Influence of Production Parameters on the Content of Polyphenolic Compounds in Extruded Porridge Enriched with Chokeberry Fruit (Aronia melanocarpa (Michx.) Elliott)

2.1 Reagents

Standards of caffeic, gallic, ferulic, isoferulic, protocatechuic, 3-OH-benzoic, 4-OH-benzoic, rosmarinic, 3,4-dihydroxyphenylacetic, vanillic, syringic, m-coumaric, o-coumaric, p-coumaric, salicylic, 3-OH-cinnamic, 3,4-dimethoxycinnamic acid and LC grade acetonitrile were purchased from Sigma–Aldrich Fine Chemicals (St. Louis, MO, USA). Gentisic, sinapic, veratric acids were from ChromaDex (Irvine, USA). LC grade water was prepared using a Millipore Direct-Q3 purification system (Bedford, MA, USA). Ethanol and methanol (reagent grade purity) used for the preparation of extracts, Folin-Ciocalteu reagents and all analytical grade reagents were obtained from Avantor Performance Materials (Center Valley, PA, USA).

2.2 Porridge production

Chokeberry fruits purchased from a local farmer (Lublin Voivodeship located in southeastern Poland) were dried in an air oven at 40°C for 12 hours. The raw material mixes used for the tests were made from corn porridge (DASCA, distributor: Awiko, Lublin, Poland) with an addition of 0, 5, 10, 15 and 20% of dried chokeberry fruit. The mixes were moistened up to 14%. The extrudates were prepared on the single-screw extruder TS-45 (ZMCh Metalchem, Gliwice, Poland) equipped with a modified plasticising system L/D = 12:1. The extrusion was carried out at a varied screw speed of the extruder plasticizing system: 80 and 120 rpm. A single-opening die of 3 mm in diameter was used. During the extrusion process, temperature changes were recorded in the individual extruder sections at different temperature profiles: 132-142°C. The obtained extrudates were milled in the laboratory mill LMN10 (TestChem, Radlin, Poland) to obtain material of less than 1 mm in granularity.

2.3 Preparation of extracts

2 gram-samples of porridge were transferred to ground-necked flasks, and 40 mL of ethanol was added. An ultrasonic bath with a thermostat was used for extraction (Bandelin Electronic, Sonorex RK 100H, Germany). The extraction protocol was previously optimized in terms of temperature, time, type of solvent and ultrasound power. The extraction process took 40 minutes at the temperature of 60°C, the ultrasound frequency of 33kHz and the power of 320W. The extracts were filtered into beakers; 40 mL of ethanol was added to the reminder, and the extraction was repeated. The obtained extracts were combined and put aside in beakers under a hood until the solvent evaporated. The dry residues were quantitatively transferred to volumetric flasks and supplemented with methanol up to 5 mL [11].

2.4 Determination of the total content of polyphenolic compounds

The total content of polyphenolic compounds in the studied extracts was determined with the Folin-Ciocalteu (FC) method designed earlier [16,17]. 100 μL of the tested extracts were placed in 5 mL volumetric flasks. Next, 900 μL of distilled water and 100 μL of Folin-Ciocalteu reagent were added. The solutions were mixed and put aside. After 4 minutes, 1mL of 7.7% sodium bicarbonate and 400 μL of distilled water were added. The content was mixed and placed in a 40°C water bath for 50 minutes. After that, the absorbance of the solutions was measured using a UV spectrometer (Genesys 10S VIS, Thermo Scientific, Waltham, MA, US) at a wavelength of 765 nm.

Next, a calibration curve of gallic acid (0.05, 0.15, 0.25, 0.35, 0.5, 1.0 mg/mL) was plotted. 100 μL of each of the calibration solutions were collected, 900 μL of distilled water and 100 μL of Folin-Ciocalteu reagent were added to them and processed as in the case of the extracts above. A solution without gallic acid was used for a blank experiment. Basedon obtained results, a calibration curve of gallic acid (y = 3.7995x + 0.0986) was plotted. It was used to calculate the total polyphenol content in the analysed extracts, expressed as the equivalents of gallic acid.

2.5 Determination of the total flavonoid content

The total flavonoid content was determined by the colorimetric method with the use of aluminium chloride. 0.5 mL of the extract were put in 10 mL volumetric flasks; next, the following were added: 0.5 mL of methanol, 4 mL of distilled water and 0.3 mL of 5% NaNO₂. The flasks were sealed and put aside for 5 minutes. After 5 minutes, 0.3 mL of 10% AlCl₃ solution was added; the content was mixed and left for 6 minutes. Then, 2 mL of 1 mol NaOH solution was added and topped up with 10 mL of distilled water. The mixture was stirred and left for 10 minutes; after this time, absorbance was measured at the wave length of 510 nm.

Next, a calibration curve of quercetin (0.08, 0.40, 0.80, 1.6, 3.2, 4.8, 6.4, 8.0 and 9.6 mg/mL) was plotted. 1mL of each solution was collected, mixed with 4 mL of distilled water; 0.3 mL of 5% NaNO₂ solution was added; the entire sample was put aside for 6 minutes and then processed as in the case of the extracts discussed above. Based on the obtained quercetin calibration curve (y = 0.1989x + 0.0908), the total flavonoid content in the extracts was calculated (as per quercetin). For a blank experiment, a solution was prepared in which aluminium chloride was replaced with distilled water; the remainder of the process remained unchanged [18].

2.6 TLC-DPPH Tests of obtained extracts

The antioxidant properties of obtained extracts were also examined by the TLC-DPPH test. For the stationary phase, 10x10 cm silica gel plates were used while for the mobile phase, a mixture of ethyl acetate, toluene, and formic acid at the ratio of 10:10:0.5 v/v/v. A standard solution of 0.1mg/mL quercetin was prepared. 10 μL aliquots of extracts were applied with the automatic TLC applicator Desaga AS -30. The plates were developed in one direction in horizontal chambers (DS II, Chromdes, Lublin, Poland). After drying, they were sprayed with a 0.1% DPPH solution. Next, the plates were scanned with a flat-bed scanner (Lide 50, Canon) after 0, 10, and 30 minutes. The results of TLC–DPPH tests were recorded in the form of JPG documents. For further analysis, the computer software Sorbfil TLC Videodensitometr (Sorbpolymer, Russia) [11,19] was used.

2.7 LC-ESI-MS/MS analysis of phenolic acids

Phenolic acids content was determined according to modified method described by Oniszczuk et al. [11]. Experiments were carried out using an Agilent 1200 Series HPLC system (Agilent Technologies, USA) connected to 3200 QTRAP Mass spectrometer (AB Sciex, USA) equipped with electrospray ionisation source (ESI). Both were controlled with Analyst 1.5 software (AB Sciex, USA), which was also used for data interpretation. Separations were carried out on a Zorbax SB-C18 column (2.1 x 100 mm, 1.8-mm particle size; Agilent Technologies, USA) at 20ºC. Gradient method was used with mobile phases: water with 0.1% HCOOH (A) and acetonitrile with 0.1% HCOOH (B). Injection volume was 3 μL, the flow rate was 250 ml/min and the gradient was as follows: 0-2 min – 25%B, 3-6 min – 35%B, 8-10 min – 55%B, 12-16 min - 75%B, 19-25 min - 25%B.

ESI operated in the negative-ion mode worked at the following conditions: capillary temperature 400 °C, curtain gas at 30 psi, nebulizer gas at 50 psi, negative ionisation mode source voltage −4500 V. Triplicate injections were made for each standard solution and sample. The analytes were identified by comparing retention time and m/z values obtained by MS and MS2 (Table 1) with the mass spectra from corresponding standards tested under the same conditions. The calibration curves obtained in MRM mode were used for quantification of all analytes. The identified phenolic acids were quantified on the basis of their peak areas and comparison with a calibration curve obtained with the corresponding standards. Linearity ranges for calibration curves were specified. The limits of detection (LOD) and quantification (LOQ) for phenolic compounds were determined at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. Samples were purified and concentrated before LC-MS analysis using SPE technique.

2.8 Solid Phase Extraction (SPE)

Solid phase extraction was carried out using SPE system (Baker spe-12G™, J.T. Baker, Deventer,Netherlands) and columns with a chemically bonded octadecyl phase (Bakerbond C18, 500 mg). 2 ml of the extract in 50% methanol was applied to each of the conditioned columns. The desired fraction was eluted with 5 mL of 50% methanol and then with 5 mL of 80% methanol. The extracts obtained were concentrated and subjected to the LC-ESI-MS/MS analysis [11].

2.9 Statistical evaluation

Statistical analysis of data was made using the MSExcel 2013 and Statistica 12 program (StatSoft Inc., USA). All analytical measurements were repeated six times for each sample and reference compounds. The obtained results were expressed as the mean values ± SD. The significance of the obtained results was determined at p<0.05 performing t-test for the applied methods.

Ethical approval: The conducted research is not related to either human or animal use.

3.1 Determination of the total content of polyphenolic compounds

Extrusion-cooking is a food processing procedure that arouses a keen interest in the food industry. The action of temperature, pressure and shear forces on moist raw material induces profound changes in the processed matter in a very short time, among them: enhanced digestibility of nutrients, inactivation of anti-nutritive factors, modified sensory characteristics. The intensity of these changes depends both on the properties of raw material and on the settings of the extrusion-cooking proper, involving the temperature or rotational speed of the extruder screw. A high degree of mixing and homogenisation leads to a decrease in diffusion barriers and the breaking down of chemical bonds, which results in a heightened reactivity of the components. As a result, this is a source of both beneficial and non-beneficial changes in the processed material [20]. Therefore, to avoid undesirable degradation of active ingredients, including one that could occur during this process, it is important to select appropriate production parameters, among which the rotational speed of the extruder screw is key.

In the opening phase of the study, the authors compared the total content of polyphenols in porridge with the different content of chokeberry fruit (0, 5, 10, 15 and 20%) obtained at 80 and 120 rpm of the extruder screw. Polyphenol compounds are popular secondary metabolites. As mentioned above, many favourable biological effects are achieved owing to their presence in functional food products. Obtained results demonstrated that the screw speed of 80 rpm results, for all the chokeberry contents used, in a higher content of polyphenols (as per gallic acid) than the speed of 120 rpm (Table 2).

The broadest group of phenolic compounds are flavonoids. They have a key role in protecting the human body against various diseases, mainly due to their high antioxidant activity. Therefore, the following stage of the research focused on the influence of screw rotational speed on the total content of flavonoids (as per quercetin). It was demonstrated that, in this case, 80 rpm showed superior results compared with 120 rpm in all other examined cases (Table 2).

According to research done by Alonso et al. [21], the main factors stimulating the transformation of input material during the extrusion-cooking process are high temperatures and mechanical aspects related to shear forces that rise along with the increase of screw rotational speed. It is possible that production at 120 rpm is too extreme and lead to the decomposition of some phenolic compounds, including flavonoids [20]. Obtained results may be interesting for consumers as the selection of appropriate extrusion-cooking parameters helps maintain adequate nutritional, sensory and, above all, health-improving properties of the tested samples. The results of the tests clearly show that a higher level of chokeberry fruit in porridge means a higher content of polyphenols and flavonoids in the samples. The highest total content of polyphenols and flavonoids was reported in porridge a 20% addition of the chokeberry while the lowest in a sample without such functional additives.

3.2 TLC-DPPH test of obtained extracts and interpretation of results using the Sorbfil TLC Videodensitometer program

Having obtained the results discussed above, the authors decided to look at the antioxidant properties of porridge produced at the speed of 80 rpm of the extruder screw. For this purpose, a TLC was performed using a stable DPPH radical. The extracts were applied to successive plates; finally, a standard quercetin solution was added and left for development. After drying, the plates were sprayed with a DPPH solution and scanned after 0, 10, 30 minutes. Next, the obtained image was converted into numerical data in the Sorbfil TLC Videodensitometer program. For this purpose, on each scan of the chromatographic plate with the visualised area of bright spots, tacks were made corresponding to the area of development of each polyphenol extract. The function of result analysis permitted a precise assessment of the colour intensity of the spots and the size of the areas under peaks (and their conversion into numerical data). The area of a standard solution of quercetin at the concentration of 0.1 mg/mL was adopted as a reference point. Its activity was marked ‘1’ [22].

The TLC-DPPH test confirmed an increase in antioxidant properties of porridge along with the growing content of chokeberry fruit (Table 3, Figure 1). Thus, a well visible trend is that a higher amount of functional raw material causes an increase in the content of polyphenols and flavonoids and an increase in antioxidant properties. Extracts with the largest, i.e. 20%, addition of chokeberry fruit exhibited superior antioxidant properties. The top free radicals scavenging activity by all extracts was reported at the beginning after 10 minutes of the process. According to Brand et al. [13], chokeberry fruits have a tremendous effect on the human body, largely due to their antioxidant properties attributable to the presence of polyphenol compounds, which, as demonstrated by the research results, remained active after the extrusion-cooking process. Such porridge are a good source of natural antioxidants, thus being capable of preventing certain diseases and improving the quality of health and life.

Figure 1

TLC–DPPH test results for the extracts of corn porridge of the varied chokeberry content; plate scanned after 30 min. Mobile phase: ethyl acetate, toluene, and formic acid at the ratio of 10:10:0.5 v/v/. Stationary phase: plates with silica gel 10 × 10 cm. 1- a standard solution of 0.1mg/mL quercetin; 2, 3, 4, 5, 6 - extract of porridge with 20%, 15%, 10%, 5%, 0% addition of chokeberry fruit, respectively.

3.3 LC-ESI-MS/MS analysis of phenolic acids

At the next stage of the research, the authors performed a quantitative analysis of free phenolic acids in porridge with a 0, 5, 10, 15 and 20% addition of chokeberry fruit; the porridge were produced at the optimum rotational speed of the extruder, i.e. 80 rpm. Prior to determination, all crude extracts were purified using the SPE method. In order to assess the accuracy of the SPE method, a recovery test was held. Crude extracts were enriched with known amounts of the standard solution having three different concentrations. Next, a solid phase extraction was performed with the same methods as used for the quantification of phenolic compounds in the samples. The recovered amounts ranged from 93.7% for isoferulic acid to 102.4% for 4-hydroxybenzoic acid, which means that using the SPE for extract purification proved most effective.

The LC-MS analysis exposed a wide variety of phenolic acids present in the tested samples. In the sample with the 20% content of chokeberry fruit, nine acids were identified (Table 4, Figure 2) . They derived from benzoic acid and included: protocatecholic acid, 4-OH-benzoic acid, gentiic acid, salicylic acid, and cinnamic acid derivatives: trans-and cis-caffeic, p-coumaric, ferulic, and isoferulic. This was the only extract where gentisic acid was determined. In the porridge without any functional additive, only five free phenolic acids were detected: protocatechuic acid, 4-OH-benzoic acid, salicylic acid, ferulic acid, and isoferulic acid. The total content of these compounds grew along with the increasing amount of chokeberry fruit in the samples and reached 18.184 μg, 35.101 μg, 39.938 μg, 41.497 μg and 45.074 μg/g of dry matter, respectively, for samples enriched with 0, 5, 10, 15 and 20% of this fruit. Also, the content of individual acids increased as more chokeberry fruit was added. We observed that porridge enriched with this functional additive become an important source of free phenolic acids, especially protocatechuic acid. Its content in samples with the addition of chokeberry fruits is very high and grows even more as more of this fruit is added. This acid plays a vital role in disease prevention, especially when we speak about diseases of affluence. Recent research have shown that it has a potential as a prevention factor in cardiovascular diseases. In the 1990s, based on the results of research on chemically induced carcinogenesis, some chemopreventive properties of this compound were reported in experimental animals. Its action is based on antioxidant properties, i.e. inhibited generation of free radicals, the ability to scavenge them and enhance the catalytic activity of endogenous enzymes taking part in the neutralisation of free radicals. Of significance is also the

Figure 2

Extracted LC-MS-MRM chromatogram of phenolic acids found in sample with 20% addition of chokeberry fruit; MRM transition are given in brackets: 1 - protocatechuic (m/z 152.9 > 107.8); 2 – trans-caffeic (m/z 178.7 > 134.9); 3 –cis-caffeic (m/z 178.7 > 134.9); 4 - 4-hydroxy-benzoic (m/z 136.9 > 93); 5 - salicylic (m/z 136.9 > 93); 6 - gentisic (m/z 152.8 > 107.9); 7 - p-coumaric (m/z 162.8 > 119); 8 - ferulic (m/z 192.8 > 133.9); 9 - isoferulic acid (m/z m/z 192.8 > 133.9).

influence of enzymes involved in the first and the second stage of carcinogen biotransformation and, most probably, the direct blocking of the locations of specific bonds of metabolised carcinogens with a DNA molecule [23,24].

A drop in the content of isoferulic acid as more of the fruit is added to porridge is interesting. It can be explained by the fact that the matrix itself, i.e. the corn porridge free of any additives, contained more of this compound that the chokeberry.

Obtained results showed that the antioxidant activity measured in the TLC-DPPH test was positively correlated with the content of all groups of compounds identified in porridge, including with free phenolic acids. Pearson’s coefficients between the addition of chokeberry fruit, the content of polyphenols, flavonoids, free phenolic acids and the antioxidant activity (measured after 10 minutes) are shown in Table 5. Very high and positive correlations were reported between the addition of chokeberry fruit and antioxidant properties (r = 0.991), between the addition of chokeberry fruit and polyphenol content (r = 0.981) and between antioxidant properties and the content of polyphenols (r = 0.967). These correlations indicate that the measured antioxidant activity of the tested porridge was mainly due to the addition of polyphenol-rich chokeberry fruit. Antioxidant properties were also closely associated with the level of flavonoids (r = 0.970) and free phenolic acids (r = 0.866). Our results are aligned with those of Madhujith et al. [25,26]. They reported that it was the level of free and not bonded and conjugated phenolic acids that significantly contributed to the antioxidant properties of the tested plant samples.

The content of free phenolic acids in the examined extracts was determined by means of calibration curves designed for each model. The limit of detection (LOD) and the limit of quantification (LOQ) were determined at the signal to noise ratio (S/N) of 3 and 10, respectively. All the tested compounds showed positive linearity. The correlation coefficients for all calibration curves were r² > 0.998. The values of LOD and LOQ as well as the linear range for all analysed compounds are shown in Table 6.

Our research has demonstrated that innovative porridge with the addition of chokeberry fruit can become a source of antioxidants that are so important for the human body. For this reason, they can prospectively make their way into the product range known as functional foods.