Impact of Package Type on Selected Health Quality Parameters of Organic, Conventional and Wild Raspberries (Rubus idaeus L.) Frozen Stored

Abstract

Functional foods and the nutritional value of products are now very relevant for consumers. Additionally, interest in biodegradable components made from natural products has recently increased. The aim of this study was to determine the effect of different package types (with addition of films made of biopolymers enriched with natural extracts of ginger, cinnamon, and turmeric) on antioxidative properties of conventional, organic, and wild-grown raspberry fruit during frozen storage for 1-, 4-, 8-, and 12-month periods. The content of total polyphenol and anthocyanins, as well as antioxidant activity, were studied. Additionally, structural (FT-IR and UV-Vis), mechanical (tensile strength and elongation), and surface wetting angle tests of used films were studied. In all crop types, polyphenol content increased at 8 and 12 months of frozen storage in packages with an addition of biopolymer films. There was a statistically significant increase in the amount of total polyphenols in the last month of storage in the package with films with added cinnamon extract. In contrast, packages with ginger-enriched films contributed to an increase in polyphenols only in organically grown and wild raspberry fruit at the 4th, 8th, and 12th month and 8th and 12th month, respectively. The addition of films with turmeric extract increased the polyphenol content in the 8th month of frozen storage of organically grown raspberry fruit and in the 12th month of wild-grown raspberry. During frozen storage of wild raspberry fruit, an increase in anthocyanin content was observed in all cases in the 12th month. This result was almost twice as high as in the control sample. The greatest rise in the content of anthocyanins occurred when packages with films with turmeric extract were applied. Tendencies presented by this paper are not clear-cut in cases of antioxidant activity of analysed raspberries. A common feature was that, in general, after 12 months of storage, raspberries from all crops had higher and statistically significant antioxidant values compared to the first month of storage. Studies on the biocomposite films have shown that the structure of chitosan and alginate polymers was partly decomposed after 12 months, and the natural extracts and raspberry crops used had different effects on the mechanical properties of the biocomposites films. Based on the results of research that has been carried out, it can be concluded that it has not been clearly proven whether the addition of polysaccharide films to packages contributes to the increase in bioactive compounds during frozen storage.

1. Introduction

Recently, there has been a growing interest in the use of biodegradable and natural components for the production of food film. Primarily, it is used to create packaging that plays an additional role in the context of environmental protection [1]. These films can also be used as coatings applied to the surface of food products, which provide a barrier against the passage of gases and water. In addition, they create an internal atmosphere, which contributes to the product preservation [2]. Current packaging, apart from its primary function of protecting food from various types of contamination, acts as active packaging, too. They work via the release of active substances into the food, so they can improve its shelf life as well as its health quality [3].

Biopolymers include natural products made of plants, living organisms, or microorganisms [4]. The main advantages of biopolymers are their renewability, toxicity, biodegradability, and bioadhesion [4,5]. These include cationic polysaccharides such as chitosan, proteins (collagen), anionic polysaccharide (alginate), or plants such as tapioca, cotton, or corn [5].

Functional foods are increasingly popular among consumers, due to their health-enhancing effects and their protection against environmental stressors [6,7]. Raspberry fruit is characterized by a high content of compounds classified as functional food constituents, which include high levels of polyphenols, total dietary fiber, and vitamins (C, B₁, B₂, B₉, E, PP) and minerals (Fe, Cu, Ca, Mg, Mn, Cu, P, and K) [6].

Research has shown that the choice of food products is of key importance for consumers. Consumers more often accepted functional foods enriched with ingredients already present in and associated with these products [7,8]. Moreover, the way the ingredients were introduced into the food was an important parameter in consumers’ choice of specific products. Less processed and naturally enriched products were more likely preferable by shoppers [8].

The price of the food product was also an important aspect for consumers. Buyers more aware of the health benefits of functional products were ready to pay more for the product than those without this knowledge; however, the price should still not be too high [8]. According to Mirosa et al. [9] and Menrad [10], the acceptable price for functional products should not exceed 40% (in China) and 30–50% in Europe. Therefore, the choice of the biopolymers used for the production of biocomposite films seems to be a good option, as these raw materials are inexpensive and broadly available [11].

Lack of knowledge and modern solutions referring to the use of biopolymers in food can cause some concern and uncertainty among consumers [8]. According to the U.S. Food and Drug Administration (FDA), the materials used to create films are considered as GRAS (generally recognised as safe), both chitosan and plant extracts [12,13]. In addition, the European Union (EU) in accordance with Annex II and Annex III to Regulation (EC) No. 1333/2008 has established that sodium alginate is a safe substance and can be used as a food additive [14]. The addition of sodium alginate is due to the fact that chitosan is rarely used alone for film formation as it has poor mechanical strength, so additives in the form of other polysaccharides are used [11].

Poland has been a major raspberry producer for many years. In 2022, our country was the second largest producer of raspberries among EU countries, with 104,900 tons [15]. Raspberry fruits are characterized by high antioxidant, anti-inflammatory, and cytotoxic activity [16]. It had been shown that they have an effect on lowering blood glucose levels and reducing cholesterol and low-density lipoprotein levels. Interestingly, these properties were also exhibited by frozen raspberries [17]. In Poland, there are conventionally and organically grown as well as wild raspberries. These crops differ in the use of fertilizers and pesticides—they are not allowed in organic farming [18].

Raspberry fruit is characterized by seasonability and perishability. The high water content contributes to faster spoilage and thus to the need for fast post-harvest processing [19]. Proper freezing reduces the possibility of unfavourable changes in the colour, smell, and taste of the fruit. The freezing process allows the long-term storage of food products and significantly reduces nutrient losses and sensory changes compared to other methods of food preservation [19,20].

The aim of this study was to determine the effect of using films based on chitosan and sodium alginate with natural extracts (ginger, turmeric, and cinnamon) on the in-crease in antioxidant potential of raspberry fruit during frozen storage for 1-, 4-, 8-, and 12-months periods. Stored raspberry fruits were examined for selected bioactive compounds (total polyphenols and anthocyanins content) as well as their antioxidant activity. Furthermore, films before and after store of fruits were analysed in terms of changes in structure, wettability, and mechanical properties.

To the best of our knowledge, this is the first study determining the effect of frozen storage through several assumed research periods in different package types (especially innovative are traditional packages with the addition of polysaccharide films enriched with different extracts) on the antioxidative properties of raspberries from different crops (conventional, organic, and wild).

What is more, it is worth noting that the polysaccharide-based edible films were enriched with plant extracts like ginger, cinnamon, and turmeric with proven high antioxidant potential. In the case of food packaging, its most important function is to protect a product from mechanical damage due to external factors as well as to extend its shelf life. For small-volume packages, the purpose of their use is to create a specific microclimate in products in order to maintain the highest quality of a product in appropriate storage conditions. In our assumption, edible inserts based on natural ingredients, additionally enriched with plant extracts, changed the way of frozen food, and the traditional packages become active packages.

This study also aimed at increasing the consumer knowledge about the biological value of the raspberry fruit from different types of cultivation and wild-grown, particularly with regard to the presence of such components as total polyphenols and anthocyanins content and antioxidant activity as well as at contributing to the selection of an optimal packaging intended for the frozen storage of such a product.

In our opinion, this paper is a response to the search for innovative food characterized by high health quality and in line with the current trend of sustainable food production.

2. Materials and Methods

2.1. Plant Materials

The experimental material consisted of raspberry fruit (Rubus idaeus L.) from three crops: conventional, ecological (organic), and wild grown. Fruits were harvested in August 2020, at the stage of harvest maturity. The conventional and organic raspberries were of the Polana variety, while the wild raspberry variety was unknown. The raspberries were harvested in different locations, shown in

Table 1. Location of obtaining plant material.

	Conventional Raspberry	Ecological Raspberry	Wild Raspberry
Localization	Village Jadowniki, Malopolska Voivodeship	Village Bębło, Malopolska Voivodeship	The countryside of Siedlec, the Siesian Voivodeship
GPS	49.9588784, 20.643984269828643	50.18166317193472, 19.78522432981779	50.68940063565632, 19.367067526363734

. The location of the organic and conventional crop excluded differences due to different climatic and soil conditions.

2.2. Biocomposites Films

2.2.1. Extraction of Active Compounds

Extraction of polyphenols from the turmeric, cinnamon, and ginger were performed. Turmeric, cinnamon, and ginger powder (produced by Dary Natury, Grodzisk, Poland) were obtained from a local biosupermarket. The 5 g of powdered samples were mixed with 100 mL of ethanol. Subsequently, they were placed in a shaker (PolyScience, Niles, IL, USA) with a water bath at room temperature overnight. The obtained mixture was filtered and stored in the fridge at 4 °C to maintain the stability of the extracts.

2.2.2. Preparation of Chitosan-Alginate Films

The films were made by dissolving sodium alginate (viscosity 15–25 cps, Sigma Aldrich, St Louis, MO, USA), (2% m/v) in distilled water and stirring at temperature of 70 °C until it reached homogeneity. To obtain a 1% (m/v) chitosan solution, chitosan (medium molecular weight, deacetylation of ≥75%, viscosity 200–800 cps, Sigma Aldrich, St Louis, USA) was solubilized by stirring in a 1% (v/v) acetic acid solution at room temperature. The time and the amount of stirring were defined when the complete solubilization was reached. The chitosan solution was filtered to remove undissolved solids. Both solutions (chitosan and sodium alginate) were mixed together in a chitosan/alginate 3:1 (m/m) ratio, glycerol (StanLab, Lublin, Poland) was added at a 1% (v/v) concentration of the final solution, and the whole mixture was stirred for 30 min at room temperature. The final solution was homogenised with laboratory homogeniser (POLYTRON PT 2500E, Kinematica, Malters, Switzerland) (15,000 rpm/min) for 5 min. Investigated films were prepared by mixing films matrix (base) with various extracts of turmeric, cinnamon, and ginger. The base sample (chitosan/alginate/glycerol) was prepared as a control sample (without any extracts). Finally, the created films were transferred into Petri dishes and dried at room temperature.

2.3. Storage

After selection and cleaning of the raspberry fruit, a representative sample of approximately 100 g was taken and placed in a 23 × 16 cm zip-lock low density polyethylene (PE-LD) bags. The “control” sample consisted of 100 g of raspberry fruit only, while the remaining samples were also packed in a 23 × 16 cm zip-lock low density polyethylene (PE-LD) bags with an addition of edible films with dimensions 10 × 10 cm: (1) the base (chitosan-alginate film) and chitosan-alginate films enriched with natural plant extracts namely (2) cinnamon, (3) ginger, and (4) turmeric). A total of 60 PE-LD packages with a zipper closure were prepared with 100 g of conventionally, organically, and wild-cultivated raspberry fruit, which may or may not have contained additional polymeric inserts based on natural ingredients. All of them were sealed using a zip-lock. After 1-, 4-, 8-, and 12-month freezing periods of storage, these samples were then successively collected for analysis. The packed fruits were then stored at −22 °C in a freezer Liebherr GTS 3612 (Bulle, Switzerland).

2.4. Analysis of Dry Mass and Antioxidant Activity

After an appropriate freezing time, the samples were crushed in a mortar. The material was then taken for the determination of dry matter and the preparation of methanol extracts for the remaining analyses.

2.4.1. Dry Matter Content

The analysis was performed with reference to Standard PN-EN-12145:2001 [21]. A fragmented, mean representative sample was dried to constant weight at normal pressure conditions at 105 °C.

2.4.2. Preparation of Methanol Extracts

Methanol extracts were prepared by shaking 5 g of mean representative raspberry fruits in 80 mL of a 70% methanol solution for 2 h at the room temperature (23 °C) in an Orbital Shaker, Incubator ES-20/60, BioSan (Riga, Latvia). The next step was to filter the samples through a paper filter. The extracts were stored in a freezer at −22 °C [22].

2.4.3. Determination of Phenolic Content

The total polyphenols content was performed according to [23]. The analysis was carried out by diluting the samples with distilled water (1:20), then to 5 mL of this diluted samples was added 0.5 mL of Folin–Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) and 0.25 mL of 25% sodium carbonate (Stanlab, Lublin, Poland). The samples were stored for 20 min and after this time absorbances were read using a RayLeigh UV-1800 spectrophotometer (RayLeigh, Beijing, China). The concentration of total polyphenols was expressed in mg of chlorogenic and gallic acid per 100 g of dry weight (mg CGA and GAE/100 g DW, respectively). Determinations were performed in triplicate, and the final result is the arithmetic mean. The absorbances of the standard for chlorogenic and gallic acid were as follows: 0.5925 and 0.511.

Polyphenol content was calculated according to the formula:

$$\mathrm{X}={\displaystyle \frac{\frac{A_p}{A_w}\cdot a\cdot b\cdot 400}{c}}$$

where:

X—polyphenol content expressed as mg of standard in 100 g of product [mg/100 g],

A_p—absorbance of the sample measured at 760 nm against methanol,

A_v—absorbance of the standard measured at 760 nm wavelength against methanol,

a—mass of standard used for determination of polyphenols [mg],

b—the volume of methanol in which the extracts were prepared [cm³],

c—the mass of the product used to prepare the methanol extract [g].

2.4.4. Preparation of Standard Solutions

From the prepared chlorogenic/gallic acid solution of 1 mg/cm³, 1 cm³ was taken into a 100 cm³ volumetric flask and made up to the mark with distilled water. Then, 5 cm³ each of the diluted solutions were pipetted into test tubes and continued as with the standard sample. Determination of standard solutions was performed in two parallel repetitions, and the result is the arithmetic mean.

2.4.5. Determination of Anthocyanins Content

The concentration of anthocyanins was determined by diluting methanol extracts with 0.1 HCl in the ratio 95:5. The samples were measured after 1:1 dilution with buffer solutions at pH 1.0 and pH 4.5 using a RayLeigh UV-1800 spectrophotometer (Beijing, China). The measurement was made at two wavelengths—524 nm and 700 nm [24]. The results were expressed as cyanidin-3-glucoside.

2.4.6. Antioxidant Capacity

Antioxidant activity was determined by the ABTS^•+ free radical method and the FRAP method. Standards curves were made using the standard Trolox solution. The results were presented as µmol Trolox equivalents per gram of dry weight (µmol Trolox/g DW).

2.4.7. ABTS Method

Appropriately diluted extracts were incubated with the ABTS reaction mixture (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) with potassium persulfate) for 6 min at 30 °C, and then the absorbance at 734 nm was measured using a spectrophotometer (UV-1800, Rayleigh, Beijing, China) [25]. Calibration curve for the ABTS^•+ method was generated by measuring the absorbance of the reaction medium at 734 nm. Trolox was used as a standard. y = 0.1445x + 0.0106, R² = 0.9956.

2.4.8. FRAP Method

Methanol extract was diluted with 70% methanol to obtain the appropriate antioxidant concentration and mixed with the reaction mixture—acetic buffer, TPTZ solution, and iron chloride solution in a ratio of 10:1:1 [26]. After 10 min, the absorbance at 593 nm was measured by RayLeigh UV-1800 spectrophotometer (Beijing, China). The computation of antioxidant activity using the FRAP method was executed by employing the equation derived from the standard curve: y = 0.0098x + 0.0265, R² = 0.9910.

2.5. Analysis of Biocomposites Films

2.5.1. Structural Properties of Chitosan–Alginate Films

UV–Vis Spectrophotometry

The UV–Vis absorption spectra of the films before and after storage were recorded using a Shimadzu 2101 spectrophotometer (SHIMADZU, UV-2101PC, Kyoto, Japan). The absorbance spectra were measured in the range of 200–800 nm using 1 mL cells (Hellma, Müllheim, Germany). Concentration of solutions were 100 μg/mL. All the spectra were performed at room temperature (23.0 ± 0.5 °C).

ATR-FTIR Spectroscopy

The infrared spectrum measurements of the films were performed with the ATR–FTIR spectrophotometer Nicolet iS5 (Thermo Scientific, Waltham, MA, USA). A MIRacle ATR accessory equipped with a ZnSe crystal was used for sampling. The FTIR spectra were recorded in the range of 4000–700 cm⁻¹ at a resolution of 4 cm⁻¹. All the spectra were made at room temperature (23.0 ± 0.5 °C).

Evaluation of the Surface Wettability

The sessile drop method was employed to evaluate contact angle between the membrane surface and a water droplet lying on its surface using the automatic drop shape analysis system DSA 25 (Kruss, Hamburg, Germany). The following liquids were: the ultra-high-quality water (UHQ, PureLab, Vivendi Water) was employed as a working liquid. Contact angle was calculated by averaging 10 measurements. The results were expressed as mean ± standard deviation (SD).

Evaluation of the Mechanical Properties

The universal testing machine Inspect Table Blue 5 kN (Hegewald & Peschke, Nossen, Germany) was used to determine tensile strength (σM) and elongation at maximum force (εM). The machine was equipped with 100 N load cell. The samples for mechanical testing were rectangular in shape (50 mm × 5 mm). The tests were performed with the pre-load force of 0.1 N and the test speed of 10 mm min⁻¹. Mechanical properties were calculated by averaging 10 measurements and were expressed as mean ± standard deviation (SD).

2.6. Statistical Analysis

Measurements were performed in triplicate. The results were subjected to one-way analysis of variance, and Duncan’s test was performed with a statistical significance of α ≤ 0.05 by Statistica software v. 13.1 PL (Dell Inc., Tulsa, OK, USA). They were expressed as the mean ± standard deviation (SD). The factors considered for one-way analysis of variance were time of storage and type of used polysaccharide films.

3. Results

3.1. Analyses on Biocomposites Films

3.1.1. UV–Vis Spectrophotometry

UV–Vis spectroscopy was used to study the absorption spectra of alginate/chitosan films (base) without (1a) and with various extracts (1b, 1c, and 1d). Figure 1 shows the UV–absorption spectrum of turmeric, ginger, and cinnamon films, before and after storage. Additionally, all of these Figures contain the spectra of added extracts. In the range of 200–300 nm, there are observed absorption peaks characteristic for polyphenol contained in the extracts. Ginger extract shows a peak at 285 nm, which indicates the presence of flavonoids in ginger [27]. Cinnamon extract has two maximum absorption peaks at 220 nm and 285 nm due to p-p* of the conjugated C=C and C=O bonds [27]. Nevertheless, turmeric extract also has two strong peaks. Thus, curcumin present in turmeric shows two bands at 245 nm and 420 nm because of the p-p* type excitation resulting from the electronic dipole [27,28].

3.1.2. ATR-FTIR Spectroscopy

Fourier-transform infrared spectroscopy of the film samples were merged in Figure 2 and shows spectra of chitosan/alginate films without (2a) and with various extracts (2b, 2c, and 2d). FTIR spectra for pure chitosan and pure alginic acid were detailed in our earlier publication [29] and are referred in many articles.

In the present study of the films, the most intense bands are those corresponding to alginate and chitosan. A broad band around 3000 to 3500 cm⁻¹ was ascribed to the strong stretching vibration of a hydroxyl group (O–H) present in polysaccharides, glycerol, and water. FTIR spectra analysis for the base indicates (extracts) that in all cases, the band at 1425 cm⁻¹ comes from the alginate carboxylic group (–COO). The characteristic chitosan bands assigned to –NH stretching vibrations (3355 cm⁻¹) and also symmetric and asymmetric stretching the of the C–H bond of the –CH₂ group originate from pyranose ring (2890 cm⁻¹, 2856 cm⁻¹, 1412 cm⁻¹, and 1320 cm⁻¹) were observed. The asymmetric vibration bands of –C–O–C bridges from the glucopyranose ring in chitosan (1145 cm⁻¹, 895 cm⁻¹) and skeletal stretching =CO (1070 cm⁻¹, 1028 cm⁻¹) assigned to characteristic bands of polysaccharides were detected [30]. Chitosan amide group bands, stretching band –C=O (1589 cm⁻¹), and bending methyl group (1382 cm⁻¹) vibrations were collected.

In the FTIR spectra of alginate and chitosan films (base), the bands of –NH stretching of amide group (1652 cm⁻¹) in chitosan and the absorption bands of carboxyl anion (1612 cm⁻¹) in alginate disappeared, suggesting the interaction between –NH³⁺ and –COO^– ions. It can be assumed that the band located around 1620 cm⁻¹ in the chitosan/alginate base film can be ascribed to the common contribution of the two types of groups [31]. These films (base) are formed through electrostatic interactions between the carboxylate groups (–COO⁻) in the alginic and the primary ammonium groups (–NH₃⁺) in chitosan. The formation of a polyelectrolyte complex (base) may be induced most likely by attractive electrostatic interactions between these two polysaccharides [31].

3.1.3. Evaluation of the Surface Wettability and Mechanical Properties

The results of static water contact angle of the foils before and after storage of raspberries are shown in Figure 3. After storage, all foils showed significantly improved wettability, indicating chemical and structural changes. In the case of base foil and material containing cinnamon, the cultivation method of the raspberries did not affect water contact angle of the foils. In turn, materials modified with ginger and turmeric showed different wettability depending on the cultivation method of the raspberries.

The results of tensile strength (σM) and elongation at maximum force (εM) of the foils before and after storage of raspberries are shown in Figure 4. The storage significantly affected mechanical properties of the materials. Moreover, the changes depended on both foil type and cultivation method of the raspberries. The contact of all types of the foils with conventionally cultivated raspberry resulted in significant lowering of the tensile strength. The foils containing cinnamon lost their integrity during storage of conventionally cultivated raspberry, making mechanical testing impossible. In the case of base foil, the contact with ecological and wild raspberries resulted in an increase in tensile strength. The storage of these two raspberries with the foil modified with cinnamon did not affect tensile strength. In turn, both ginger- and turmeric-containing materials showed similar pattern of changes in tensile strength. Namely, ecological raspberry induced reduction in tensile strength, while wild fruit caused a significant increase in tensile strength. For all types of foils, storage resulted in significant reduction in the elongation at maximum force. As with wettability, the changes in the elongation observed for the base foil and the one containing cinnamon did not depend on the raspberry cultivation method. In contrast, for materials modified with ginger and turmeric, differences were observed depending on the method of cultivation of the fruit.

3.2. Antioxidant Activity

This paper presents the total content of polyphenols, anthocyanins, and antioxidant activity in frozen stored raspberry fruit from the three crops examined. On the other hand, as for the results obtained for fresh raspberry fruit, they were described in detail in a previous study [32].

3.2.1. The Content of Total Polyphenols

In each raspberry fruit crop, a decrease in total polyphenol content after the first month of refrigerated storage was observed when compared to fresh fruit, as shown in

Table 2. The content of polyphenol in frozen raspberry fruits stored with sodium alginate and chitosan films (expressed as mg of chlorogenic acid per 100 g⁻¹ of dry weight).

Raspberry Fruits	The Kind of Packaging	The Time of Frozen Storage
		1 Month		4 Month		8 Month		12 Month
		Total Polyphenols [mg CGA 100 g−1 DW]	Changes [%] *	Total Polyphenols [mg CGA 100 g−1 DW]	Changes [%] **	Total Polyphenols [mg CGA 100 g−1 DW]	Changes [%] **	Total Polyphenols [mg CGA 100 g−1 DW]	Changes [%] **
Conventional	control	900.76 ± 9.95 a,A	−30.22	987.78 ± 31.48 ab,A	9.66	1152.57 ± 32.18 bc,AB	27.95	1300.56 ± 127.49 c,BC	43.69
	base	917.65 ± 49.80 a,A	1.88	886.03 ± 19.20 a,A	−3.45	1456.28 ± 155.52 b,C	58.70	1090.15 ± 196.07 a,AB	18.12
	cinnamon	1179.79 ± 3.43 b,A	−30.98	735.15 ± 168.12 a,A	−37.69	1044.54 ± 41.36 b,A	−11.46	1360.99 ± 32.24 c,C	15.36
	ginger	1246.64 ± 22.83 b,BC	38.40	806.44 ± 145.20 a,A	−35.31	1147.42 ± 108.65 b,AB	−7.96	1075.68 ± 132.39 ab,A	−13.95
	turmeric	1336.13 ± 55.91 b,C	48.33	825.70 ± 21.68 a,A	−38.20	1326.93 ± 72.91 b,BC	−0.69	1048.47 ± 165.16 ab,A	−21.76
Organic	control	1219.36 ± 43.10 a,C	−27.14	1216.45 ± 80.29 a,AB	−0.24	1266.84 ± 52.60 a,A	3.89	1546.15 ± 20.42 b,B	26.80
	base	815.63 ± 16.34 a,B	−33.11	1192.31 ± 157.41 ab,AB	46.18	1450.75 ± 333.66 b,A	77.87	1525.50 ± 144.18 b,B	87.03
	cinnamon	658.26 ± 83.37 a,A	−46.01	1390.77 ± 94.29 b,B	111.28	1384.87 ± 31.37 b,A	110.39	1530.28 ± 155.26 b,B	132.47
	ginger	851.49 ± 35.82 a,B	−30.17	1114.05 ± 33.91 b,A	30.84	1256.31 ± 33.78 b,A	47.54	1324.77 ± 127.45 b,A	55.58
	turmeric	1222.45 ± 0.19 a,C	0.25	1302.73 ± 37.02 ab,AB	6.57	1483.61 ± 196.87 b,A	21.36	1337.20 ± 7.65 ab,A	9.39
Wild	control	1324.77 ± 86.15 ab,A	−18.76	1208.33 ± 44.30 a,C	−8.79	1444.28 ± 192.21 b,A	9.02	2480.66 ± 34.68 c,D	87.25
	base	1003.40 ± 254.10 a,A	−24.26	960.11 ± 36.88 a,A	−4.31	1439.24 ± 209.44 b,A	43.44	1820.77 ± 11.29 c,A	81.46
	cinnamon	1071.73 ± 5.93 b,A	−19.10	880.83 ± 17.97 a,A	−17.81	1548.15 ± 0.17 c,A	44.45	2122.79 ± 52.74 d,B	98.07
	ginger	1202.94 ± 21.10 a,A	−9.20	1097.58 ± 134.20 a,B	−8.76	1270.08 ± 118.30 a,A	5.58	2362.38 ± 84.18 b,C	96.38
	turmeric	1211.39 ± 212.47 a,A	−8.56	1265.73 ± 72.02 a,C	4.49	1140.40 ± 190.85 a,A	−5.86	1799.29 ± 42.56 b,A	48.53

Results expressed as mean ± SD obtained from the analysis of three individual samples (n = 3). Mean values presented as letters (a–d) within individual periods of freezing storage (rows) are significantly different (Duncan test p ≤ 0.05). Mean values presented as letters (A–D) within application of the specific polysaccharide films (columns) are significantly different (Duncan test p ≤ 0.05). * change in the content of total polyphenols in relation to fresh raspberry fruit samples. ** change in the content of total polyphenols in relation to storage after the first month of storage of raspberries.

and

Table 3. The content of polyphenol content in frozen raspberry fruits stored with sodium alginate and chitosan films (expressed as mg of gallic acid per 100 g⁻¹ of dry weight).

Raspberry Fruits	The Kind of Packaging	The Time of Frozen Storage
		1 Month		4 Month		8 Month		12 Month
		Total Polyphenols [mg GAE 100 g−1 DW]	Changes [%] *	Total Polyphenols [mg GAE 100 g−1 DW]	Changes [%] **	Total Polyphenols [mg GAE 100 g−1 DW]	Changes [%] **	Total Polyphenols [mg GAE 100 g−1 DW]	Changes [%] **
Conventional	control	1112.19 ± 10.07 a,A	−26.81	1234.01 ± 33.17 ab,B	10.95	1369.13 ± 32.77 bc,AB	23.10	1527.89 ± 130.16 c,B	37.38
	base	1137.99 ± 50.33 a,A	−25.11	1146.17 ± 20.80 a,AB	0.76	1708.00 ± 160.76 b,C	50.09	1284.27 ± 204.53 a,A	12.85
	cinnamon	1423.02 ± 2.44 bc,B	−6.36	945.24 ± 173.54 a,A	−33.57	1253.36 ± 42.48 b,A	−11.92	1578.98 ± 34.00 c,B	10.96
	ginger	1498.54 ± 23.38 b,BC	−1.39	1025.65 ± 153.64 a,AB	−31.56	1349.62 ± 112.12 b,AB	−9.94	1283.80 ± 136.89 ab,A	−14.33
	turmeric	1581.00 ± 58.24 b,C	4.04	1070.57 ± 22.86 a,AB	−32.28	1573.56 ± 76.29 b,BC	−0.47	1261.39 ± 169.87 a,A	−20.22
Organic	control	1496.26 ± 41.80 a,C	−22.60	1502.39 ± 83.56 a,AB	0.41	1534.66 ± 54.51 a,A	2.57	1807.22 ± 21.39 b,B	20.78
	base	1052.96 ± 19.77 a,B	−45.53	1465.66 ± 164.12 ab,AB	39.19	1737.75 ± 347.26 b,A	65.03	1775.27 ± 149.28 b,B	68.60
	cinnamon	903.73 ± 86.44 a,A	−53.25	1688.97 ± 99.08 b,B	86.89	1662.24 ± 30.68 b,A	83.93	1759.52 ± 161.78 b,B	94.70
	ginger	1091.24 ± 38.86 a,B	−43.55	1361.38 ± 38.56 b,A	24.76	1533.72 ± 40.55 b,A	40.55	1551.43 ± 132.95 b,A	42.17
	turmeric	1505.84 ± 1.81 a,C	−22.10	1574.73 ± 35.57 ab,AB	4.58	1751.67 ± 203.54 b,A	16.33	1571.78 ± 8.00 ab,A	4.38
Wild	control	1544.47 ± 89.09 ab,A	−16.54	1393.63 ± 46.02 a,C	−9.77	1665.67 ± 199.30 b,AB	7.85	2752.02 ± 34.99 c,D	78.19
	base	1189.67 ± 263.05 a,A	−35.71	1128.95 ± 38.24 a,A	−5.10	1657.51 ± 222.39 b,AB	39.33	2080.82 ± 11.70 c,A	74.91
	cinnamon	1268.55 ± 6.15 b,A	−31.45	1049.80 ± 18.88 a,A	−17.24	1820.40 ± 0.55 c,B	43.50	2409.13 ± 55.53 d,B	89.91
	ginger	1399.97 ± 21.88 a,A	−24.35	1273.71 ± 138.84 a,B	−9.02	1482.63 ± 127.79 a,AB	5.90	2662.11 ± 86.37 b,C	90.15
	turmeric	1426.55 ± 220.06 a,A	−22.91	1472.41 ± 74.85 a,C	3.21	1347.36 ± 202.43 a,A	−5.55	2084.15 ± 43.82 b,A	46.10

Results expressed as mean ± SD obtained from the analysis of three individual samples (n ≥ 3). Mean values presented as letters (a–d) within individual periods of freezing storage (rows) are significantly different (Duncan test p ≤ 0.05). Mean values presented as letters (A–D) within application of the specific polysaccharide films (columns) are significantly different (Duncan test p ≤ 0.05). * change in the content of total polyphenols in relation to fresh raspberry fruit samples. ** change in the content of total polyphenols in relation to storage after the first month of storage of raspberries with appropriate films.

. The values were 30.22, 27.14, and 18.76% for chlorogenic acid and 26.81, 22.60, and 16.54% for gallic acid in conventionally, organically, and wild-grown fruit, respectively.

Our studies showed that refrigerated storage of wild and organically grown raspberry fruit resulted in a significant increase in polyphenol content after 12 months of storage. When storing raspberry fruit in a zip-lock bag without polysaccharide film, a statistically significant increase was observed after 12 months of frozen storage compared to the first month in each crop type.

In all crop types, polyphenol content increased at 8 and 12 months of frozen storage with films based on sodium alginate and chitosan. In the films with added cinnamon extract, there was a statistically significant increase in the amount of polyphenols in the last month of storage. In contrast, films with the addition of ginger contributed to an increase in polyphenols only in organically grown and wild raspberry fruit at the 4th, 8th, and 12th month and 8th and 12th month, respectively. The addition of turmeric extract increased the polyphenol content in the 8th month of frozen storage of organically grown raspberry fruit and in the 12th month of wild grown raspberry.

The changes in the polyphenol content of the samples examined during frozen storage seems not to be dependent on the use of polysaccharide films.

3.2.2. The Content of Anthocyanins

A decrease in anthocyanin content was observed only in the frozen-stored and organically grown raspberry, and this was by 12.94% compared to fresh raspberry fruit (

Table 4. The content of anthocyanins in frozen raspberry fruits stored with sodium alginate and chitosan films (mg 100 g⁻¹ DW).

Raspberry Fruits	The Kind of Packaging	The Time of Frozen Storage
		1 Month		4 Month		8 Month		12 Month
		Total Anthocyanins [mg 100 g−1 DW]	Changes [%] *	Total Anthocyanins [mg 100 g−1 DW]	Changes [%] **	Total Anthocyanins [mg 100 g−1 DW]	Changes [%] **	Total Anthocyanins [mg 100 g−1 DW]	Changes [%]**
Conventional	control	195.33 ± 13.34 b,AB	23.54	177.60 ± 20.01 ab,A	−9.08	175.07 ± 2.21 ab,A	−10.37	161.99 ± 6.26 a,A	−17.07
	base	178.96 ± 9.41 a,A	9.41	165.72 ± 0.76 a,A	−7.40	246.27 ± 18.35 b,D	37.61	170.46 ± 25.28 a,AB	−4.75
	cinnamon	206.63 ± 7.86 a,ABC	30.69	164.90 ± 46.60 a,A	−20.19	181.42 ± 3.60 a,AB	−12.20	201.12 ± 13.05 a,B	2.67
	ginger	234.20 ± 7.35 b,C	48.13	169.21 ± 25.59 a,A	−27.75	192.57 ± 3.31 a,B	−17.77	168.53 ± 18.87 a,AB	−28.04
	turmeric	217.82 ± 25.34 b,BC	37.77	154.53 ± 0.81 a,A	−29.06	215.12 ± 6.58 b,C	−1.24	198.75 ± 31.25 b,B	−8.75
Organic	control	221.82 ± 24.63 a,C	−12.94	238.19 ± 48.25 a,A	7.38	209.88 ± 1.13 a,A	−5.39	219.44 ± 5.04 a,B	−1.08
	base	182.63 ± 8.07 a,B	−28.32	259.63 ± 83.42 a,A	42.16	236.66 ± 16.70 a,B	29.58	220.86 ± 14.70 a,B	20.93
	cinnamon	207.63 ± 0.55 a,BC	−18.51	287.58 ± 19.64 c,A	38.50	239.45 ± 1.43 b,B	15.33	257.68 ± 14.95 bc,C	24.10
	ginger	147.08 ± 9.24 a,A	−42.28	245.92 ± 3.38 c,A	67.21	223.39 ± 7.20 bc,AB	51.89	195.41 ± 16.43 b,A	32.86
	turmeric	268.09 ± 1.66 d,D	5.21	249.32 ± 8.27 c,A	−7.00	225.38 ± 0.63 b,AB	−15.93	192.86 ± 3.55 a,A	−28.06
Wild	control	265.80 ± 45.60 b,C	30.92	172.92 ± 5.69 a,C	−34.94	203.34 ± 29.39 a,B	−23.50	315.09 ± 0.42 b,D	18.54
	base	157.47 ± 14.33 a,A	−22.44	142.78 ± 5.17 a,B	−9.33	222.13 ± 23.93 b,B	41.06	223.26 ± 16.02 b,A	41.78
	cinnamon	179.45 ± 4.14 b,AB	−11.61	111.78 ± 6.69 a,A	−37.71	216.16 ± 7.70 c,B	20.46	252.52 ± 5.50 d,B	40.72
	ginger	206.51 ± 13.82 b,B	1.72	157.23 ± 22.38 a,BC	−23.86	202.43 ± 8.26 b,B	−1.97	284.86 ± 10.25 c,C	37.94
	turmeric	172.82 ± 21.18 a,AB	−14.88	166.42 ± 11.62 a,C	−3.70	147.85 ± 17.25 a,A	−14.45	250.55 ± 12.72 b,B	44.97

Results expressed as mean ± SD obtained from the analysis of three individual samples (n ≥ 3). Mean values presented as letters (a–d) within individual periods of freezing storage (rows) are significantly different (Duncan test p ≤ 0.05). Mean values presented as letters (A–D) within application of the specific polysaccharide films (columns) are significantly different (Duncan test p ≤ 0.05). * change in the content of anthocyanins in relation to fresh raspberry fruit samples. ** change in the content of anthocyanins in relation to storage after the first month of storage of raspberries with appropriate film.

). After one month of frozen storage, anthocyanin content in wild and conventionally grown raspberry fruit increased by 30.92 and 23.54%, respectively.

The use of ginger and turmeric films led to a rise in anthocyanins in conventionally grown fruit in the 1st, 8th, and 12th (turmeric and cinnamon) months of storage, compared to the control in each month of storage. The highest increase in anthocyanins was noted during the eighth month (baseline film).

After the fourth month of frozen storage of conventionally grown fruit and those growing wild, a decrease in nutritional value was observed. A statistically significant increase in anthocyanin content was recorded when freezing organic fruit with a film of cinnamon extract and ginger, compared to the first month of freezing. An increase in nutritional value by 38.50% and 67.21%, respectively, was noticed in the organic crop with the cinnamon and ginger films compared to the first month. Fruit with these films also demonstrated the highest increase in anthocyanins at the 8th and 12th month of frozen storage, as compared to fresh raspberry.

During frozen storage of wild raspberry fruit, an increase in anthocyanin content was observed in all cases at the 12th month. This result is almost twice as high as in the control sample—raspberry fruit frozen in zip-lock bags for 12 months. The greatest rise in the content of bioactive constituents occurred when films with turmeric extract were applied.

Depending on the crop, a trend towards an increase in anthocyanin content can be observed with the use of film with ginger and turmeric (compared to the control) and cinnamon and ginger (compared to the storage period).

3.2.3. ABTS and FRAP Method

The results of antioxidant activity obtained by the FRAP method and the free radical scavenging capacity of ABTS showed similar trends in changes of antioxidant compounds in the examined material, as shown in

Table 5. The antioxidant activity of frozen raspberry fruits stored with sodium alginate and chitosan films (µmol Trolox g⁻¹ DW) evaluated by using ABTS assay.

Raspberry Fruits	The Kind of Packaging	The Time of Frozen Storage
		1 Month		4 Month		8 Month		12 Month
		ABTS [µmol Trolox g−1 DW]	Changes [%] *	ABTS [µmol Trolox g−1 DW]	Changes [%] **	ABTS [µmol Trolox g−1 DW]	Changes [%] **	ABTS [µmol Trolox g−1 DW]	Changes [%] **
Conventional	control	100.93 ± 0.95 a,A	−13.28	119.30 ± 0.98 b,B	18.21	112.05 ± 1.39 b,A	11.02	172.73 ± 5.10 c,B	71.14
	base	107.01 ± 4.33 a,B	−8.06	117.19 ± 3.07 a,B	9.51	136.87 ± 6.33 b,B	27.90	146.13 ± 7.37 b,A	36.56
	cinnamon	125.73 ± 0.73 c,C	8.02	95.29 ± 8.25 a,A	−24.21	111.93 ± 0.29 b,A	−10.98	167.15 ± 3.14 d,B	32.94
	ginger	131.16 ± 0.33 b,D	12.69	102.24 ± 7.68 a,A	−22.05	111.34 ± 0.08 a,A	−15.11	149.94 ± 7.16 c,A	14.32
	turmeric	132.52 ± 1.04 b,D	13.85	107.41 ± 0.66 a,AB	−18.94	131.46 ± 6.40 b,B	−0.80	154.43 ± 9.53 c,A	16.54
Organic	control	135.31 ± 0.32 a,C	13.80	143.04 ± 3.34 b,BC	5.72	199.89 ± 1.90 d,A	47.73	191.49 ± 3.98 c,C	41.52
	base	107.08 ± 2.00 a,B	−9.94	138.08 ± 6.03 b,B	28.95	228.66 ± 9.71 d,C	113.54	189.07 ± 9.38 c,BC	76.56
	cinnamon	100.39 ± 4.87 a,A	−15.57	161.27 ± 2.11 b,D	60.64	204.46 ± 2.65 d,AB	103.66	180.67 ± 4.37 c,B	79.97
	ginger	108.34 ± 0.14 a,B	−8.88	129.69 ± 0.18 b,A	19.70	213.97 ± 1.53 d,B	97.50	166.64 ± 7.25 c,A	53.81
	turmeric	147.22 ± 0.06 a,D	23.81	146.94 ± 0.43 a,C	−0.19	203.45 ± 0.57 c,AB	38.19	171.14 ± 1.35 b,A	16.25
Wild	control	172.53 ± 0.65 b,C	43.73	106.56 ± 0.78 a,C	−38.23	242.44 ± 17.87 c,B	40.52	324.56 ± 3.55 d,B	88.12
	base	141.18 ± 9.13 b,A	17.61	93.89 ± 1.96 a,AB	−33.50	242.51 ± 9.75 c,B	71.77	288.18 ± 5.72 d,A	104.12
	cinnamon	156.64 ± 0.00 b,B	30.49	90.74 ± 1.39 a,A	−42.07	279.61 ± 2.32 c,C	78.51	325.91 ± 6.53 d,B	108.06
	ginger	166.75 ± 1.33 b,BC	38.91	97.84 ± 5.30 a,B	−41.32	220.84 ± 11.79 c,A	32.44	348.40 ± 7.50 d,C	108.94
	turmeric	157.94 ± 6.46 b,B	31.57	119.92 ± 2.16 a,D	−24.07	208.36 ± 11.70 c,A	31.94	321.82 ± 9.36 d,B	103.77

Results expressed as mean ± SD obtained from the analysis of three individual samples (n ≥ 3). Mean values presented as letters (a–d) within individual periods of freezing storage (rows) are significantly different (Duncan test p ≤ 0.05). Mean values presented as letters (A–D) within application of the specific polysaccharide films (columns) are significantly different (Duncan test p ≤ 0.05). * change the antioxidant activity in relation to fresh raspberry fruit samples. ** change in the antioxidant activity in relation to storage after the first month of storage of raspberries with appropriate film.

and

Table 6. The antioxidant activity of frozen raspberry fruits stored with sodium alginate and chitosan films (µmol Trolox g⁻¹ DW) evaluated by using FRAP assay.

Raspberry Fruits	The Kind of Packaging	The Time of Frozen Storage
		1 Month		4 Month		8 Month		12 Month
		FRAP [µmol Trolox g−1 DW]	Changes [%] *	FRAP [µmol Trolox g−1 DW]	Changes [%] **	FRAP [µmol Trolox g−1 DW]	Changes [%] **	FRAP [µmol Trolox g−1 DW]	Changes [%] **
Conventional	control	226.74 ± 0.90 a,A	−0.94	263.54 ± 7.78 a,C	16.23	265.11 ± 5.13 a,A	16.92	361.62 ± 33.79 b,AB	59.48
	base	222.93 ± 10.34 a,A	−2.60	237.84 ± 3.73 a,BC	6.69	355.57 ± 48.43 b,C	59.50	310.54 ± 56.87 ab,A	39.30
	cinnamon	278.68 ± 3.99 b,B	21.76	190.10 ± 25.73 a,A	−31.78	283.17 ± 3.78 b,AB	1.61	379.84 ± 5.19 c,B	36.30
	ginger	298.49 ± 9.12 b,BC	30.41	212.03 ± 23.88 a,AB	−28.97	286.62 ± 6.09 b,AB	−3.98	316.47 ± 40.46 b,AB	6.02
	turmeric	311.68 ± 14.25 b,C	36.17	218.58 ± 9.84 a,AB	−29.87	327.47 ± 11.95 b,BC	5.07	304.39 ± 49.11 b,A	−2.34
Organic	control	279.97 ± 6.03 a,C	−7.15	302.73 ± 17.96 b,A	8.13	380.94 ± 3.31 d,A	36.07	343.89 ± 6.86 c,B	22.83
	base	200.78 ± 0.72 a,B	−33.41	292.78 ± 37.04 ab,A	45.82	405.50 ± 73.90 c,A	101.96	346.68 ± 35.21 bc,B	72.67
	cinnamon	158.72 ± 17.14 a,A	−47.36	366.30 ± 18.46 b,B	130.79	390.37 ± 1.80 b,A	145.96	362.23 ± 23.06 b,B	128.23
	ginger	197.13 ± 5.97 a,B	−34.62	298.62 ± 3.36 b,A	51.48	412.04 ± 6.47 c,A	109.02	296.12 ± 30.51 b,A	50.22
	turmeric	320.99 ± 3.79 b,D	6.46	326.16 ± 0.99 b,AB	1.61	397.36 ± 2.40 c,A	23.79	300.90 ± 2.48 a,A	−6.26
Wild	control	284.78 ± 21.14 a,A	−11.38	273.02 ± 5.39 a,BC	−4.13	378.81 ± 52.61 b,B	33.02	598.44 ± 4.82 c,D	110.14
	base	230.82 ± 43.12 a,A	−28.17	224.45 ± 5.81 a,A	−2.76	399.49 ± 65.72 b,B	73.07	443.62 ± 2.23 b,B	92.19
	cinnamon	246.17 ± 0.96 b,A	−23.39	199.85 ± 4.53 a,A	−18.82	420.00 ± 5.51 c,B	70.61	517.59 ± 6.29 d,C	110.25
	ginger	277.63 ± 3.25 a,A	−13.60	253.19 ± 33.76 a,B	−8.80	350.60 ± 22.34 b,AB	26.28	575.78 ± 53.40 c,D	107.39
	turmeric	246.45 ± 40.83 ab,A	−23.31	296.64 ± 12.10 b,C	20.36	302.15 ± 43.79 b,A	22.60	236.10 ± 25.18 a,A	−4.20

Results expressed as mean ± SD obtained from the analysis of three individual samples (n ≥ 3). Mean values presented as letters (a–d) within individual periods of freezing storage (rows) are significantly different (Duncan test p ≤ 0.05). Mean values presented as letters (A–D) within application of the specific polysaccharide films (columns) are significantly different (Duncan test p ≤ 0.05). * change the antioxidant activity in relation to fresh raspberry fruit samples. ** change in the antioxidant activity in relation to storage after the first month of storage of raspberries with appropriate film.

.

A notable effect was seen in the first month of freezing the fruit from conventionally grown raspberries—all these samples exhibited increased antioxidant activity compared to the control.

The results showed that raspberries from specific crops behave differently in terms of changes in bioactive components during storage. A common feature was that after 12-month storage, raspberries from all crops had higher and statistically significant values of antioxidants (ABTS) compared to the first month of storage. A similar relationship can also be noticed at the eighth month, with the exception of raspberry fruit from the conventional crop.

In the eighth month of storage, there was a noticeable increase in antioxidant compounds in the base films (conventional and organic raspberry) and in the films with added turmeric extract (conventional raspberry), ginger (organic raspberry), and cinnamon (wild-grown raspberry), compared to the control sample.

Moreover, the wild raspberry fruit behaved differently from the rest of the crop. In the fourth month of storage, their antioxidant activity decreased compared to the first month of storage, followed by an increase at the eighth and twelfth months—even amounting to more than 100 per cent at the twelfth month in the control samples.

After examining antioxidant compounds by means of the FRAP method, similar conclusions can be drawn, i.e., an increase in these components was in the eighth and twelfth month of storage; however, not in all samples as when they were determined using ABTS. A statistically significant increase of antioxidant components was observed in the eighth month of storage of conventional raspberry fruit in basal film and in the film with turmeric extract, compared to the control. This was also proved by the ABTS analysis.

We could also observe a relationship occurring in raspberry fruit frozen with films with added turmeric extract from all crops in the 12th month of storage, i.e., a decrease in antioxidant activity measured by FRAP.

There was a replicating trend of an increase in antioxidant compounds with the length of storage of the raspberry fruit, with the greatest rises in the eighth and twelfth months and also in the fourth month (organic raspberry, film with cinnamon and ginger extract).

4. Discussion

Raspberry fruit has a number of components that show health-promoting effects and act as functional foods [33]. Due to the high content of polyphenolic compounds, including anthocyanins, and other bioactive compounds (carotenoids, vitamin C, and macro- and micronutrients), an increase in consumption and production of raspberries is observed [17]. The content of these compounds contributes to their antioxidant, anti-inflammatory, and antimicrobial activities [34]. Moreover, the activity of functional components is particularly high in the fruit [6].

In this study, biopolymer films made of natural biodegradable ingredients (chitosan and sodium alginate) were used with added natural plant extracts. It has been confirmed that biopolymers can serve as a carrier for polyphenols. Natural plant extracts used as an additive to biopolymer films can provide an additional source of antioxidant substances [3]. Incorporating natural extracts into the packaging material appears to be a more beneficial solution than applying it directly onto the examined product, since direct contact with the food is reduced [35]. Furthermore, when introducing ingredients with a functional effect into the packaging of a food product, we can expect that the product will acquire new properties and also increase its health-promoting value due to the possible migration of the introduced constituents [6,36]. The use of natural extracts may also contribute to extending the shelf life of the product, due to their antimicrobial and antibacterial properties [3]. A potential disadvantage of using natural extracts may be their intense odour and taste [3].

In this study, raspberries from three crops are investigated: conventional, organic, and wild grown. These crop types differ from each other, which may affect the content of bioactive components in them. This was shown in previous studies providing an overview of the basic composition, the content of minerals and bioactive substances, as well as selected heavy metals in these raspberries [32,37]. The investigated crops differed the most in terms of application and type of fertilizer. In organic crops, the use of synthetic fertilisers and pesticides is forbidden [18]. On the other hand, wild-growing raspberries are not controlled and supervised by regulations. Therefore, the soil they are growing in is not examined for the content of substances in the soil, including heavy metals. As a result, there may be differences in the bioactive compounds in the fruit depending on its origin [16,38].

In our study, a completely different trend in bioactive compounds was observed depending on the type of crop. The content of total polyphenols in the first month of frozen storage of raspberry fruit (control) was lower than that found in fresh fruit. With the length of the storage period (8 and 12 months), the content of these compounds was increasing. In contrast, a different trend was observed in the content of anthocyanins. The amounts increased or decreased throughout storage, depending on the type of crop and the biocomposites used. This fact is also confirmed by the literature [39,40,41]. This phenomenon is due to unfavourable conditions for polyphenols, such as low temperature or the formation of ice crystals during the freezing process. The latter process can have a twofold effect—destroying compounds contained in tissues, as well as releasing them by damaging plant tissues [19]. The other occurring phenomenon is the inhibition of polyphenol-degrading enzymes, e.g., polyphenol oxidase [16,20]. An unusual phenomenon was that after 4-month storage at −22 °C, there was a noticeable trend towards a decrease in antioxidant compounds, i.e., anthocyanins (conventional and wild raspberry) and polyphenols (conventional and wild raspberry). In turn, González et al. [42] showed that the total polyphenol and anthocyanin content of four raspberry varieties (Heritage, Autumn, Zeva and Rubi) during frozen storage for 3, 6, 9, and 12 months was different for each variety and month of storage. No joint trend in changes in total polyphenols and anthocyanin contents during frozen storage was observed, which agree with our findings. As Mullen et al. (2002) [43] report, antioxidant activity increases by 3.45%, when raspberries are stored at −30 °C due to easier extraction of antioxidant compounds after cell disruption occurring during the freezing process [44].

The assessment of the direction of changes occurring in antioxidants during frozen storage is a difficult issue. It is influenced by many variables, starting with the freezing technique and thus the extent of ice crystal formation, as well as the method of thawing. Environmental conditions and cultivation method, the plant variety, and its degree of maturity are also affecting factors [19]. The lower polyphenol content may result from the increased interaction of these compounds with oxidative enzymes, which are formed when cells are destroyed during the freezing and ice crystal formation process [19]. Degradative enzymes, not decomposed before the freezing process, start changes in the structure and colour of the fruit and in the content of antioxidant compounds [19]. The above changes are also affected by the freezing method used. According to Ergün et al. [45], quick freezing of strawberry fruit caused less adverse changes in the colour, structure, and antioxidant activity of the fruit than static freezing.

During freezing with the films, raspberry fruits were in contact with only one side of the experimental material, which may explain differences in the content of bioactive compounds. Therefore, it is worth considering whether to carry out studies involving spray or dip application of films [46]. Research proves that the use of gel-based coatings and alginate with additives increases the amount of antioxidants in fresh fruit pieces [47,48].

Examination of fresh biocomposites films and after storage together with raspberries after 12 months shows what changes occurred in them after that period. As observed in a Figure, absorbance spectrum of base (1a) differ significantly from the remaining spectra. It depends on the overlapping spectra of mixed polysaccharides. In the same Figure 1a, one can observe a strong peak at ~270–280 nm. Since these samples did not contain extracts, these peaks are likely due to anthocyanins contained in raspberries. A typical UV–Vis spectrum of an anthocyanin shows two basic maximum peaks of absorbance (as well as all flavonoids) [49]. The first peak is being recorded at UV region (260–280 nm) and the other one at 490–550 nm which corresponds to visible region [49]. This is related to their long chromophore of eight conjugated double bonds and presence of a positive charge on the heterocyclic oxygen ring, observed in acidic conditions [50,51]. In our spectra analysis, only the peak characteristic for the UV range was observed. It is worthy to know that spectra and intensity of peaks depends on pH [52] and type of anthocyanins predominant in a sample [53].

The other spectra displayed a significant peak at around 285 nm. These data confirm the successful interaction between extracts and base film. Slight shifts were recorded in the absorption peak of samples. The shifts in band positions indicate the involvement of the polyphenol and anthocyanins aromatic resonance electrons in the interaction with the host molecules (base) [54]. The main anthocyanins found in raspberries composed of aglycone cyanidin. This structure has several hydroxyl substitutions and is highly unstable [53]. The interaction with host polysaccharides and guest (polyphenols from the extracts) causes a shift in UV–Vis spectrum.

In the ATR-FTIR spectrophotometry study, there was a very limited number of changes between the obtained absorption spectra for base film and films with addition of extracts before storage. A comparative evaluation of the FTIR bands indicated that most of the bands were similar in their band characteristics such as peak position and broad nature. The synthesized alginate/chitosan films with extracts had no significant differences from the FTIR spectrum of the of base films with slight changes (small bands shifts).

The FTIR spectra analysis was used to identify the functional groups and carried out to observe the changes in the films after 12-month storage with raspberries in a freezer. In all of the samples, many structural changes were observed in the films. The bands characteristic to polysaccharides (1145 cm⁻¹, 895 cm⁻¹) and the glucopyranose ring (2890 cm⁻¹, 2856 cm⁻¹, 1412 cm⁻¹, and 1317 cm⁻¹) disappeared. This suggests that the structure of chitosan and alginate polymers were partly decomposed. Simultaneously, the presence of new bands was recorded. These bands could be attributed to polyphenols and anthocyanins contained in raspberries. As a consequence of low pH in plants, cyanidin-3-glucoside is present. Due to the acidic nature of the berries, all berries contain glycosides of cyanidin [55]. Especially, raspberry contains cya-3-glu, cya-3-rut, cya-3-sop [55,56].

The intense broad band at 3300 cm⁻¹ was assigned to –OH stretching vibrations presence in these compounds [57,58]. The absorbance band at ~ 1050 cm⁻¹ was due to the stretching vibration of the C-O-C esters, which are typical of flavonoid compounds [58,59,60].

The intense band at around 1600–1610 cm⁻¹ could be related to the flavylium cationic form of the anthocyanins [61]. Another researcher found that a strong absorption between peaks at 1600 cm⁻¹ and 1700 cm⁻¹ corresponds to the bending vibration of C=H aromatic rings [62]. Moreover, bands within the range 1636–1647 cm⁻¹ [60,62] and 1700 cm⁻¹ are attributed to the C=C and C=O groups for aromatic rings, respectively [58], which indicates the presence of aromatic compounds.

The topic of functional foods is becoming increasingly popular among consumers due to the search for products that improve health. As a result, the market offers an increasing range of functional food products [6]. The use of biopolymers in food products seems justified due to their low cost, non-toxicity, and biodegradability. A significant advantage and additional benefit of food products packaged in active packaging is an increase in the health quality of food due to an increase in antioxidant substances [63]. It is also important to spread knowledge about functional food and its safety, as well as to gain understanding and lack of concern among consumers [8,64].

5. Conclusions

In the present study, it is not possible to formulate general and simple conclusions relating to all analysed raspberry fruits and components and antioxidant activity, due to the very large number of individual results, often with opposite trends.

There was a statistically significant increase in total polyphenols in the last month of storage in a foil package with the addition of cinnamon extract. When freezing wild raspberry fruit, an increase in anthocyanin content was observed in all cases in the 12th month. This was almost twice as high as in the control sample. The greatest increase in anthocyanin content occurred when using foil packaging with turmeric extract. Overall, after 12 months of storage, raspberries from all crops had statistically significantly higher antioxidant activity, compared to raspberry fruit after the first month of freezer storage.

Studies on the biocomposites films have shown that the structure of chitosan and alginate polymers was partly decomposed after 12 months, and the natural extracts and raspberry crops used had different effects on the mechanical properties of the biocomposite films. Current studies reveal that the components used to produce biocomposite films are considered safe for human consumption. In addition, the cost of producing polysaccharide films is not high, and potential benefits have been shown.

Thus, further research is required to determine the suitability of using polysaccharide films with the added natural extracts to enhance the nutritional value of frozen stored fruit.