulphate. The ABTS•<sup>+</sup> solution was diluted to an absorbance of 0.7 <sup>±</sup> 0.05 at 734 nm. Then, 0.03 mL of extract was mixed with 2.97 mL of ABTS•<sup>+</sup> solution and left for 6 min at 25 ◦C. Next, the absorbance was measured at 734 nm using the UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). The results of antiradical capacity were expressed as Trolox equivalents in μmol per g d.s.

The DPPH method was carried out with the method described by Yen and Chen [23]. Then, 0.50 mL of extract was mixed with ethanol (1.5 mL) and DPPH•<sup>+</sup> solution (0.5 mL), and left for 10 min at 25 ◦C. Next, the absorbance was measured at 517 nm using the UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). The results of antiradical activity were expressed as Trolox equivalents in μmol per g d.s.

#### 2.7.2. Reducing Potential

The FRAP test was made on the basis of the method described by Benzie and Strain [24]. First, 0.1 mL of extract was prepared with 0.9 mL of clean H2O with 3 mL of ferric complex. Next, after 10 min, the absorbance was checked at 593 nm using the UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). The results of reducing activity were expressed as Trolox equivalents in μmol per g d.s.

#### 2.7.3. Ability to Inhibit the Activity of COX 1 and COX-2

The effect of the bread extract on COX-1 and COX-2 (cyclooxygenase-1 and cyclooxygenase-2) activities was tested using COX Inhibitor Screening Assay Kit (Cayman, No. 560131). One unit of inhibitor activity (IU) was defined as the activity inhibiting 1 unit of enzyme activity. The results were expressed in kIU per g of d.s.

#### 2.7.4. Ability to Inhibit the Activity of Lipoxygenase (LOX)

The LOX inhibitory assay was made on the basis of the method described by Axelroad et al. [25] with modifications. LOX activity was tested by BioTek Microplate Readers in absorbance at 234 nm. The reaction mixture contained 0.245 mL 1/15 mol/L phosphate buffer, 0.002 mL of lipoxygenase solution (167 U/mL), and 0.005 mL of inhibitor solution. After preincubation of the mixture at 30 ◦C for 10 min, the reaction was initiated by adding 0.008 mL 2.5 mmol/L linoleic acid. One unit of LOX activity was defined as the activity oxidizing 0.12 μmole of linoleic acid per 1 min at reaction conditions. One unit of inhibitor activity (IU) was defined as the activity inhibiting 1 unit of enzyme activity. The results were expressed in kIU per g of d.s.

#### 2.7.5. Ability for Inhibit Acetylcholinesterase Activity (AChE)

The effect of the bread extract on AChE was tested by AChE Assay Kit (Sigma-Aldrich, No. CS0003). One unit of inhibitor activity (IU) was defined as the activity inhibiting 1 unit of enzyme activity. The results were expressed in kIU per g of d.s.

#### 2.7.6. Activity of α-Amylase Inhibitors

α-Amylase inhibitor (αA) activity was made on the basis of the method described by Jakubczyk et al. [26]. α-Amylase from hog pancreas (50 U/mg) was dissolved in the 100 mM phosphate buffer (containing 6 mM NaCl, pH 7.0). To measure the α-amylase inhibitory activity, a mixture of 25 μL of α-amylase solution and 25 μL of sample was firstly incubated at 40 ◦C for 5 min. Then, 50 μL of 1% (*w*/*v*) soluble starch (dissolved in 100 mM phosphate buffer containing 6 mM NaCl, pH 7) was added. After 10 min, the reaction was stopped by adding 100 μL of 3,5-dinitrosalicylic acid (DNS) and was heated for 10 min. The mixture was then made up to 300 μL with double distilled water and absorbance 540 nm was measured using BioTek Microplate Readers. One αA inhibitory unit (AIU) was as the activity of αA that inhibited one unit of enzyme. αA was as AIU/mg of sample.

#### 2.7.7. Activity of α-Glucoamyalse Inhibitors

α-Glucoamyalse inhibitor (αG) activity was made on the basis of the method described by Jakubczyk et al. [26]. Firstly, 10 μL of α-glucosidase (1 U/mL) and 20 μL 1% saccharose were added to 0.5 mL of 0.1 mol/L phosphor buffer, pH 6.8. The reaction was incubated at 37 ◦C for 5 min, stopped by adding 100 μL of 3,5-dinitrosalicylic acid (DNS), and heated for 10 min. The mixture was then made up to 300 μL with double distilled water and absorbance 540 nm was measured. For the αGIA measurement, 10 μL of α-glucosidase (1 U/mL) and 50 μL of the sample were added to 0.45 mL of 0.1 mol/L phosphor buffer pH 6.8. After the incubation at 37 ◦C for 5 min, 20 μL of 1% saccharose was added. The reaction was incubated at 37 ◦C for 50 min, stopped by adding 100 μL of 3,5-dinitrosalicylic acid (DNS), and heated for 10 min. The absorbance was tested at 540 nm using BioTek Microplate

Readers (Bad Friedrichshall, Germany). One αG inhibitory unit (αGAU) was as the activity of αG that inhibited one unit of enzyme. αGA was as AIU/mg of sample.

#### 2.7.8. Activity of Lipase Inhibitors

Lipase inhibitory (LP) activity was made on the basis of the method described by Jakubczyk et al. [26]. First, 2 μL, 100 mg mL−<sup>1</sup> was added to 5 μL of the sample and 142 μL of 100 mM potassium phosphate buffer, pH 7.5. After preincubation at 30 ◦C for 3 min, the reaction was initiated by mixing the reaction mixture with 1 μL of a 100 mM pNPA solution in dimethyl sulfoxide (DMSO). The absorbance was tested at 405 nm using BioTek Microplate Readers. Lipase One LP inhibitory unit (AIU) was as the activity of inhibitor that inhibited one unit of enzyme. LP was as IU/mg of sample.

#### 2.7.9. Theoretical Approach

For a clear picture of the relationships between the activity of sample and bioaccessibility of their phenols, as well as pro-healthy properties, the following parameters were described by Gawlik-Dziki et al. [17].

Phenolics bioaccessibility index (ACP), which is an indication of the bioaccessibility of phenolic compounds [15]:

$$\text{ACP} = \text{C}\_{\text{D}} / \text{C}\_{\text{R}}$$

where CD = amount of components after simulated gastrointestinal digestion and CR = amount of components after chemical extraction (raw extract).

The biological bioaccessibility index (BAC), which is an indication of the bioaccessibility of antioxidative compounds [15]:

$$\mathbf{BAC} = \mathbf{A}\_{\mathbb{R}} / \mathbf{A}\_{\mathbb{D}}$$

where AD = extract after simulated gastrointestinal digestion and AR = raw extract.

#### *2.8. Relative Digestibility (RD)*

#### 2.8.1. Relative Digestibility Proteins

The RD of proteins was expressed as the differences in the content of free amino groups (FAGs) evaluated for the samples after the in vitro digestion [27]. The amount of FAG was evaluated using the TNBS method [28]. Rye bread (20 μL) was prepared with 0.2 M NaH2PO4 buffer (0.980 mL; pH 8.0) and 0.1% TNBS (0.5 mL). After 30 min, the absorbance was tested at 340 nm and the amount of FAG was as L-leucine standard (μg per mL).

#### 2.8.2. Relative Digestibility Starch

The RD of starch was as the difference in the content of reducing sugars (RS) evaluated for the samples after the in vitro digestion [27]. The amount of RS was evaluated by DNSA. Bread (0.2 mL) was prepared with H2O (0.3 mL) and DNSA reagent (0.5 mL). Next, the substance was incubated at 100 ◦C/10 min. After that, the absorbance was tested at 540 nm. The amount of RS was as maltose standard (μg per mL).

#### *2.9. Statistical Analysis*

All experimental results were mean ± SD of two parallel experiments (*N* = 18 for bioactive compounds, enzymatic activity, and relative digestibility; *N* = 20 for colour parameters). Extractions were repeated three times for all analyzed samples. One and multi-way analysis of variance (ANOVA), Duncan's multiple range, as well as median test were analyzed by Statistica 12.5 (Kraków, Poland).

#### **3. Results and Discussion**

#### *3.1. Sensory Evaluation and Colour Parameters*

The sensory assessment of rye breads demonstrated that the use of pure fruit powder (FP), encapsulated fruit powders with maltodextrin (FPM), and encapsulated fruit powders with inulin (FPI) had an insignificant effect on loaf appearance (Figure 1)—the highest scores were given to BRP3, BRM3, and BRI3, as well as to BRM4 and BRI4 breads, whereas the lowest ones were given to the breads with 6% content of the functional additive. In turn, statistically significant differences were noted in terms of aroma and taste, mainly in the breads with 5% and 6% contents of FP, FPM, and FPI compared with the breads with 1% and 4% contents of the functional additive. This happened probably because this content of the additive contributed to the bitter aftertaste and fruity aroma that merged with the acid flavor of rye bread. Deterioration of the sensory traits of breads with berry fruit can also result from a high content of procyanidin polymers that may cause a bitter and astringent aftertaste [13,29]. The highest scores were given by panelists to the breads with 3% and 4% contents of the powders, which were the most effective in making the rye bread taste intensity milder. Considering crumb porosity and elasticity, no statistically significant differences were noted, except for the breads with fruit powder levels of 5% and 6%, in which the values of these parameters slightly decreased. Summing up, the highest score in the overall acceptability assessment was given by the panelists to the bread with 3% content of the functional additive, regardless of functional additive type, as well as to the product with 4% content of the microencapsulated additive. The panelists evaluated this product as more desirable than the control bread. For this reason, the test can be successfully used for the production of sourdough rye breads. In turn, the microencapsulation of the functional additives allowed increasing its content in bread to 4%. This was also confirmed in a study that demonstrated that breads with the addition of a microencapsulated extract from *Garcinia cowa* fruit were scored higher by panelists than the crude fruit extract [13]. Rye breads could also be successfully supplemented with 5% of tomato pomace additive, which improved their taste values [4]. Other investigations have confirmed the feasibility of fortifying rye breads with saffron at levels of 0.08% and 0.12% [30] as well as with grape pomace at the level up to 6% [6], with both additives ensuring product acceptability by consumers.

Saskatoon berry fruit added as a supplement to rye bread (pure powder (FP) and powders encapsulated with maltodextrin (FPM), and inulin (FPI)) significantly diversified the colour of the crumb and crust of rye bread (Table 1). After preparation of rye bread, it was observed that, with the higher amount of FP, FPM, and FPI in rye product production, the L\* parameter was lower, which indicates that the enriched rye breads were darker. Thus, BRC was brighter compared with breads with functional additives. In addition, the higher the fruit powder used, the higher the value of the a\* parameter and the lower the value of the b\* parameter. In turn, the use of encapsulated powder masked the red colour of the fruit powder used. Similar observations of colour were noted by Ezhilarasi et al. [13] for wheat bread supplemented with a fruit powder made from *Garcinia cowa*. The ΔE parameter was calculated for rye bread enriched with additives from pure FP and encapsulated FPM, as well as FPI, and the higher the proportion of additives, the higher the ΔE parameter value. The values of ΔE parameter for crumb bread with 1% and 2% of additives were <5 units, which indicates no difference to distinguish the colours of the two products. Meanwhile, the highest saturation of crust colour was noted in the sample with 6% of additives. Similar observations of change of colour were obtained by Bajerska et al. [30] through the use of saffron to make rye bread. In the case of pure and encapsulate fruit powder, no significant difference in color was noted.

**Figure 1.** Sensory evaluation of rye bread fortified with fruit powder (**A**), fruit powders covered with maltodextrin (**B**), and fruit powder covered with inulin (**C**).


**Table 1.** The colour parameter of enriched rye bread. FP, pure fruit powder; FPM,

encapsulated

 fruit powders with

maltodextrin;

 FPI,

encapsulated

 fruit powders

*Antioxidants* **2020** , *9*, 614

374

#### *3.2. Phenolic Compounds and Their Relative Bioaccesibility*

The available literature provides sparse reports on rye bread enrichment with plant material [4–6,29]. The effect of rye bread fortification through the addition of pure fruit powder (FP), encapsulated fruit powders with maltodextrin (FPM), and encapsulated fruit powders with inulin (FPI) on the profile and content of polyphenolic compounds is presented in Table 2. Protective effects of carriers on polyphenolic compounds were demonstrated. The content of polyphenolic compounds in powders additionally protected by carriers after freeze-drying was two times higher compared with pure powders (Table S1) [18,19]. Additionally, enriched products in microencapsulated powders after bread baking contained on average a 1.8 times higher content of polyphenolic compounds than breads enriched with pure powder compared with the content of these compounds before baking [18,19]. The total content of polyphenols in BRC reached 127.2 mg/100 g d.s. Already at 1% FP, the mean content of polyphenols was three times higher compared with the control bread, while the 6% addition of crude FP caused a 13-fold increase. In the breads enriched with microencapsulated FPM and FPI, the content of phenols was by ca. 10% and 12% higher than in the breads with crude FP. This could be owing to the protection of these compounds during bread baking. Many authors have confirmed that carriers used in the microencapsulation process protected bioactive compounds from degradation [7,31,32], while the high temperature used during bread baking could contribute to the degradation of phenols in the breads with crude FP. Compared with the breads fortified with crude FP, greater protection of compounds was noted in the group of anthocyanins and in the group of phenolic acids, which reached 11% and 8% as well as 8% and 15% in the breads with FPM and FPI, respectively. These compounds are particularly unstable at high temperatures [18,31,33] and require additional protection that can be ensured by the microencapsulation process. In addition, our observations concerning bread fortification were consistent with earlier reports addressing rye bread enrichment with saffron [30], tomato pomace [4], green tea [5], and grape pomace [6], which demonstrated that the supplementation of rye breads contributed to an increase in the content of polyphenolic compounds.







Valuesdetected; BC—control rye bread; BR1–BR5—breads with fruit powders (1–6%); FP—additives fruit powders; FPM—additives of encapsulated fruit powders with maltodextrin; FPI—additives of encapsulated fruit powders with inulin.

The use of the functional additive, especially in the form of FPM and FPI, significantly improved the content of polyphenolic compounds in the products examined; however, relative bioaccesibility assessment in the simulated digestive system in vitro was conducted for better identification of the fortification effect in the products with the highest level of the additive (3%) acceptable by consumers (Table 3). Compared with the chemical extracts, the results obtained after simulated in vitro digestion demonstrated that the control sample had a 1.4-fold higher content of phenolic compounds, including a 1.3-fold higher content of flavan-3-ols, as well as a 3.4-fold higher content of phenolic acids, and a 1.2-fold lower content of flavonols. The relative bioaccesibility index computed for BRC showed that phenolic acids were more bioaccessible than the other groups of polyphenolic compounds. In turn, compared with the chemical extracts, the samples of bread enriched with FP obtained after in vitro digestion had a 1.3-fold higher content of polyphenolic compounds, including 5.5-fold and 1.2-fold higher contents of anthocyanins and flavonols, as well as 1.7-fold and 1.2-fold lower contents of phenolic acids and flavan-3-ols, respectively. Analogously, the breads fortified with FPM contained 2-fold higher amount of polyphenols, including 6.1, 1.1, 1.4, and 1.3 times more anthocyanins, flavan-3-ols, phenolic acids, and flavonols, respectively, compared with the chemical extracts. In turn, the breads supplemented with FPI were also characterized by a 2-fold higher content of phenols, including 6.4-fold, 1.2-fold, and 1.3-fold higher contents of anthocyanins, flavan-3-ols, and phenolic acids, respectively, as well as by a 1.2 times lower content of flavonols. The relative bioaccesibility index estimated for the enriched rye bread demonstrated that anthocyanins were highly bioaccessible, and that flavan-3-ols and phenolics were poorly bioaccessible upon the use of FP. The highest relative bioaccesibility index was computed for the breads with FPM addition followed by those with FPI addition, which may indicate that these carriers, and maltodextrin in particular, contributed to greater release of the compounds tested during in vitro digestion. This observation can be explained by better protection of these compounds during bread baking. Besides, it has been described earlier that the digestion process itself can enhance the release of phenolic compounds [34,35], while the low relative bioaccesibility of the compounds could suggest interactions with bread matrix components, which had earlier been reported for the wheat bread enriched with green coffee grains or with broccoli sprouts [17]. The results obtained may also suggest that the use of the powdered functional additive increased the content of phenols, which affected a higher relative bioaccesibility of the rye bread examined, which is indicative of the additive's effectiveness. In contrast, the microencapsulated functional additive was more effective than the additive without the carrier in increasing the potential relative bioaccesibility of the compounds tested. This was also confirmed by Vitaglione et al. [14], who demonstrated that the fortification of wheat bread with microencapsulated curcumin contributed to greater relative bioaccesibility of bioactive compounds compared with the non-microencapsulated material. According to the authors above, this could be owing to the impaired interactions between the compounds tested and bread matrix. However, the mechanisms of action of the microencapsulated functional additives during simulated in vitro digestion of rye bread require further extensive research. Such attempts will be undertaken in the future.


**Table3.**Potentiallybioaccessiblephenoliccompoundsinthecontrolandenrichedryebread.

Values are expressed as the mean (n = 18) ± standard deviation. Mean values bearing different letters in the same row denote statistical difference (a > b > c ... etc.). BRC—rye bread control; BRP3—rye bread with 3% of fruit powder; BRM3—rye bread with 3% of fruit powders with maltodextrin; BRI3—rye bread with 3% of fruit powders with inulin; FP—additives fruit powders; FPM—additives of encapsulated fruit powders with maltodextrin; FPI—additives of encapsulated fruit powders with inulin; PC—sum of phenolic compounds; ANT—sum of anthocyanins; FL—sum of flavonols; PA—sum of phenolic acid; F3O—sum of flavan-3-ols (monomers and oligomers); F3O1—B-type procyjanidin dimer; F302—epigallocatechin; F3O3—B-type procyanidin dimer; F3O4—(-)-Epicatechin; F3O5—(+)-Catechin; FL1—kampferol-3- *O*-galactoside; FL2—kampferol-3- *O*-glucoside; FL3—quercetin-3- *O*-arabinoglucoside; FL4– quercetin-3- *O*-rutinoside; FL5—quercetin-3- *O*-robinobioside; FL6—quercetin-3- *O*-galactoside; FL7—quercetin-3- *O*-glucoside; FL8—quercetin-3- *O*-arabinoside; FL9—quercetin-3- *O*-xyloside; FL10—quercetin-3- *O*-deoxy-hexoside; ANT1—cyanidin-3-O-galactoside; ANT2—cyanidin-3-O-glucoside; ANT3—cyanidin-3-O-arabinoside; ANT4—cyanidin-3-O-xyloside; PA1—protocatechuic acid; PA2—vanilic acid; PA3—caffeic acid; PA4—3- *O*-caffequinic acid; PA5—ferulic acid; PA6—5-*O*-*p*-coumaroylquinic acid; PA7, 3-*O*-*p*-coumaroylquinic acid; PA8—4-*O*-*p*-coumaroylquinic acid.

#### *3.3. Pro-Healthy Potency and Their Bioaccesibility*

The antioxidant activity of BRC analyzed in ABTS, DPPH, and FRAP tests reached 13.09, 3.04, and 21.00 μmol Trolox/g d.s., respectively (Table 4), and was consistent with the literature data [3]. The addition of Saskatoon berry fruit powder improved the antioxidant potential of the fortified breads compared with BRC. Even the 1% addition of FP, FPM, and FPI to rye bread increased its antiradical activity and reducing properties by 5%, 7.5%, and 6%; 6%, 7%, and 6.5%; as well as by 6%, 8%, and 6.2%, respectively. In turn, the 6% addition of FP, FPM, and FPI increased both the reducing (FRAP) and antiradical potential (ABTS and DPPH) by 39%, 46%, and 46.5%; 39%, 46%, and 47%; as well as by 40%, 47.5%, and 48%, respectively. The increased antioxidant potential of the supplemented rye breads could be owing to sourdough fermentation, because—as reported by Banu et al. [2]—it can increase the extractability of polyphenols from both the additive and rye flour. The baking process of rye bread can also contribute to an increase of its antioxidant potential owing to the appearance of Maillard reaction products [36]. Compared with the use of crude FP, rye bread fortification with microencapsulated FPM and FPI contributed to the greater protection of compounds exhibiting antioxidant potential by 9% and 8% on average in the FRAP assay, by 8% and 7% on average in the ABTS assay, and by 13% and 8% on average in the DPPH assay, respectively. However, there were no statistically significant differences between the microencapsulated additives. Earlier studies have demonstrated rye bread supplementation with grape pomace at the level of 10% to cause a 10-fold increase in its reducing potential [6]. In turn, rye bread fortification with 0.16% of saffron resulted in a 1.6-fold increase in its antiradical activity [30]. A green tea extract added at the level of 1.1% caused a 13-fold increase in the antioxidant activity of rye bread compared with the control sample [5]. As in the study by Mildner-Szkudlarz et al. [6], the higher value of the antioxidant potential was strongly correlated with the amount of polyphenolic compounds, that is, *r*<sup>2</sup> = 0.801 in the FRAP test and 0.837 in the ABTS test. A similar observation was noted in this work where strong Pearson correlation between polyphenols and antioxidant activity was *r*<sup>2</sup> = 0.928 for FRAP assay, *r*<sup>2</sup> = 0.929 for ABTS assay, and *r*<sup>2</sup> = 0.892 for DPPH assay. In addition, in accordance with the findings reported by Ezhilarasi et al. [13], the microencapsulated functional additives from Garcinia fruits ensured 2-fold greater protection of the antioxidant activity in wheat bread compared with the non-microencapsulated ones. The use of microencapsulated red grape seeds also caused a two times higher antioxidant potential of wheat cookies compared with the product containing a crude additive from grape seeds [37]. The higher antiradical and reducing properties can be owing to the greater protection of bioactive compounds during high-temperature baking [13,37].

However, the antioxidant potential of the bioaccessible fraction of the breads with 3% content of the additive was significantly higher compared with that of the chemical extracts, that is, by 20%, 23%, and 50% in the case of BRP3; by 63%, 27%, and 64% in the case of BRM3; and by 54%, 26%, and 53% in the case of BRI3, while measured with the FRAP, ABTS, and DPPH tests, respectively (Table 5). The analyses demonstrated that in vitro digestion could affect the release of polyphenols from a bread matrix, thereby leading to an increase in their antioxidant activity [33]. Similar results were obtained upon wheat bread enrichment with flaxseed hulls [38] and green coffee extracts [35]. The value of the relative bioaccesibility index computed for the antioxidant activity pointed to high in vitro relative bioaccesibility of these breads. In the FRAP test, the highest value of the relative bioaccesibility index was demonstrated for BRI3 (2.36), whereas the lowest one was for BRC (0.71), while in the ABTS and DPPH test, the highest value of this index was shown for the products containing the functional additive microencapsulated with maltodextrin (3.15 and 2.78). In turn, earlier studies have shown that interactions of bioactive compounds with the matrix of food products can reduce their antioxidant potential [27], which was noted in the case of BRC in our study. In turn, the highest relative bioaccesibility of the microencapsulated functional additives can be owing to the better release of compounds with the antioxidant activity during in vitro digestion or to the protective effect on the formation of complexes with rye bread components. Similar observations were made for the wheat bread enriched with microencapsulated curcumin [14].


**Table 4.** The antioxidant activity of rye bread fortification with functional additives without and with carriers.

<sup>1</sup> Values are expressed as the mean (n = 18) ± standard deviation. Mean values bearing different letters in the same row denote statistical difference (a > b > c ... etc.). BC—control rye bread; BR1–BR6—breads with fruit powders (1–6%); FP—additives fruit powders; FPM—additives of encapsulated fruit powders with maltodextrin; FPI—additives of encapsulated fruit powders with inulin; ABTS—2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid; DPPH—2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl.


**Table 5.** Ability to inhibit the activity of enzymes related to metabolic syndrome.

<sup>1</sup> Values are expressed as the mean (n <sup>=</sup> 18) ± standard deviation. Mean values bearing different letters in the same row denote statistical difference (a > b > c ... etc.). EBD—extracts buffer digestion; PBE—potentially bioaccesible fraction; BRC—rye bread control; BRP3—rye bread with 3% of fruit powder; BRM3 —rye bread with 3% addition of fruit powder microcapsulated with maltodextrin; BRI3—rye bread with 3% addition of fruit powder microcapsulated inulin; LOX— lipoxygenase; COX— cyclooxygenases.

Many of the previous studies have demonstrated the in vitro digestion to act as an extractor of compounds with potential relative bioaccesibility and capable of inhibiting enzymes responsible for the induction of inflammatory conditions, including the activity of lipoxygenase (LOX) [17,39]. The effect of in vitro digestion on the selected activities of bread with the optimal content of the functional additive (3%) is presented in Table 5. The highest ability to inhibit LOX activity in the buffered extracts was determined in BRP3 samples, and it was 1.2 times higher compared with BRC. There were no statistically significant differences in the capability for LOX activity inhibition between various forms of the additive (BRC, BRM3, and BRI3). The in vitro digestion caused an increase in the ability to inhibit LOX activity by ca. 29%, with the highest increase noted for BRP3 (over 50%). The relative bioaccesibility index pointed to a high relative bioaccesibility of the supplemented breads, that is, 2.04 (BRP3) 1.25 (BRM3), and 1.37 (BRI3), whereas poor relative bioaccesibility was demonstrated for BRC (0.56). It can be hypothesized that the breads fortified with the fruit powder can suppress the generation of reaction oxygen species (ROS) at the lipoxygenase pathways, thus leading to the inhibition of inflammatory conditions in the body, particularly in the gastrointestinal tract wherein the ROS can lead to the development of carcinogenic lesions [40].

In addition, the breads with the functional additives were determined for their capability to inhibit cyclooxygenases (COX), compared with BRC. The addition of crude FP to bread caused a 2-fold increase in the ability to inhibit COX-1 activity, whereas no such activity was demonstrated in BRM3 and BRI3 products (Table 5). The in vitro digestion caused a 2.5-fold suppression in COX-1 inhibiting activity of BRC, whereas no such activity was observed in the breads fortified with functional additives. In turn, the anti-inflammatory activity analyzed against the inhibition of COX-2 activity in the fortified breads BRP3, BRI3, and BRM3 was 1.3-fold, 2.1-fold, and 2.5-fold higher, respectively, compared with BRC. In the potentially bioaccessible fraction, the best COX-2 inhibiting effect was achieved for BRM3 and BRI3. The potential to inhibit the activity of COX-2 is significant as it is activated in the event of the inflammatory reaction of the body [41] and is responsible for the synthesis of prostaglandin E2, which promotes the development of carcinogenic lesions [42]. The high anti-inflammatory activity against COX-2 can be owing to a higher content of polyphenolic compounds in the products. It was confirmed in the study conducted by Moschon et al. [43], who noted that the microencapsulated products characterized by a higher content of phenols had a higher biological value, including the anti-inflammatory activity.

The use of Saskatoon berry fruits microencapsulated with inulin had only a negligible effect on AChE activity inhibition (an increase by 5%) compared with BRC, whereas no AChE inhibition was noted in the breads with 3% addition of FP and FPM. In turn, the negligibly higher ability to inhibit the activity of this enzyme determined in the products with FPI addition can be owing to the health-promoting properties of inulin [44]. Unfortunately, this activity was not detected in the fractions obtained after in vitro digestion.

Investigations conducted so far have proved that polyphenolic compounds can inhibit the activities of digestive hydrolases, including the activity of pancreatic lipase, being responsible for dietary fat absorption, as well as activities of α-glucosidase (αG) and α-amylase (αA) responsible for the hydrolysis of carbohydrates [10,12]. All additives used for bread fortification were able to inhibit αA activity. The highest inhibition was demonstrated for BRP3 and BRM3, and it was 2.8 and 2.6 times higher compared with BRI3, and 5.3 and 5.6 times higher compared with BRC (Table 6). The control bread exhibited a marginal ability to inhibit αG. This activity was a dozen times higher in BRP3, BRM3, and BRI3. In turn, the ability to inhibit the activity of pancreatic lipase was demonstrated for BRM3 and BRI3, and it was 1.3 and 1.2 times higher compared with BRC, and 1.3 and 1.4 times higher compared with BRP3. It can be concluded that the use of functional additive had a positive effect on the breads' ability to inhibit activities of αA and αG. In contrast, only the microencapsulated additives inhibited the activity of pancreatic lipase. The high inhibition of digestive enzymes can be owing to the presence of polyphenolic compounds in the additives. This was confirmed by Zhang et al. [45], who noted a strong correlation between the amount of polyphenols and the anti-diabetic activity of lentil. However, an undesirable effect was observed in the rye breads fortified with the microencapsulated powder, which involved the reduction of the potential to inhibit activities of αG and αA compared with FP. It is likely that the carrier can be responsible for masking enzyme inhibitors (polyphenols) and inhibiting their interactions. Considering the phenolic profile of the analyzed breads, it can be speculated that they effectively influence the inhibition of hyperglycemia and obesity, likewise in the study conducted by Zhao et al. [12], who demonstrated that powders from *Amelanchier alnifolia* Nutt. could alleviate hyperlipidemia, hyperglycemia, and blood vessel inflammation induced by high-saccharose and high-fat diet. In addition, the research carried out by Bajerska et al. [30] proved that rye bread enrichment with saffron could lead to the enhanced secretion of insulin and reduced blood levels of glucose and triglycerides. Furthermore, as shown by the literature data [46], the sourdough

rye bread itself displays high anti-diabetic and anti-obesity potentials and, according to results of our study, these effects can be enhanced by rye bread fortification. Nevertheless, these metabolic effects need to be further explored in future research.


**Table 6.** Enzymatic in vitro inhibition activity of enriched rye bread.

<sup>1</sup> Values are expressed as the mean (n <sup>=</sup> 18) ± standard deviation. Mean values bearing different letters in the same row denote statistical difference (a > b > c ... etc.). BRP3—rye bread with 3% of fruit powder; BRM3—rye bread with 3% of fruit powders with maltodextrin; BRI3—rye bread with 3% of fruit powders with inulin.

#### *3.4. Relative Digestibility of Starches and Proteins*

Functional additives with a high content of polyphenols usually reduce the relative bioaccesibility of nutrients. Considering the above, analyses were conducted to determine the effect of the functional additives on the relative digestibility of starch and proteins (Table 7). In the case of starch digestibility, it was found to decrease upon rye bread enrichment with FP, FPM, and FPI, with the most significant decrease determined in BRP3 (ca. 25%) and BRM3 (ca. 21%). Similar observations were made upon wheat bread enrichment with green coffee powder [35]. The fortification of wheat bread with sorghum flour also led to a ca. 40% decrease in the relative digestibility of starch [47]. Investigations conducted so far have shown that the reduced digestibility of starch and protein can be attributed to polyphenolic compounds present in the plant material, chlorogenic acid in particular [48]. In turn, the fruit powder with the addition of maltodextrin and inulin only slightly decreased protein digestibility (by ca. 16% and 14%, respectively), whereas the use of FP increased it by 27%. Reduced digestibility of protein and starch was also reported upon pasta enrichment with carob flour [27]. In turn, the decreased digestibility of proteins in the in vitro analyses can be owing to the interactions between bioactive compounds of plant origin and components of the bread matrix, leading to the formation of complexes that can be either completely excreted with digested products or inhibit activities of digestive enzymes in the gastrointestinal tract [27]. In addition, the reduction in the relative digestibility of protein can be owing to the fact that rye bread may be a better substrate for digestive enzymes [2].

**Table 7.** Relative digestibility of starch and proteins.


<sup>1</sup> Values are expressed as the mean (n <sup>=</sup> 18) ± standard deviation. Mean values bearing different letters in the same row denote statistical difference (a > b > c ... etc.). BRC—rye bread control; BRP3—rye bread with 3% of fruit powder; BRM3—rye bread with 3% addition of fruit powder microcapsulated with maltodextrin; BRI3—rye bread with 3% addition of fruit powder microcapsulated inulin.

#### **4. Conclusions**

The enrichment of rye breads with fruits offers an effective method for the improvement of their biological value. The addition of Saskatoon berry fruit powders to rye bread caused a significant increase in the content of their polyphenols and their antioxidant activity, compared with the control products. The higher the content of the functional additive, the higher the content of antioxidant compounds and their particular groups. Bread supplemented with 3% of the fruit powder, regardless of its form, was acceptable in terms of its sensory attributes and colour. The simulated in vitro digestion showed that anthocyanins of the supplemented rye bread were highly bioaccessible compounds, whereas the least bioaccessible turned out to be flavan-3-ols. Among the functional additives studied, the highest value of the antioxidant potential and the highest relative bioaccesibility of flavonols, flavan-3-ols, and phenolic acids were achieved in BRM3. The addition of fruits caused an insignificant reduction in the relative digestibility of starch and proteins. Bread fortification led to the enhanced capability for the inhibition of α-glucosidase and α-amylase activities, whereas in the case of BRM3 and BRI3 analyses, it additionally showed the ability to inhibit the activity of pancreatic lipase and cyclooxigenase-2 as well as a low inhibitor activity against acetylcholinesterase. On the basis of the above results, it can be concluded that the Saskatoon berry fruit powders, especially these subjected to the microencapsulation process, are valuable and prospective functional additives that increase the attractiveness and nutritive value of rye bread.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/7/614/s1, Table S1: The content of phenolic compounds in pure fruit powder, encapsulated fruit powders with maltodextrin, and encapsulated fruit powders with inulin [mg/100 g d.s.].

**Author Contributions:** Conceptualization, S.L.; prepared of sample, S.L. and E.P.; methodology, S.L. and M.S.; ´ writing—original draft preparation, S.L.; writing—editing, S.L.; writing—review, S.L.; project administration, S.L.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Wrocław University of Environmental and Life Sciences; grant name "Innovative scientist", grant number B030/0032/20.

**Acknowledgments:** The work was created in a leading research team 'Food&Health' (S.L.).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Peptides from Di**ff**erent Carcass Elements of Organic and Conventional Pork—Potential Source of Antioxidant Activity**

### **Paulina Keska 1, Sascha Rohn 2, Michał Halagarda <sup>3</sup> and Karolina M. Wójciak 1,\***


Received: 3 August 2020; Accepted: 3 September 2020; Published: 7 September 2020

**Abstract:** The growing consumer interest in organic foods, as well as, in many cases, the inconclusiveness of the research comparing organic and conventional foods, indicates a need to study this issue further. The aim of the study was to compare the effects of meat origin (conventional vs. organic) and selected elements of the pork carcass (ham, loin, and shoulder) on the meat proteome and the antioxidant potential of its peptides. The peptidomic approach was used, while the ability of antioxidants to scavenge 2,2 -azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS), to chelate Fe(II) ions, and to reduce Fe(III) was determined. Most peptides were derived from myofibrillary proteins. The meat origin and the element of the pork carcass did not have a significant effect on the proteome. On the other hand, the pork origin and the carcass element significantly affected the iron ion-chelating capacity (Fe(II)) and the reducing power of peptides. In particular, pork ham from conventional rearing systems had the best antioxidant properties in relation to potential antioxidant peptides. This could be a factor for human health, as well as for stabilized meat products (e.g., toward lipid oxidation).

**Keywords:** antioxidant peptides; element of pork carcasses; spectrometric analysis

#### **1. Introduction**

The food consumption pattern has changed over the years. Currently, consumers appreciate foods that, in addition to favorable sensory properties, are characterized by high nutritional value and potential health benefits [1,2]. Therefore, an increasing interest in organic foods can be observed [3,4]. Nonetheless, many consumers still do not believe in the favorable characteristics of organic food products. Moreover, the research findings regarding the extensive, positive effects of the consumption of such foods are not comprehensive enough; in many cases, they are also inconclusive [5–8], which indicates the need to study this issue further.

Meat consumption, although discussed sometimes quite controversially, is a good source of many highly valuable nutrients [9]. Meat has a complex physical structure and chemical composition, highly sensitive to enzymatic activities. Glycolysis and rigor mortis, occurring in the muscles within the first 24 h of slaughtering, are significant processes that can regulate proteolysis and affect the peptide profile of muscle tissue. Proteins involved in these biochemical processes are subject to complex metabolic regulations that significantly contribute to the final quality of the meat. Moreover, the potential resulting peptides not only have nutritional value, but also carry further benefits for

human health [10]. Meat-derived peptides have been shown to have antioxidant, antihypertensive, antidiabetic, antimicrobial, opioid, anticoagulant, and other bioactive effects. Regulation of the immune, gastrointestinal, and neurological responses of these bioactive peptides is an important basis for the prevention of noncommunicable diseases such as hypertension, obesity, diabetes, and other metabolic disorders [11]. In addition to nutritional value, natural antioxidants might also be beneficial for stabilizing products with regard to rancidity via lipid peroxidation [12].

Before being released from parent proteins, peptides remain latent and do not show any bioactive effect. Their potential activity is only activated after the release of specific amino-acid sequences, through gastrointestinal digestion or food processing (e.g., drying, curing, fermentation, or enzymatic hydrolysis). The literature data indicate processed meat products as good sources of peptides with biological activity, particularly dry-cured or fermented meats [13–16]. For example, the antiradical activity of protein extracts from dry-cured pork loins over a 12-month period of aging was confirmed. Moreover, antiradical activity was further observed in peptic and pancreatic hydrolysates after simulated gastrointestinal digestion [14]. However, the chemical composition of meat also promotes oxidation processes. Ingredients susceptible to oxidation, i.e., polyunsaturated fatty acids, cholesterol, proteins, and pigments, should be in balance with endogenous antioxidant substances. Endogenous antioxidant systems consist of nonenzymatic compounds such as tocopherols, ascorbic acids, carotenoids, and ubiquinols, as well as enzymes (superoxide dismutase, catalase, and glutathione peroxidase). Furthermore, peptides such as anserine and carnosine have high antioxidant activity, acting as free-radical scavengers and metal-ion chelators. Peptide formation via hydrolytic reactions is the main technique used to create antioxidants from proteins, as the resulting peptides have significantly advanced antioxidant potential compared to intact proteins [17,18].

According to the literature, meat peptides can contribute to maintaining the oxidative stability of meat tissue [18–20]. Lipids are particularly vulnerable to oxidative factors, especially during meat processing. The carbonyl-based compounds known as secondary lipid oxidation products are characterized by cytotoxic and genotoxic properties [17]. In addition to exogenous/added antioxidants or endogenous small-molecular antioxidants (e.g., vitamins), lipid oxidation in meat and meat products can also be inhibited by proteins as a result of biologically designed mechanisms (such as iron-binding proteins and antioxidant enzymes) or via some non-specific mechanisms. The activity of these mechanisms can be intensified when the conformation of proteins is denatured. Previous studies showed that <3.5 kDa peptides have strong antioxidant activity. They can function as inhibitors of lipid oxidation, leading to color change or discoloration during prolonged storage in uncured roasted beef with acid whey [15,21]. Moreover, the role of proteins, as important components of muscle tissue, in developing the sensory profile of meat products was emphasized. Among them, myofibrillar proteins proved to be particularly good flavor precursors, i.e., active peptides and amino acids. The suppressing (sourness and sweetness) and enhancing (salty and umami) role of peptides was also presented in pork meat based on an in silico approach [22]. Apart from improving the shelf-life and organoleptic qualities of meat, controlling lipid-based oxidative degradation contributes to preventing the negative effects of oxidative stress, which is generally caused by the excessive accumulation of reactive oxygen species (ROS). It is an imbalance between the production and accumulation of ROS and endogenous defense mechanisms (e.g., enzymes, vitamins) in cells and tissues. Consequently, the limited capacity of the biological system to detoxify these reactive products can lead to disease-causing pathological conditions. Importantly, according to some literature reports, elevated levels of oxidative stress play a dominant role in initiating many cardiovascular diseases, diabetes mellitus, and other metabolic disorders [23]. Antioxidants are effective in combating the effects of oxidative stress. In this context, digestive amino acids, peptides, and proteins from different food sources can also act as antioxidants to protect cells and organisms from oxidative damage [17,18].

The composition of oxidation-promoting factors and antioxidants may vary between the meat of different animal species [24,25] or gender [26]. In addition, the animal's diet plays a significant role in modifying the concentration of antioxidants or pro-oxidative factors [27–30]. Therefore, different biomolecular compositions of meat from slaughter animals can affect the functionality and use of a particular muscle tissue for specific applications. Thus, it is necessary to know and understand the differences in the biomolecule profiles between different carcass elements of the animal muscles. The understanding of any differences or similarities between them, determined based on the abundance in protein composition and related peptides, can be helpful in achieving this goal. Moreover, the antioxidant potential of peptides from selected elements of organic vs. conventional pork carcasses has not been compared before.

Therefore, the aim of the present study was to investigate the impact of organic and conventional rearing systems on protein stability and peptide formation with regard to their antioxidant activity in pork meat available for consumers. Furthermore, various pork tissues such as ham, loin, and shoulder, in relation to their potential to be used as starting materials for obtaining high-quality functional ingredients or meat products, were characterized.

#### **2. Materials and Methods**

#### *2.1. Meat Samples*

The research was conducted on organic and conventional pork meat. The material consisted of three elements of pork meat: loin, ham, shoulder. The meat, intended for consumer market, was bought in the same, organically certified slaughterhouse, which enabled the elements to be cut out fresh from the same carcass. Eighteen meat samples (organic and conventional) were selected. The samples were vacuum-packed and transported, in refrigerated conditions, without any exposure of light, directly to the analytical laboratory. The analysis of the samples was carried out 24 h after the slaughter. Before the analysis, the whole-muscle samples were purified by removing external fat and membranes. The experiment was repeated three times. The research aimed at simultaneous verification of all the conditions connected to farming (organic/conventional), as well as their effect on the difference in protein degradation and formation of bioactive peptides with antioxidant activity from meat available for consumers. However, an interview conducted with the manager of the slaughterhouse made it possible to determine the following:


#### *2.2. Peptide Extraction and LC–MS*/*MS Identification*

The peptides were isolated from the meat samples according to the procedures provided by Mora et al. [13]. Briefly, muscles (15 g) were homogenized with 100 mL of 0.01 N HCl for 5 min. The homogenate was centrifuged (2200 rpm for 20 min at 4 ◦C). The supernatant was decanted and filtered through glass wool, and then deproteinized by adding three volumes of ethanol and centrifuged, retaining the previously defined conditions. The obtained supernatant was then dried in a vacuum evaporator (Rotavapor R-215, BüchiLabortechnik AG, Flawil, Switzerland). The dried extract was dissolved in 0.01 N HCl, filtered through a 0.45 μM nylon membrane filter (Millipore, Bedford, MA, USA) and stored at −60 ◦C prior to further use. The peptides were analyzed by liquid chromatography coupled with tandem electrospray mass spectrometry (LC–MS/MS). The samples were concentrated and desalted on an RP-C18 pre-column (Waters Corp., Milford, MA, USA), and further peptide separation was achieved on an RP-C18 nano-Ultra Performance column (Waters) using a 180 min linear acetonitrile gradient (0–35%) at a flow rate of 250 nL·min<sup>−</sup>1. The outlet of the column was directly connected to a mass spectrometer (Orbitrap Velos, Thermo Fisher Scientific Inc. Waltham, MA, USA). The raw data files were preprocessed using Mascot Distiller software (version 2.4.2.0, Matrix Science Inc., Boston, MA, USA). The obtained peptide masses and their fragmentation pattern were compared with the protein sequence database (UniProt KB, www.uniprot.org) using the Mascot search engine (Mascot Daemon v. 2.4.0, Mascot Server v.2.4.1, Matrix Science, London, UK). The "mammals" option was chosen as the taxonomy constraint parameter. The following search parameters were applied: enzyme specificity, none; peptide mass tolerance, 5 ppm; fragment mass tolerance, 0.01 Da. The protein mass was left unrestricted, and mass values were monoisotopic with a maximum of two missed cleavages allowed. Methylthiolation, oxidation, and carbamidomethylation were set as fixed and variable modifications. The sequences of peptides from unknown original proteins were not listed. The peptide identification was performed using the Mascot search engine (Matrix Science), with a probability-based algorithm. An expected value threshold of 0.05 was used for the analysis (all peptide identifications had less than 0.05% chance of being a random match).

#### *2.3. The Identification of Bioactive Peptides—In Silico Analysis*

The peptides identified in meat samples were investigated as a source of bioactive peptides in relation to the information about peptides previously identified in the literature, using the BIOPEP-UWM database (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep; access: December 2019) [34].

#### *2.4. Evaluation of Bioactive (Antioxidant) Peptides—In Vitro Analysis*

#### 2.4.1. Ability to Scavenge 2,2 -Azino-bis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS)

The ability of the extracts obtained to scavenge free radicals was tested using the method of Re et al. [35] with the free-radical ABTS. The degree of reduction of ABTS• was determined spectrophotometrically at 734 nm. The scavenging ability was determined using the following formula:

ABTS radical-scavenging activity (%) = (1 − A2/A1) × 100,

where A1 is the absorbance of the control sample, and A2 is the absorbance of the sample.

2.4.2. Ability to Chelate Fe(II) Ions

The study on the ability to chelate Fe(II) ions by compounds contained in sample extracts was conducted according to the method of Decker and Welch [36]. The absorbance of the colored complex was measured spectrophotometrically at 562 nm.

Fe(II)-chelating activity (%) = (1 − A2/A1) × 100,

where A1 is the absorbance of the control sample, and A2 is the absorbance of the sample.

#### 2.4.3. Fe(III) Reduction Power (FRAP)

The FRAP method according to Oyaizu [37] involves the reduction of the reagent (Fe(III)) in stoichiometric excess relative to antioxidants. A spectrophotometric method with measurements at 700 nm was used. A higher absorbance value indicates a higher ability to reduce the test substance.

#### *2.5. Statistical Analysis*

Statistical analysis was carried out using Statistica 13.1 (StatSoft, Cracov, Poland) and Microsoft Office Excel 2013 software. Based on a two-factor analysis of variance (meat element, rearing system), homogeneous groups were separated using the Tukey test. The differences were considered statistically significant at *p* < 0.05. All of the data were presented as mean ± standard error. In the case of multidimensional analyses, a hierarchical grouping of data based on identified peptides was performed, and the results were presented as a heat map and dendrogram, using the Ward method and Euclidean distance. Data were normalized prior to the analysis.

#### **3. Results**

#### *3.1. Protein Degradation and Peptide Formation*

In this study, organic vs. conventional pork and the culinary element of the pork carcass (ham, loin, or shoulder) were compared, taking into account protein stability and peptide formation. Potential differences in the abundance of endogenous peptides were evaluated with an LC–MS-based peptidomic approach. In total, 4178 peptide sequences were identified. The analyses were performed in triplicate, and only the sequences present in three independent replicates were selected for further analysis. Eventually, 646 peptides were chosen for the subsequent stages of the analysis.

The distribution of identified peptides with regard to their protein origin is shown in Figure 1. A diverse number of protein fragments were identified, with no clear tendency depending on the type of muscle. Nevertheless, Picard et al. demonstrated a relationship between the protein profile and the muscle type [38]. The authors revealed relationships between proteins differing in contractile and metabolic properties (acting as biomarkers of tenderness and intramuscular fat content) compared in five bovine muscles [38]. According to Picard and Gagaou, a muscle-type effect depends on the biological function of proteins [39]. The authors emphasized that, in longissimus thoracis muscle, tenderness-related proteins mainly correspond to contractile, structural, and heat-shock proteins, while, in semitendinosus muscle, the tenderness-related proteins are mainly involved in metabolism. In the present study, the rearing system did not affect the protein profile distinctly. This observation is consistent with descriptions by Picard et al., who reported that the abundance of very few proteins from bovine meats was modified by rearing practices [40]. Moreover, factors associated with diet composition had weak effects on protein abundances determined by proteomics [39]. These data indicate that the impact of rearing practices on the proteome is muscle-type-dependent. As expected, several significant proteins were identified. Protein chains were degraded to produce a large number of different peptides, which confirmed proteolytic activity postmortem.

The highest diversity of proteins as peptide precursors was obtained in organically produced shoulder meat, where the highest percentage of the most different proteins (named "other" according to the designations in Figure 1) was classified. The protein generating the highest number of peptides was nebulin (in order, conventionally produced shoulder meat (44.26%) > conventional loin (37.85%) > organic loin (28.68%) > organic ham (20.74%) > conventional ham (6.58%)). Other myofibrillar proteins, especially structural proteins such as desmin, titin, or troponin were also the main sources of peptides in the analyzed meat samples (Figure 1). According to the literature, myosin, nebulin, actin, tropomyosin, tropomodulin, and troponin (assigned a role in stabilizing and determining highly ordered muscle structure), and nebulin, tropomyosin, and tropomodulin ("protein rulers" to precisely regulate the connection of myosin and actin fibers) are very sensitive to proteolysis [41].

**Figure 1.** Distribution in percentages of identified peptides according to the origin of proteins in the analyzed muscle tissue (abbreviations: H-O: organic ham, L-O: organic loin, S-O: organic shoulder, H-C: conventional ham, L-C: conventional loin, S-C: conventional shoulder).

Previous studies of postmortem protein degradation in pig muscle, using peptide profiling and amino-acid sequence analysis of protein fragments, showed that fragments of myofibrillary protein primarily undergo postmortem degradation, before severe deterioration catalyzed by cathepsins takes place. Taylor et al. reported that nebulin, titin, vinculin, and desmin are significantly degraded within three days of death in the semimembranosus muscle, which is consistent with the present report [42]. Furthermore, Huff Lonergan reported the postmortem μ-calpain-induced degradation of titin, nebulin, filamin, desmin, and troponin-T from bovine longissimus thoracis [43,44]. Sarcoplasmic proteins (among others, glyceraldehyde-3-phosphate dehydrogenase, myosin, and PDZ (PSD95, Dig, ZO-1) and LIM (Lin11, Isl-, Mec-3) domain protein-3) gave smaller amounts of peptides relative to myofibrillary proteins (Figure 1). Previous studies consistently showed that myofibrillar proteins were more easily hydrolyzed than sarcoplasmic proteins during postmortem meat tenderization. Nevertheless, sarcoplasmic protein fragments similar to those from the present study were also reported as occurring in tenderization processes [45,46].

Various methods of analyzing meat tissue were presented in the literature, regarding the presence of tissue-specific proteins, as well as their proteolysis or denaturation due to storage and processing. As an example, Sarah et al. discovered porcine-specific peptides as potential markers for meat species using LC–QTOF-MS [47]. In addition, Kim et al. [48] proposed a proteomic method for the authentication of meat species such as raw beef, pork, and poultry (chicken and duck), using a protein-based approach, including one-dimensional (1D) gel electrophoretic separation and LC–MS/MS analysis. The authors showed that troponin I, enolase 3, 1-lactate dehydrogenase, and triose phosphate isomerase may be useful markers for distinguishing mammalian meat from poultry [48]. They also emphasized that species-specific peptides determined by LC–MS/MS allow each species to be identified independently from the same protein. Furthermore, Mi et al. [49] defined the differences between Tibetan and Duroc (Landrace × Yorkshire) pork, using a label-free quantitative proteomics approach. Therefore, in the present study, an attempt was made to assess whether specific peptides show a difference between different elements of pork carcasses, but from the same species. The impact of conventional or organic pork origin was also considered. The general aim of this approach consisted of profiling the changes in peptide composition. The final peptide composition was identified, and, among the selected peptides, only five identical peptides from muscle tissue proteins occurred in each variant analyzed (Table 1), suggesting large differences in the peptide profiles.


**Table 1.** Peptide sequences identified in all analyzed samples.

<sup>1</sup> The period indicates cutting points; <sup>2</sup> position in parental protein.

A qualitative comparison of the unique and common peptides identified in the three muscle types is shown in the Venn diagrams (Figure 2). The analyzed samples had a different peptide profile, depending on the meat origin (conventional vs. organic). The obtained LC–MS/MS spectra made it possible to identify the peptides characteristic for conventional (Table 2) or organic (Table 3) meat.

In addition, the samples collected from different elements of pork carcasses had a different peptide profile (Tables S1–S3, Supplementary Materials). The highest diversity of peptides was obtained for ham samples, characterized by the highest number of individual sequences (105 peptides and 261 peptides for organic ham and conventional ham, respectively) with a low number of common sequences (i.e., 33 peptides). The most similar analysis results were obtained for loins, for which 93 common peptide sequences were determined. Considering the pork origin (organic vs. conventional), there was no clear trend in the number of peptides. A higher variety of peptides, characteristic for the element of pork carcasses, was noted for the organic meat samples (organic loin, 166 peptides; organic shoulder, 64 peptides), while the smallest number of specific sequences (typical for these samples only) was obtained for organic ham (i.e., 51 peptides).



1 Periods indicate cutting points; 2 position in parental protein; 3 stimulating vasoactive substance release or glucose uptake-stimulating peptide; 4 other activity: DPP-III inhibitor, IH, PE; 5 other activity: DPP-III inhibitor, YK, DA (2) 6, KA; 6 the number in parentheses indicates the number of identified peptides if more than one; 7 other activity: CaMPDE (calmodulin-dependent phosphodiesterase) inhibitor and renin inhibitor, IR; DPP-III inhibitor, KA; 8 other activity: dipeptidyl carboxypeptidase inhibitor, PPPA; 9 other activity: DPP-III inhibitor, RV; 10 other activity: DPP-III inhibitor, GE; 11 other activity: antiamnestic (prolyl endopeptidase inhibitor) and antithrombotic, GP; regulating stomach mucosal membrane activity, GP; 12 other activity: DPP-III inhibitor, PE; rennin inhibitor, FT; 13 other activity: bacterial permease ligand, KK; DPP-III inhibitor, LR, YK, KA; rennin inhibitor, LR; 14 other activity: antiamnestic (prolyl endopeptidase inhibitor), antithrombotic, and regulating stomach mucosal membrane activity, GP; 15 other activity: DPP-III inhibitor, RV; 16 other activity: antiamnestic (prolyl endopeptidase inhibitor), antithrombotic, and regulating stomach mucosal membrane activity, PG; DPP-III inhibitor, MR, YK, RV, PE.


**Table 3.** List of peptide sequences common for organic meat.

1Periods indicate cutting points; 2 position in parental protein; 3 the number in parentheses indicates the number of identified peptides if more than one; 4 stimulating vasoactivesubstance release or glucose uptake-stimulating peptide; 5 other activity: dipeptidyl carboxypeptidase inhibitor, PPPA; 6 other activity: antiamnestic (prolyl endopeptidase inhibitor)and antithrombotic, GP; regulating, DY, GP; bacterial permease ligand, KK; 7 other activity: DPP-III inhibitor, YK, DA (2), KA; 8 other activity: dipeptidyl carboxypeptidase inhibitor,PPPA; 9 other activity: antiamnestic (prolyl endopeptidase inhibitor), antithrombotic, and regulating, GP; 10 other activity: bacterial permease ligand, KK; activating ubiquitin-mediatedproteolysis, LA; DPP-III inhibitor, LA; 11 other activity: dipeptidyl carboxypeptidase inhibitor, PPPA; 12 other activity: bacterial permease ligand, KK; 13 other activity: antiamnestic(prolyl endopeptidase inhibitor), antithrombotic, and regulating stomach mucosal membrane activity, GP; 14 other activity: antiamnestic (prolyl endopeptidase inhibitor), antithrombotic,and regulating stomach mucosal membrane activity, GP; 15 other activity: antiamnestic (prolyl endopeptidase inhibitor), antithrombotic, and regulating stomach mucosal membraneactivity,GP;16 otheractivity:antithrombotic,PPK;dipeptidylcarboxypeptidaseinhibitor,PPPA,PPAP.

#### *Antioxidants* **2020**, *9*, 835

**Figure 2.** Venn diagram showing number of peptides obtained in the pork meat tissue (abbreviations: H-O: organic ham, L-O: organic loin, S-O: organic shoulder, H-C: conventional ham, L-C: conventional loin, S-C: conventional shoulder).

The resulting dataset was also used to assess the similarity between the samples, using multidimensional hierarchical clustering of objects. For data visualization, a heat map with a color scale was introduced to encode existing values from the smallest to the largest (a higher color intensity at each scale represents a higher change in the number of each place). The highest intensity of green color (and, hence, the smallest abundance of given peptide sequences) was characterized for conventional ham and organic loin, which corresponds to the results presented in Figure 2. These batches contained the largest number of individual, characteristic peptide sequences. The highest values marked on the heat map in red were obtained for conventional shoulder, followed by organic ham and organic shoulder, indicating the smallest diversity of peptide sequences among all analyzed variants. These assays simultaneously formed a common cluster on the dendrogram shown in Figure 3.

**Figure 3.** Heat map of abundance levels (**left**) and dendrogram (**right**) obtained as a result of hierarchical clustering based on proteomic data. The changes in abundance of statistically significant (*p* < 0.05) spots among meat tissue models were analyzed. Fold change: negative values (decreasing abundance), 0 (no differences), positive values (increasing abundance); abbreviations: H-O: organic ham, L-O: organic loin, S-O: organic shoulder, H-C: conventional ham, L-C: conventional loin, S-C: conventional shoulder.

Differences in the peptide content between particular muscle types may probably result from their chemical composition. Oxidative muscles generally contain more lipids than glycolytic muscles. As reported by Bonnet et al., the abundance of myosine-1- or triosephosphate isomerase was appropriately distinguished between the lean or fat muscle groups observed, when using proteomics [50]. Bazile et al. identified proteins with abundance differing depending on carcass and muscular dispositions in longissimus thoracis from cows [51]. Seven proteins involved in glycolysis or gluconeogenesis were the least abundant, while 14 proteins related to oxidative metabolism, slow-type muscle, or retinoic acid metabolism were the most abundant in the high-adiposity group.

#### *3.2. Antioxidant Properties of Peptides—In Silico Analysis*

In this study, peptides were identified with high precision and a mass tolerance lower than 5 ppm. The length of identified peptides ranged from 7–50 amino acids (50 amino acids: only one peptide from creatine kinase at chain position 331–381, M-type; Uniprot ID Q5XLD3, data not shown). The number and type of peptide sequences were compared depending on the type of carcass element and rearing system, and the results are presented graphically in Figures 4 and 5.

**Figure 4.** The distributions of peptides based on their molecular weight (abbreviations: H-O: organic ham, L-O: organic loin, S-O: organic shoulder, H-C: conventional ham, L-C: conventional loin, S-C: conventional shoulder).

**Figure 5.** The distributions of peptides based on their number of amino-acid residues (abbreviations: H-O: organic ham, L-O: organic loin, S-O: organic shoulder, H-C: conventional ham, L-C: conventional loin, S-C: conventional shoulder).

Short-chain peptides had higher antioxidant activity than their proteins and polypeptides of origin, as also suggested by Zhu et al. [52]. A common feature of antioxidant peptides is 4–16 amino acids and a molecular weight of about 400–2000 Da [19]. In the present study, sequences predominantly contained 11–20 amino acids (Figure 4), with a molecular weight ranging from 1500 to 2000 Da (Figure 5).

The obtained peptide sequences were evaluated with regard to a potential biological activity using an in silico approach. In particular, the antioxidant potential properties of peptides were considered. To reduce the search area, peptides common for the muscle types tested (Supplementary Materials) and the meat origin (conventional vs. organic; Tables 2 and 3, respectively) were selected for further analysis.

The main purpose of the in silico approach was to determine the antioxidant capacity of meat peptides. However, among the analyzed sequences, low levels of antioxidant peptides were recognized. Only a few peptide sequences, such as dipeptides (AY, AH, EL, HH, HL, KD, IR, KP, LK, LY, LH, MM, SE, TW, WY, VY) and tripeptides (LHV, IKK, VKL, VKV, PEL, PHQ, SDF, FVP, GAA, GAH), as well as one four-amino acid sequence (YVGD) were found. Their location and source of origin are presented in Tables 3 and 4, Tables S1–S3. Nevertheless, in this study, peptides acting as a potential antioxidant were obtained primarily from myofibrillary proteins (nebulin and rarely titin, regardless of the rearing system applied). K ˛eska and Stadnik [14] indicated higher values of the in vitro antioxidant activity of myofibrillary proteins compared to water-soluble (sarcoplasmic) proteins in the ABTS test in dry-cured pork loin. These results are in line with the previous observations that the total number of bioactive peptides predicted to be released after in silico pepsin or pancreatin hydrolysis of selected porcine myofibrillar proteins ranged from six peptides for troponin C, skeletal muscle troponin C (TNNC2) to 112 for myosin-2 Myosin-2 (MYH2), preceded by nebulin (NEB) with 109 peptides per protein molecule. Of these, 1, 13, and 11 two- or three amino-acid peptides with antioxidant properties were observed (for TNNC2, MYH2, and NEB, respectively) [20].


**Table 4.** The antioxidant properties of peptides based on in vitro analysis. ABTS: 2,2 -azino-bis-3 -ethylbenzthiazoline-6-sulfonic acid; FRAP: Fe(III) reduction power.

Abbreviations: O: organic rearing, C: conventional rearing. The results are presented as mean ± SD (standard deviation); a–c means in the same row with different letters differ significantly (*p* < 0.05); A, B means in the same column with different letters differ significantly (*p* < 0.05); NS, not significant; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

Some food-derived peptides were reported to be multifunctional, because they can provide two or more health-promoting effects. As shown in Tables 2 and 3, the analyzed peptides may have more than one bioactivity, mainly acting as dipeptidyl peptidase-IV (DPP-IV) and angiotensin-I converting enzyme (ACE-I) inhibitors. Both groups of biologically active peptides can act against the effects of non-communicable diseases. The DPP-IV inhibitors belonging to one of these groups are involved in the regulation of blood glucose levels and are, therefore, strong antidiabetic agents. A similar bioactive action attributed also to another group of peptides termed "glucose uptake-stimulating peptides" was detected in this study. In turn, the ACE-I inhibitor, by inhibiting the conversion of angiotensin, reduces the negative effects of hypertension. All peptides analyzed in this study can act as DPP-IV inhibitors as well as ACE-I inhibitors (except for one peptide sequence (V.IIIIIIIIII.I), as shown in Table 3). As an example, based on the in silico analysis, dipeptide AY has antioxidant, cardioprotective, and antidiabetic effects. In addition, the potential of proteins as precursors of dipeptidyl peptidase-III (DPP III) inhibitors was also noted. The peptidase with DPP-III-inhibiting

activity has a high affinity for cleavage of opioid peptides such as endomorphins and encephalin. These opioid peptides regulate a variety of physiological functions, such as signal transduction, gastrointestinal motility, immune and hormonal functions, and pain modulation.

According to the report by Khaket et al. [53], the β-subunit of chicken hemoglobin and annexin A5 showed a high inhibitory potential for DPP-III in in silico studies. However, as noted by Galleo et al. [16], only a few studies identified peptides that inhibit DPP-III from meat proteins. Therefore, the information gathered in this study regarding pork as a potential source of DPP-III is of particular interest. The presence of these biopeptides was identified in nebulin (YK, DA, KA, IH, PE, LR MR, RV), as well as in astitin (RV) and desmin (GE) (Tables 2 and 3). Furthermore, several peptides with biologically active properties, such as renin inhibition, calmodulin-dependent phosphodiesterase (CaMPDE) inhibition, dipeptidyl carboxypeptidase inhibition, antiamnestic and antithrombotic activity, regulation of the stomach mucosal membrane activity, bacterial permease ligand ability, and activation of ubiquitin-mediated proteolysis, were reported in unique biopeptides (Tables 2 and 3).

#### *3.3. Antioxidant Properties of Peptides—In Vitro Analysis*

The effectiveness of antioxidant compounds depends on various mechanisms. Consequently, a single test cannot cover all of the different modes of action of different food systems, especially in complex tissues such as meat. Therefore, three different tests were used to assess the differences between conventional and organic meats, as well as between selected pork elements. The variance analysis indicated that all of the effects of the rearing system or muscle type on the total antioxidant activity were significant (*p* < 0.05). The obtained values of radical-scavenging activity measured by the ABTS test were similar, ranging from 41.89% for organic loin to 33.92% for organic shoulder. The rearing method did not significantly affect the radical-scavenging activity of peptides in the ABTS test. This observation is also consistent with other descriptions in the literature, where meat extracts were also analyzed [27,28]. However, the meat origin (organic vs. conventional) and the carcass element significantly affected the iron-ion-chelating capacity (Fe(II)) and the reducing power. Descalzo et al. [54] reported that meat samples from animals reared on pasture had a higher content of antioxidant vitamins (α-tocopherol, β-carotene, and ascorbic acid) than the meat obtained from grain-fed animals. This observation became the basis for estimating the antioxidant potential of meat samples from various feeding systems. The authors further showed that the pasture rearing system had a greater reduction potential than in the case of grain-fed animal samples according to the FRAP assessment; however, there were no differences between these groups in the ABTS test. Importantly, the results indicated the non-enzymatic antioxidants as a cause of differences in antioxidant properties in the samples of meat from animals reared on pasture or fed on grains [28].

As presented in Table 4, the antioxidant properties were higher in the conventional meat samples, while the largest differences were noted in ham samples, i.e., 4.46% for the iron chelate efficiency (*p* < 0.05) and ΔA700 = 0.175 for reducing power activity (*p* < 0.05). Based on the results of the FRAP tests, the smallest differences between organic and conventional pork were noted for shoulder batches.

To better understand how the carcass elements (ham, loin, or shoulder) collected from animals from a conventional or organic farming system were associated with the presence of antioxidant peptides, the changes in their antioxidant properties were quantified using various tests. By means of hierarchical clustering, the peptides were grouped according to their antioxidant trends. The hierarchical cluster analysis (HCA) was used in this study to calculate the multidimensional Euclidian distances between the observations (antioxidant activity). Using a stepwise algorithm (Ward's linkage criterion), observations behaving similarly across the initial variables were linked, and the results were graphically shown in a clustering tree (Figure 6).

The groups of observations behaving similarly were gathered in clusters. The derived dendrogram made it possible to distinguish three groups: cluster 1, organic loins and conventional ham; cluster 2, organic ham and conventional loins; cluster 3, organic and conventional shoulders (the most separated group). As observed in this study, conventional ham and organic loin were characterized by the highest

variety of peptides. This confirms the hypothesis that the quantity and quality of peptides determine their contribution to act as additional antioxidant compounds against oxidation. On the basis of the results obtained, it can be stated that the antioxidant activity of peptides from pig muscles corresponds to their specific (individual) sequences.

**Figure 6.** Dendrogram resulting from Ward's method of hierarchical cluster analysis of antioxidant activity.

As the role of food-derived antioxidants is significant, research on them is still widely carried out. Despite the fact that studies on living organisms are important in verifying the biological properties of peptides, there are little data on the direct antioxidant potential of food peptides on cell biology at the living organism level. So far, there have only been a few studies on food-origin antioxidant peptides in animal models. The administration of egg-white hydrolysate to spontaneously hypertensive rats for 17 weeks was shown to improve plasma antioxidant properties [55]. In turn, Ebaid et al. [56] showed that administering 100 mg/kg body weight of whey protein to streptozotocin-induced diabetic rats reduced several indicators of oxidative stress, such as malondialdehyde, nitric oxide, and ROS levels, while it also reduced proinflammatory cytokines and increased the level of glutathione [56]. Cellular assays are more commonly used as indirect methods to assess the protective effect of antioxidants against oxidative stressors and to elucidate the mechanism of action of peptides in cells. Thus, Katayama, Xu, Fan, and Mine, using human intestinal epithelial cells (Caco-2) as an intestinal epithelia model, reported that oligophosphopeptides derived from hen egg yolk exert an antioxidant effect, causing the upregulation of glutathione-induced biosynthesis accompanied by increased glutathione reductase activity [57]. This observation was also accompanied by inhibition of the production of proinflammatory cytokines, contributing to antioxidative protection against hydrogen peroxide-induced damage in Caco-2 cells. Moreover, the antioxidant peptide from fish skin gelatin hydrolysate increased the expression of cellular antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) in human hepatoma (Hep3B) cells [58]. Numerous reports showed that food-derived peptides can retain antioxidant activity in simulating gastrointestinal digestion and then retain their properties in further studies of the cellular pathway (as a simulated uptake step) in the Caco-2 cell monolayer [59–61]. Research results confirmed that peptides from food can cross the gastrointestinal barrier and exert antioxidant effects. Thus, upon oral ingestion of peptide-rich foods, such as meat, different in vivo antioxidant efficacy can be produced.

#### **4. Conclusions**

The obtained results indicate some influence of the meat origin (organic vs. conventional) on the health characteristics of pork. Moreover, an in-depth mapping of the peptidome of pig carcass elements obtained from these two farming methods provides (at the molecular level of the protein) important information on how the peptide profile is being shaped. Based on the LC–MS/MS analysis of the different carcass elements, myofibrillary proteins (such as nebulin, titin, or desmin) were identified as the main sources of peptides. However, the meat origin (organic vs. conventional), as well as the element of the pork carcass (ham, loin, or shoulder), did not have a significant effect on the proteome. On the other hand, the pork origin and the carcass element significantly affected the iron-ion-chelating capacity (Fe(II)) and the reducing power of peptides. In particular, pork ham from the conventional rearing system, which had the best antioxidant properties due to the presence of peptides, can be recommended for daily consumption to people who care about their health. Analyses also showed that this meat element may be a source of peptides that support the treatment of noncommunicable diseases such as hypertension and diabetes, but additional research is needed to further confirm this aspect.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/9/835/s1, Table S1. The list of peptide sequences common for conventional and organic ham; Table S2. The list of peptide sequences common for conventional and organic loins; Table S3. The list of peptide sequences common for conventional and organic shoulders.

**Author Contributions:** Conceptualization, K.M.W. and P.K.; methodology, P.K.; formal analysis, M.H., K.M.W., and P.K.; investigation, K.M.W., S.R., M.H., and P.K.; writing—original draft preparation, P.K.; writing—review and editing, K.M.W., P.K., M.H., and S.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was financed under the program of the Minister of Science and Higher Education (Poland) under the name "Regional Initiative of Excellence" in 2019–2022, project number 029/RID/2018/19, funding amount 11 927 330.00 PLN.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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