1. Introduction
Poland is a very important producer of fruit and fruit processing products, such as juices, pulps, and jams, which results in an increase in waste associated with their processing. Currently, manufacturers strive for waste-free production, and waste is increasingly managed. In large amounts, these products are used as compost or added to animal food. Sometimes, for a new participant in the production chain, a by-product may become the main raw material, which is a source of valuable, previously ignored, or not fully obtained ingredients. Some waste may have health-promoting properties; others may be used as functional additives, replacing synthetic compounds, or supporting the functioning of the human body. Reusing waste has many advantages, including reducing the costs of waste disposal, reducing seasonality, and improving the level of hygiene. Such behavior brings not only financial and environmental benefits but is also consistent with the concept of a circular economy [
1]. During the production of juices, the waste mass consists of pomace, the amount of which can amount to 25% of the raw material [
2,
3]. Fruit pomace is still rich in bioactive compounds, primarily polyphenols and dietary fiber, which can be recovered [
4,
5]. Polysaccharides included in fiber can perform technological functions in food and can be used as fillers, thickeners, and structure stabilizers. In particular, pectins have the ability to bind metals and create stable networks through bonds between Ca
2+ ions and free carboxyl groups. Fiber from fruit is also used as a functional additive in ice cream, snacks, bakery products, and meat products [
6]. Dried and powdered pomace is often used in the food industry, such as the confectionery and bakery industries [
7]. According to Tarko et al. [
8], pomace, thanks to its use in ethanol production, is an important source of biofuel, and, in the case of apple pomace, it can be a breeding ground for bacteria producing lactic acid.
One of the by-products of the fruit industry in Poland is rose fruits pomace, which is obtained in the juice production process [
9].
Rosa rugosa is a representative of the
Rosaceae family with the most appreciated nutritional value among the representatives of this family found and cultivated in Poland.
Rosa rugosa can occur in a variety of areas because it is resistant to unfavorable environmental conditions, such as poor soil or water restrictions. In Poland, it occurs both in the natural environment and in cultivation and breeding. The commercial planting of wild rose in Poland consists of 1200 ha of
Rosa rugosa, with a yield of approximately 3000 tons per year [
9]. This plant is widely used in various industries. Due to its inedible interior, the fruit cannot be eaten fresh. The rose is used for medicinal purposes and in food processing to produce juices, purees, jams, preserves, confectionery fillings, nectars, tinctures, wines, fruit, and herbal teas [
10,
11,
12,
13,
14,
15,
16]. It has been proven that rose fruits extracts not only contain high levels of bioactive substances such as vitamin C or polyphenols but also have antibacterial, antiviral, antihypertensive, anti-inflammatory, and antidiabetic properties [
9,
17,
18,
19]. Preparations of this type may inhibit the growth of bacteria and fungi considered pathogenic and contaminating food. As reported in the literature, rose and rose pomace extracts limit the development of microorganisms such as
Escherichia coli,
Staphylococcus aureus,
Staphylococcus epidermidis,
Klebsiella pneumoniae,
Enterococcus faecalis,
Bacillus subtilis,
Bacillus cereus, and
Candida albicans [
9,
17,
20,
21,
22]. Marmol et al. [
10] showed that
Rosa rugosa inhibits the growth of pathogenic bacteria without changing the growth of lactic acid bacteria. Kamijo [
23] showed that phenolic compounds from the tannin group of
Rose rugosa seem to be a promising prebiotic because they selectively inhibit the growth of pathogenic bacteria. Thanks to the scientific evidence proven and presented so far, we can confidently classify such plant extracts as natural food preservatives [
18].
One of the simplest methods of obtaining bioactive ingredients is the extraction of plant material with a solvent. Post-production waste from plant materials from various industries is also perfect for this purpose. Most often, pomace is used, which may also contain large amounts of valuable bioactive ingredients. The extraction process involves the separation of a specific ingredient from the mixture using a solvent by diffusion. The effectiveness of this process depends primarily on the type of solvent but also on the temperature, extraction time, solid-to-solvent ratio, number of extractions, and partial size of the sample material [
24,
25]. The most important criterion for effective extraction is the appropriate adjustment of the solvent. The most commonly used solvents for extracting active substances from plant material are water, ethanol, methanol, acetonitrile, acetone, hexane, and chloroform. The appropriate selection of the extractant should be based on knowledge of the chemical nature of the extracted substance and the solvent. The temperature used is also a very important element in the extraction process because increased temperature affects the loss of labile compounds. Polar solvents are most often used to extract polyphenolic compounds and vitamin C from plant material [
26,
27,
28]. The recovery of phenolic compounds during extraction is closely related to their chemical structure. An example of this is gallic acid, which is the most soluble in water because its molecule has four hydroxyl groups and one carboxyl group. The study by Arize et al. [
29] showed that water, due to its higher polarity coefficient, washes out more polyphenols from the solid than ethanol. According to Pompeu [
30], increasing the share of ethanol in the solvent mixture results in better extraction due to the reduction of the dielectric constant of the solution, which is directly related to the reduction of the energy needed to separate the solvent molecules. Based on research, it is known that chlorogenic acid and flavonoids are better extracted with ethanol [
31,
32]. In comparison, aqueous acetone is best for extracting higher molecular weight flavanols, and methanol is best for extracting lower molecular weight polyphenols [
33]. Ethanol has less toxicity compared to acetone, methanol, and other organic solvents [
34]. The use of extraction solvents in industry requires consideration of, among others, factors such as solvent residue in the product, its removal, and environmental pollution [
35]. The most recommended solvent for extraction is water, due to features such as non-toxicity, ecological nature, low cost of use, high extraction potential, and no restrictions on human consumption [
33]. According to the European Pharmacopoeia, only class 3 solvents such as ethanol and acetone can be used in pharmaceutical products because they are less toxic and pose less risk to human health [
36]. In contrast, solvents such as acetonitrile, chloroform, hexane, and methanol must be limited in pharmaceutical products due to their inherent toxicity. The concentration limits of these products for acetonitrile, chloroform, hexane, and methanol are 400, 60, 290, and 3000 ppm, respectively [
36]. To the best of our knowledge, there is no study investigating the effect of different solvents on the recovery of phenolic compounds and L-ascorbic acid from rose fruits pomace waste.
The aim of this study was to investigate the effects of temperature, time extraction, and different solvents, including water, methanol, ethanol, and acetone, and the combination of these organic solvents with water in a ratio of 50:50 (v/v) on the recovery of total phenolic compounds (measured by Folin–Ciocalteu) and L-ascorbic acid (measured by the HPLC method) of rose fruits (Rosa rugosa) pomace.
2. Materials and Methods
2.1. Chemicals and Reagents
Anhydrous sodium carbonate and Folin–Ciocalteu reagent were purchased from Chempur (Piekary Śląskie, Poland). Gallic acid anhydrous (GAE), L-ascorbic acid, oxalic acid, m-phosphoric acid, DL-dithiothreitol (DTT), ethanol, acetone, and methanol were purchased from Sigma-Aldrich (Poznań, Poland). All reagents were of analytical grade.
2.2. Plant Material
Fresh fruits (including seeds) of Rosa rugosa were harvested at a plantation of the company “Polska Róża” located in Kotlina Kłodzka in September 2020 in Poland. The cultivation of Rosa rugosa was carried out in accordance with the cultivation recommendations for this species, and no events occurred that could have affected this cultivation.
2.3. Preparation of Rose Fruits Juice on a Laboratory Scale
Fruits were homogenized by Thermomix® (Vorwerk & Co. KG, Wuppertal, Germany). The pulp was subsequently macerated for 24 h with 0.2 g/kg macerating enzyme pectinase (Rohapect 10 L, AB Enzymes GmbH, Darmstadt, Germany) at room temperature. After this time, the juice was pressed on an automatic press (Bucher Unipektin, Niederweningen, Switzerland).
2.4. Freeze-Drying of Rose Fruits Pomace
Directly after juice pressing, a portion of the pomace was frozen and freeze-dried (Christ, Alpha 1-2 LD Plus, Osterode am Harz, Germany). Lyophilization lasted 45 h at a temperature of −56 °C and a pressure of 1.03 mbar. The obtained dried material was crushed in a Retsch GM 200 homogenizer. The obtained powder was transferred to vacuum packaging bags, closed tightly, and stored in a shaded place.
2.5. Preparation of Extracts from Rose Fruits Pomace Using Classic Extraction by Shaking with a Solvent
A three-way experiment with a completely randomized design was set up. Solvent combinations were created in a cross-arrangement. The following solvents were used for extraction: water, ethanol, methanol, acetone, and methanol: water (50:50,
v/
v), ethanol: water (50:50,
v/
v), and acetone: water (50:50,
v/
v). Seven types of extracts were prepared by shaking with different solvents. The extraction process was performed with a solvent-to-sample ratio of (100:1
v/
w). The solvent-to-sample ratio was selected based on the literature data [
26,
27]. Approximately 0.5 g of freeze-dried pomace was weighed exactly into Falcon tubes with a screw cap, and 50 mL of an appropriate solvent was added. A single extraction was carried out on an LLG-uni THERMIX 2 pro thermal shaker at a speed of 500 rpm at three different temperatures of 25, 45, and 65 °C and in four different time ranges: 15, 30, 45, and 60 min. Reference samples were prepared in the same manner as tested samples but were not shaken and heated, and their extraction time was 60 min. After extraction, the individual extracts were centrifuged at 5000 rpm for 5 min, filtered through a 0.45 µm PTFE filter, and then the resulting clear supernatants were evaporated with nitrogen using a nitrogen gas generator (Peak Scientific Instruments Ltd., Scotland, UK). After evaporation, the extracts were dissolved in 50 mL of water, transferred to plastic vials, and stored at −20 °C until analyzed.
2.6. Determination of Total Phenolic Content
The total phenolic content (TPC) of the extract solution was determined by the Folin–Ciocalteu reagent’s method using a UV1650PC spectrophotometer (Shimadzu, Kyoto, Japan) [
37]. Total phenolic content was expressed as mg gallic acid equivalents (GAE) per g of freeze-dried pomace. The equation obtained from the calibration curve of gallic acid in the range of 5–50 mg/100 mL was y = 0.0372x + 0.0826 (r = 0.9965). The test was performed in triplicate.
2.7. Determinations of L-Ascorbic Acid (AA) Content
L-ascorbic acid (AA) (as the sum of AA and L-dehydroascorbic acid (DHAA) after its reduction to AA) was determined using high-pressure liquid chromatography coupled with a UV-VIS detector (Prominence HPLC system, Shimadzu, Kyoto, Japan) according to the method previously stated in the literature with our own modification [
11,
38]. One mL of extract was transferred to a volumetric flask and diluted to 10 mL with 2% of oxalic acid. Then, the obtained solution was diluted in a 1:1 ratio with 1% DTT (DL-dithiothreitol). The solution was left in a dark place for 1 h to reduce DHAA (L-dehydroascorbic acid) to AA (L-ascorbic acid). This reaction solution was filtered through a 0.45 µm PTFE syringe filter into a chromatographic vial. The sample volume of 20 µL from each chromatographic vial was injected into the HPLC system. The separation was carried out using an Onyx Monolithic C18, 100 × 4.6 mm column (Phenomenex, Torrance, CA, USA), at 25 °C. 0.1% aqueous solution of m-phosphoric acid was used as the eluent at a flow rate of 1.0 mL/min. The analysis was performed in an isocratic system. L-ascorbic acid was determined at 254 nm.
Blank samples were prepared in the same way as the analytes. All results were expressed in mg/g of freeze-dried pomace. The test was performed in triplicate.
The reliability of the HPLC-method was validated for linearity, sensitivity (limit of detection, LOD, and limit of quantification, LOQ), precision, and recovery. Precision was determined by six measurements of an AA standard. The LOD and LOQ were defined as 3 times and 10 times the signal-noise ratio, respectively. Recovery was determined in seven representative extracts (water, methanol, ethanol, acetone, 50% methanol, 50% ethanol, and 50% acetone) from rose fruits pomace. Two levels of AA were added to the tested rose fruits pomace (5 and 20 mg/g), and the recovery level was determined twice for each prepared extract.
2.8. Statistical Analysis
Statistica 13.3 (TIBCO Software Inc., Carlsbad, CA, USA) was used to perform the statistical analysis. The obtained experimental results were subjected to calculations of mean values, standard deviations, and multivariate ANOVA. The significance of statistical differences was verified using the Tukey test with a significance level of p ≤ 0.05. We also used principal components analysis (PCA) as a multi-trait method describing the relationship among polyphenols and L-ascorbic acid content at study temperatures across a combination of time and solvents.