**The Qualitative and Quantitative Compositions of Phenolic Compounds in Fruits of Lithuanian Heirloom Apple Cultivars**

**Aurita Butkeviˇciut¯ e˙ 1,\*, Mindaugas Liaudanskas 1,2, Darius Kviklys 2,3, Dalia Gelvonauskiene˙ 2 and Valdimaras Janulis <sup>1</sup>**


Academic Editors: Jan Oszmianski and Sabina Lachowicz

Received: 22 October 2020; Accepted: 10 November 2020; Published: 11 November 2020

**Abstract:** As the interest in heirloom cultivars of apple trees, their fruit, and processed products is growing worldwide, studies of the qualitative and quantitative composition of biological compounds are important for the evaluation of the quality and nutritional properties of the apples. Studies on the variations in the chemical composition of phenolic compounds characterized by a versatile biological effect are important when researching the genetic heritage of the heirloom cultivars in order to increase the cultivation of such cultivars in orchards. A variation in the qualitative and quantitative composition of phenolic compounds was found in apple samples of cultivars included in the Lithuanian collection of genetic resources. By the high-performance liquid chromatography (HPLC) method flavan-3-ols (procyanidin B1, procyanidin B2, procyanidin C2, (+)-catechin and (−)-epicatechin), flavonols (rutin, hyperoside, quercitrin, isoquercitrin, reynoutrin and avicularin), chlorogenic acids and phloridzin were identified and quantified in fruit samples of heirloom apple cultivars grown in Lithuania. The highest sum of the identified phenolic compounds (3.82 ± 0.53 mg/g) was found in apple fruit samples of the 'Koštele' cultivar ˙

**Keywords:** apple; phenolic compounds; genetic resources; HPLC-DAD

#### **1. Introduction**

Apple trees are among the old cultivated fruit trees in the world [1]. Domestic apple trees (*Malus domestica* Borkh.) were starting to be grown 4000–10,000 years ago in the orchards of Central Asia [2]. Until the end of the 19th century, domestic apple trees were grown in the orchards of manors and monasteries [3,4]. Later on, local farmers started growing apple trees of the traditional cultivars in their orchards. During this period, a number of traditional apple cultivars of genetic resource heritage were bred in Lithuania, including 'Lietuvos pepinas', 'Montvilinis', 'Popierinis', 'Rudeninis dryžuotasis', 'Žemaiˇciu˛ grietininis', etc. [3]. It is expedient to preserve these apple cultivars, as their value has not been assessed yet.

The results of studies on the qualitative and quantitative composition of the fruit samples of apple cultivars grown in industrial orchards have been presented in scientific literature, but research data on the chemical composition of the fruit of heirloom apple cultivars grown in genetic heritage collections are fragmented [5]. The qualitative and quantitative composition of phenolic compounds in apples grown in Lithuanian industrial orchards has been investigated [5]. Secondary metabolites are phenolic compounds such as flavanols, flavonols, hydroxycinnamic acids, dihydrochalcones and anthocyanins were identified in fruit of the heirloom apple cultivars grown in Portuguese and Polish orchards [6,7]. Fruit samples of heirloom apple cultivars grown in Italian orchards and collections were found to have higher levels of phenolic compounds compared to those found in samples of apples of cultivars grown in industrial orchards [8,9].

Phenolic compounds have a wide range of biological effects: they act as strong antioxidants, scavenging free radicals [9,10], inhibit inflammatory processes [11,12] and the multiplication of bacteria [13] and they also have anti-cancer [14,15] and anti-aging effects [16–18]. The recommended daily diet should include apples, their processed products, and dietary supplements for the prevention of diabetes, asthma, cancer, and neurodegenerative and chronic cardiovascular diseases [11,15,19].

Industrial horticulture has been developing highly intensively during recent decades [1]. New apple cultivars are bred in industrial orchards, taking into account the needs of consumers in different regions of the world [1,20]. The introduction of new apple cultivars by fruit tree breeders reduced the demand for heirloom cultivars of apples grown in orchards [1,21]. Numerous cultivars of apple-specific trait donors have been used to create new apple cultivars grown in industrial orchards. Therefore, the characteristics of fruit trees of these cultivars (adaptability to biotic and abiotic factors, fruit taste, texture of the flesh, etc.) based on genetic factors are "harmonized". Consumers are missing the traditional cultivars due to the variety of their fruit taste, aroma, consistency and suitability for unique national heritage products. Many countries, including Lithuania, have signed the Convention on Biological Diversity [22], the TREATY agreement [23] and the Nagoya Protocol [24] to assess the genetic uniqueness, distinctiveness and risk of extinction of heirloom varieties of garden plants. The signatories of the mentioned documents are committed to collecting, preserving and researching the diversity and economic, biological and medical value of the genetic resources of heirloom varieties of agricultural plants.

Currently, consumers are looking for high-quality products with a known composition and health benefits [25]. Research into the qualitative and quantitative composition of biologically active compounds is important in assessing the quality and nutritional properties of apples. In Lithuania, apple cultivars belonging to the collection of genetic resources are grown in private orchards. In order to study the genetic heritage of heirloom cultivars with the aim of promoting their cultivation in orchards, it is important to carry out detailed studies on the variability of the chemical composition of phenolic compounds with biological effects. Data on qualitative and quantitative chemical composition will provide new scientific knowledge about variability of the qualitative and quantitative composition of biologically active compounds in the fruit of apple trees of heirloom cultivars.

The aim of the study was to investigate variability of the qualitative and quantitative composition of phenolic compounds in the samples of heirloom apple cultivars, to substantiate the cultivation of heirloom cultivars in Lithuanian orchards, and to preserve the genetic heritage of heirloom cultivars.

#### **2. Results and Discussion**

#### *2.1. Qualitative and Quantitative Analysis of Phenolic Compounds of Apple of Heirloom Cultivars*

Scientific literature provides data on the variability of the chemical composition of phenolic compounds in fruit samples of apple trees grown in industrial orchards [26]. The total amount of phenolic compounds found in fruit samples of apple cultivars 'Aldas', 'Auksis', 'Ligol', and 'Šampion' grown in Lithuanian industrial orchards ranged from 1.64 mg/g to 5.75 mg/g [27,28]. In Lithuania as well as in many other countries, collections and private orchards contain heirloom apple cultivars that form the heritage of genetic resources. Fruit of the heirloom apple cultivars are characterized by a high nutritional value and are of different shapes, colors, sizes and organoleptic properties

(crispness, juiciness, and sweetness) [29]. Studies of the chemical composition of the fruit of heirloom apple cultivars are patchy.

The sum of the identified phenolic compounds in the samples of heirloom apple cultivars included in the collection of the Lithuanian heritage of genetic resources varied from 0.15 ± 0.01 mg/g to 3.82 ± 0.53 mg/g (Figure 1). The highest sum of the identified phenolic compounds (3.82 ± 0.53 mg/g) was detected in samples of the 'Koštele' apple cultivar, which di ˙ ffered statistically significantly from the amount of these compounds detected in apple fruit samples of the other studied cultivars (*p* < 0.05) (Figure 1). The lowest content of the sum of the identified phenolic compounds (0.15 ± 0.01 mg/g) was found in fruit samples of the 'Birutes pepinas' cultivar, and it did not di ˙ ffer statistically significantly from that detected in fruit samples of 'Beržininku˛ ananasinis', 'Danu˛ karaliene Luiza', 'Montvilinis', ˙ 'Panemunes baltasis', 'Pilkasis alyvinis', 'Raudonasis alyvinis' or 'Žemaiˇci ˙ u˛ grietininis' apple cultivars (Figure 1).

**Figure 1 Figure 1.** Variability of the sum of the identified phenolic compounds in fruit samples of heirloom apple cultivars.

Studies of the fruit samples of heirloom apple cultivars grown in the orchards of the Marche region of Italy showed that the total amount of phenolic compounds ranged from 0.82 mg/g to 3.60 mg/g [30]. The total amount of phenolic compounds in fruit samples of heirloom apple cultivars grown in Brazilian orchards ranged from 0.46 mg/g to 1.58 mg/g [31]. Meanwhile, the total amount of phenolic compounds in fruit samples of heirloom apple cultivars grown in orchards of the Piedmont region of Italy ranged from 0.45 mg/g to 5.00 mg/g [8]. The sum of the identified phenolic compounds in fruit samples of heirloom apple cultivars grown in Lithuanian orchards was higher than that detected in fruit samples of the heirloom apple cultivars grown in the orchards of the Italian Marche region or Brazil, but lower

than that found in fruit samples of the heirloom apple cultivars grown in the orchards of the Italian region of Piedmont.

## 2.1.1. Variation of the Amount of Flavan-3-ols

Flavan-3-ols (procyanidin B1, procyanidin B2, procyanidin C2, (+)-catechin, and (−)-epicatechin) identified in fruit samples of heirloom apple cultivars grown in Lithuania accounted for 30% of the total amount of the identified and quantified phenolic compounds. The flavan-3-ol content in fruit samples of heirloom apple cultivars ranged from 0.03 ± 0.001 mg/g to 1.40 ± 0.05 mg/g (Figure 2). The flavan-3-ol content in fruit samples of heirloom apple cultivars grown in Croatian orchards was found to vary from 0.02 mg/g to 0.69 mg/g [26]. The flavan-3-ol content in fruit samples of heirloom apple cultivars grown in Italian orchards ranged from 0.02 mg/g to 0.66 mg/g [32]. The flavan-3-ol content in fruit samples of heirloom red-fleshed apple cultivars grown in Spanish orchards was found to range from 0.016 mg/g to 0.022 mg/g, while the flavan-3-ol content in samples of heirloom white-fleshed apples ranged from 0.09 mg/g to 0.20 mg/g [33]. The content of flavan-3-ols in fruit samples of heirloom apple cultivars of the Lithuanian genetic heritage collection was found to be higher than that in fruit samples of heirloom apple cultivars grown in Croatian, Italian, or Spanish orchards.

**Figure 2 Figure 2.** Variability of the amount of flavan-3-ols in fruit samples of heirloom apple cultivars.

The predominant compound of the flavan-3-ol group in fruit samples of heirloom apple cultivars grown in Lithuania was (−)-epicatechin. The highest amount of (−)-epicatechin (0.53 ± 0.01 mg/g) was found in fruit samples of the 'Koštele' cultivar, and it was statistically significantly ( ˙ *p* < 0.05) different from the amount of (−)-epicatechin detected in fruit samples of other apple cultivars (Figure 2). The content of (−)-epicatechin in fruit samples of heirloom apple cultivars grown in Polish orchards

was found to range from 0.05 mg/g to 2.79 mg/g [34]. The amount of (−)-epicatechin in fruit samples of heirloom apple cultivars grown in Italian orchards ranged from 0.09 mg/g to 0.53 mg/g [8]. The data obtained by Polish and Italian researchers corroborate the results of our research.

The highest amounts of (+)-catechin (0.14 ± 0.01 mg/g) were found in fruit samples of the 'Koštele'˙ apple cultivar (Figure 2). The analysis of the fruit samples of heirloom apple cultivars showed a statistically significant (*p* < 0.05) difference in (+)-catechin content. The amount of (+)-catechin in fruit samples of heirloom apple cultivars grown in Italian orchards ranged from 0.02 mg/g to 0.05 mg/g [9]. Meanwhile, the amount of (+)-catechin in fruit samples of heirloom apple cultivars grown in Polish orchards ranged from 0.01 mg/g to 0.72 mg/g [35]. Fruit samples of heirloom apple cultivars grown in Lithuanian orchards were found to contain higher amounts of (+)-catechin, compared to that in fruit samples of heirloom apple cultivars grown in Italian orchards, but lower than that found in fruit samples of heirloom apple cultivars grown in Polish orchards. Procyanidins are among the most common flavan-3-ols found in samples of heirloom apple cultivars [8]. The highest amounts of procyanidin B2 (0.72 ± 0.18 mg/g), procyanidin C2 (0.14 ± 0.03 mg/g) and procyanidin B1 (0.06 ± 0.01 mg/g) were found in apple fruit samples of the 'Virginijos rožinis' cultivar (*p* < 0.05) (Figure 2). Procyanidin B2 predominated among the procyanidins identified and quantified in apple samples of heirloom cultivars grown in Lithuania. The amount of procyanidin B2 in fruit samples of heirloom apple cultivars grown in Polish orchards ranged from 0.07 mg/g to 2.00 mg/g [35]. Meanwhile, the amount of procyanidin B2 in fruit samples of heirloom apple cultivars grown in Italian orchards ranged from 0.018 mg/g to 2.09 mg/g [8]. The amount of procyanidin C1 in fruit samples of heirloom apple cultivars grown in Polish orchards ranged from 0.0006 mg/g to 0.97 mg/g [35]. The amount of procyanidin B1 in fruit samples of heirloom apple cultivars grown in Italian orchards ranged from 0.005 mg/g to 0.34 mg/g [8] and from 0.006 mg/g to 0.014 mg/g [32]. The results of studies on the variability of procyanidin content in fruit samples of heirloom apple cultivars from the collection of the Lithuanian heritage of genetic resources are confirmed by the data of research conducted by Polish and Italian scientists.

According to their amount in fruit samples of heirloom apple cultivars grown in Lithuanian orchards, the compounds of the flavan-3-ol group can be arranged in the following order: (−)-epicatechin>procyanidin B2>procyanidin C2>(+)-catechin>procyanidin B1. Studies of the qualitative and quantitative composition of compounds of the flavan-3-ol group are valuable due to the antioxidant properties of this group of compounds [10] and their glucose regulating effects [34].

#### 2.1.2. Variation of the Amount of Flavonols

The following flavonols were identified and quantified in fruit samples of heirloom apple cultivars from the collection of the Lithuanian heritage of genetic resources: rutin, hyperoside, quercitrin, isoquercitrin, reynoutrin, and avicularin. They comprised 13% of all the identified and quantified phenolic compounds. The content of flavonols in fruit samples of heirloom apple cultivars ranged from 0.04 ± 0.001 mg/g to 0.47 ± 0.12 mg/g (Figure 3). Studies of fruit samples of heirloom apple cultivars grown in Croatian orchards showed that flavonol levels ranged from 0.20 mg/g to 1.22 mg/g [26]. Meanwhile, flavonol levels in fruit samples of heirloom apple cultivars grown in Austrian orchards ranged from 0.67 mg/g to 5.66 mg/g [19]. The results of our study are corroborated by research data obtained by Croatian and Polish researchers.

**Figure 3 Figure 3.** Variability of the amount of flavonols in fruit samples of heirloom apple cultivars.

3 Hyperoside was the predominant compound of the flavonols group in fruit samples of heirloom apple cultivars grown in Lithuania. The highest amount of hyperoside (0.19 ± 0.01 mg/g) was found in apple fruit samples of the 'Koštele' cultivar, and it was statistically significantly di ˙ fferent from hyperoside content in apple samples of other studied cultivars (*p* < 0.05) (Figure 3). The amount of hyperoside in fruit samples of heirloom apple cultivars grown in Italian orchards ranged from 0.0003 mg/g to 0.002 mg/g [8]. The amount of hyperoside found in fruit samples of heirloom apple cultivars grown in Lithuania was higher than that detected in fruit samples of heirloom apple cultivars grown in Italian orchards. The highest amount of avicularin (0.11 ± 0.01 mg/g) was found in apple fruit samples of the 'Lietuvos pepinas' cultivar (Figure 3). Avicularin content differed statistically significantly between fruit samples of heirloom apple cultivars (*p* < 0.05). The highest amount of quercitrin (0.06 ± 0.002 mg/g) was found in apple fruit samples of the 'Koštele' cultivar, and it was ˙ statistically significantly different from that found in apple samples of other cultivars (*p* < 0.05) (Figure 3). The amount of quercitrin found in fruit samples of heirloom apple cultivars grown in Italian orchards ranged from 0.005 mg/g to 0.043 mg/g [8]. The amount of quercitrin in fruit samples of heirloom apple cultivars grown in Lithuanian orchards was higher, compared to the amount found in fruit samples of heirloom apple cultivars grown in Italian orchards. The highest amounts of isoquercitrin (0.06 ± 0.002 mg/g) and reynoutrin (0.04 ± 0.002 mg/g) were found in apple fruit samples of the 'Lietuvos pepinas' cultivar (Figure 3). The amount of isoquercitrin differed statistically significantly between fruit samples of differed heirloom apple cultivars (*p* < 0.05). There was no statistically significant difference in the amount of reynoutrin between apple fruit samples of the 'Sierinka' and 'Koštele' cultivars ( ˙ *p* > 0.05). Among the fruit samples of heirloom apple cultivars grown in Lithuania, the highest amount of rutin (0.04 ± 0.002 mg/g) was found in fruit samples of the 'Paprastasis antaninis'

cultivar, and it did not differ statistically significantly from that found in apple samples of 'Pilkasis alyvinis', 'Beržininku˛ ananasinis', 'Golden russet', 'Jono pepinas', 'Koštele', 'Sierinka', 'Tabokin ˙ e', ˙ 'Baltasis alyvinis', or 'Lietuvos pepinas' cultivars (*p* > 0.05) (Figure 3). The amount of rutin found in fruit samples of heirloom apple cultivars belonging to the collection of the Lithuanian heritage of genetic resources was higher compared to the amount of rutin (0.004 mg/g) found in fruit samples of heirloom apple cultivars grown in Italian orchards [9].

According to their amount in fruit samples of heirloom apple cultivars belonging to the collection of the Lithuanian heritage of genetic resources, the compounds of the flavonols group can be arranged in the following order: hyperoside>avicularin>quercitrin>isoquercitrin>reynoutrin>rutin. Studies of the qualitative and quantitative composition of the compounds of the flavonols group are important due to their antioxidant [34], anti-inflammatory [11], and antiallergic properties [36].

## 2.1.3. Variation of the Amount of Chlorogenic Acid

Phenolcarboxylic acids are an important group of secondary metabolites in apples. It is important to determine the variability of their quantitative composition in fruit samples of heirloom apple cultivars. Chlorogenic acid was the predominant compound, making up 50% of the total amount of the identified and quantified phenolic compounds. The amount of chlorogenic acid in fruit samples of heirloom apple cultivars ranged from 0.01 ± 0.001 mg/g to 2.35 ± 0.03 mg/g (Figure 4).

**Figure 4 Figure 4.** Variability of the amount of chlorogenic acid in fruit samples of heirloom apple cultivars.

The highest amount of chlorogenic acid (2.35 ± 0.03 mg/g) was detected in apple fruit samples of the 'Lietuvos pepinas' cultivar (Figure 4). The content of chlorogenic acid in fruit samples of heirloom apple cultivars differed statistically significantly (*p* < 0.05). The lowest amount of chlorogenic

acid (0.01 ± 0.001 mg/g) was found in apple fruit samples of the 'Montvilinis' cultivar (Figure 4). The amount of chlorogenic acid in fruit samples of heirloom apple cultivars grown in orchards in the Tuscan region of Italy was found to vary from 0.12 mg/g to 0.63 mg/g [9]. Meanwhile, the amount of chlorogenic acid in fruit samples of heirloom apple cultivars grown in orchards in the Piedmont region of Italy ranged from 0.13 mg/g to 2.08 mg/g [8]. Fruit samples of heirloom apple cultivars from the collection of the Lithuanian heritage of genetic resources contained higher amounts of chlorogenic acid compared to those found in fruit samples of heirloom apple cultivars grown in Italian orchards.

## 2.1.4. Variation of the Amount of Phloridzin

Compounds of the dihydrochalcone group are naturally prevalent in the vegetative and generative organs of plants of the apple (*Malus L*.) genus, while in other plant species, they are almost undetectable [33]. Fruit samples of heirloom apple cultivars were found to contain the dihydrochalcone group compound phloridzin, which accounted for 7% of the total amount of the identified and quantified phenolic compounds. The amount of phloridzin in apple samples ranged from 0.02 ± 0.002 mg/g to 0.30 ± 0.005 mg/g (Figure 5).

**Figure 5 Figure 5.** Variability of the amount of phloridzin in fruit samples of heirloom apple cultivars.

The highest content of phloridzin (0.30 ± 0.005 mg/g) was found in apple fruit samples of the 'Golden russet' cultivar (Figure 5), which did not differ statistically significantly only from the amounts found in apple fruit samples of the 'Jono pepinas' cultivar. The lowest amount of phloridzin (0.02 ± 0.002 mg/g) was found in apple fruit samples of the 'Beržininku˛ ananasinis' cultivar (Figure 5). The amount of phloridzin in fruit samples of heirloom apple cultivars grown in orchards in the Piedmont region of Italy was found to range from 0.001 mg/g to 0.26 mg/g [8]. The amount of

phloridzin in fruit samples of heirloom apple cultivars grown in orchards in the Garfagnana region of Italy ranged from 0.01 mg/g to 0.05 mg/g [32]. Fruit samples of heirloom apple cultivars from the collection of the Lithuanian heritage of genetic resources contained higher amounts of phloridzin compared to those found in fruit samples of heirloom apple cultivars grown in Italian orchards.

#### *2.2. Hierarchical Cluster Analysis of Phenolic Compounds of Apple of Heirloom Cultivars*

Hierarchical cluster analysis of heirloom apple cultivars was performed, the results of which are presented in Figure 6. Based on the variability of the quantitative composition in apple samples of heirloom cultivars, phenolic compounds were distributed into clusters.

**Figure 6.** Dendrogram of the similarity of apple samples in terms of the amounts of phenolic compounds. Flavonols were distributed into three clusters (**A**), chlorogenic acid was distributed into four clusters (**B**), (−)-epicatechin, (+)-catechin, and phloridzin were distributed into four clusters (**C**), and compounds of the procyanidin group were distributed into four clusters (**D**).

Fruit samples of heirloom apple cultivars assigned to cluster I (3, 4, 6, 7, 8, 14, 15, 17, 18, 21 and 22) were found to contain lower than average amounts of flavonols. Fruit samples of heirloom apple cultivars assigned to cluster II (1, 2, 5, 9, 12, 16 and 20) were found to contain average amounts of flavonols. Meanwhile, fruit samples of heirloom apple cultivars assigned to cluster III (10, 11, 13 and 19) were found to contain higher than average amounts of flavonols (Figure 6A). Fruit samples of heirloom apple cultivars assigned to cluster I (1, 2, 3, 4, 6, 7, 12, 14, 15, 16, 17, 20, 21 and 22) were found to contain lower than average amounts of chlorogenic acid. Fruit samples of the heirloom apple cultivar assigned to cluster II (18) had average amounts of chlorogenic acid. Meanwhile, fruit samples of heirloom apple cultivars assigned to cluster III (5, 8, 9 and 13) had higher than average amounts of chlorogenic acid. The highest amounts of chlorogenic acid were found in fruit samples of heirloom apple cultivars assigned to cluster IV (10, 11 and 19) (Figure 6B).

Fruit samples of heirloom apple cultivars assigned to cluster I (1, 2, 3, 4, 6, 11, 12, 14, 15, 16, 17, 18, 20 and 22) were found to contain lower than average amounts of (-)-epicatechin, (+)-catechin and phloridzin. Fruit samples of heirloom apple cultivars assigned to cluster II (8 and 9) were found to contain average amounts of (−)-epicatechin, (+)-catechin and phloridzin. Higher than average levels of (−−)-epicatechin, (+)-catechin and phloridzin were found in fruit samples of heirloom apple cultivars assigned to cluster III (5, 7, 13, 19 and 21). The highest levels of (−)-epicatechin, (+)-catechin and phloridzin were found in fruit samples of the heirloom apple cultivar assigned to cluster IV (10) (Figure 6C). Fruit samples of heirloom apple cultivars assigned to cluster I (1, 2, 5, 7, 14, 16, 18, 20 and 22) were found to contain average amounts of procyanidin B1, procyanidin B2 and procyanidin C2. Fruit samples of heirloom apple cultivars assigned to cluster II (3, 4, 6, 11, 12, 15 and 17) contained lower than average amounts of procyanidins. Higher than average amounts of procyanidins were found in fruit samples of heirloom apple cultivars assigned to cluster III (8, 9, 10, 13 and 19). The highest amounts of procyanidin B1, procyanidin B2, and procyanidin C2 were found in fruit samples of heirloom apple cultivars assigned to cluster IV (21) (Figure 6D).

#### *2.3. Principal Component Analysis of Phenolic Compounds of Apple of Heirloom Cultivars*

In this study, we analyzed the main components of phenolic compounds in fruit samples of heirloom apple cultivars. Two main components were used for the analysis, as they explain 80.19% of the total variability in the study data (Figure 7).

**Figure 7 Figure 7.** Analysis of the main components of phenolic compounds in apple samples.

The amounts of isoquercitrin (0.939), hyperoside (0.930), avicularin (0.930), quercitrin (0.922), chlorogenic acid (0.902) and reynoutrin (0.815) strongly positively correlated with the first component, which describes 48.53% of the total data variability, while the correlation of the amounts of rutin (0.707) and phloridzin (0.691) with this component was strongly positive (Figure 7). The amounts of procyanidin C2 (0.889), procyanidin B1 (0.887), (−)-epicatechin (0.882), procyanidin B2 (0.878) and (+)-catechin (0.805) very strongly positively correlated with the second component, which describes 31.66% of the dispersion (Figure 7).

Recently, there has been a growing interest in the genetic resources of heirloom cultivars, they are more widely grown, and there is an increasing number of studies on the qualitative and quantitative variability of the composition of their fruit. Fruit samples of some heirloom apple cultivars were found to have a richer quantitative and qualitative composition of phenolic compounds. Heirloom apple cultivars can be used for the selection of new apple cultivars. Apple trees of heirloom cultivars are becoming more popular, and higher levels of biologically active compounds are detected in their fruit [9]. Fruit samples of heirloom apple cultivars grown in Lithuania were found to contain 2.6 times higher amounts of flavonols, 7 times higher amounts of dihydrochalcones, 1.2 times lower amounts of phenolic acids and 1.5 times lower amounts of flavan-3-ols compared to those detected in fruit samples of heirloom apple cultivars grown in Polish orchards [34]. Quantitative differences can be explained by the competitive interaction between the enzymes anthocyanidin reductase and anthocyanidin synthase during flavonoid synthesis, which results in a slower synthesis of flavan-3-ols and their lower accumulation in apples [2].

The results of our study provided new knowledge about apple cultivars from the collection of the Lithuanian heritage of genetic resources and the variability of the qualitative and quantitative composition of the phenolic compounds found in their fruit. The highest sum of the identified phenolic compounds (3.82 ± 0.53 mg/g) was found in apple fruit samples of the 'Koštele' cultivar. The sum ˙ of the identified phenolic compounds was higher than that (0.86 mg/g) found in fruit samples of heirloom apple cultivars grown in Germany [36]. Fruit samples of heirloom apple cultivars included in the collection of the Lithuanian heritage of genetic resources contained higher amounts of phenolic compounds compared to those detected in fruit samples of the 'Jonagold' cultivar grown in the orchards of Lhasa (Italy), Rokietnica (Poland), and Randwijk (the Netherlands) regions (respectively, 2.21 mg/g, 2.69 mg/g, and 3.81 mg/g). However, this amount was lower than that (4.76 mg/g) found in the samples of apples grown in the orchards of the Wielu ´n region of Poland [37]. In fruit samples of heirloom apple cultivars grown in Lithuania, chlorogenic acid comprised the greatest part of the phenolic compounds. The highest content of chlorogenic acid (2.35 ± 0.03 mg/g) was found in apple fruit samples of the 'Lietuvos pepinas' cultivar. Polish researchers indicated that chlorogenic acid might account for 64–94% of all the identified and quantified phenolic acids in apple fruit samples [34]. Chlorogenic acid in fruit and vegetables determines their sensory properties and has anti-mutagenic and antioxidant effects [34]. The compound phloridzin belonging to the dihydrochalcone group was found in fruit samples of heirloom apple cultivars. Its highest amounts (0.30 ± 0.005 mg/g) were found in apple fruit samples of the 'Golden russet' cultivar. Phloridzin is a biologically active compound with a wide range of biological effects. It regulates blood glucose levels [38,39], antioxidant and anti-aging effects [11,40]. Qualitative and quantitative analysis of dihydrochalcone group compounds is important, as they can be used as chemotaxonomic markers in the taxonomy of apple species as well as for the identification and quality assessment of apple products [6].

#### **3. Materials and Methods**

#### *3.1. Plant Materials*

The study included 22 heirloom apple cultivars, of which 21 (except for 'Golden russet') are included in the List of the National Plant Genetic Resources (Table 1).

The apple trees were grown in the Collection of the Apple Tree Genetic Resources at the Institute of Horticulture (in Babtai town), a division of the Lithuanian Research Centre for Agriculture and Forestry (henceforth, LAMMC). Coordinates: 55◦600 N, 23◦480 E. The study was conducted during 2019–2020.



**Table 1.** Origin and properties of the heirloom apple cultivars of Lithuania [4,41].

### *3.2. Chemicals and Solvents*

All solvents, reagents, and standards used were of analytical grade. Acetonitrile and acetic acid were obtained from Sigma-Aldrich GmbH (Buchs, Switzerland), ethanol was obtained from AB Stumbras (Kaunas, Lithuania), hyperoside, rutin, quercitrin, phloridzin, procyanidin B1, procyanidin B2 and chlorogenic acid standards were purchased from Extrasynthese (Genay, France), reynoutrin, (+)-catechin and (−)-epicatechin–from Sigma-Aldrich GmbH (Steinheim, Germany), and avicularin, procyanidin C1 and isoquercitrin–from Chromadex (Santa Ana, CA, USA). Purified deionized water used in the tests was prepared with the Milli-Q® (Millipore, Bedford, MA, USA) water purification system.

#### *3.3. Preparation of Samples*

For the analysis, twenty apples at the optimal maturity stage were picked from different parts of the tree crown. Whole apples were immediately frozen in a freezer (at −35 ◦C) with air circulation. Subsequently, these frozen samples were lyophilized with a ZIRBUS sublimator 3 × 4 × 5/20 (ZIRBUS technology, Bad Grund, Germany) at a pressure of 0.01 mbar (condenser temperature: −85 ◦C). The lyophilized samples were ground to fine powder using a Retsch 200 mill electric grinder (Haan, Germany). Loss on drying before the analysis was determined by drying the apple lyophilisate in a laboratory drying oven to complete the evaporation of water and volatile compounds (temperature: 105 ◦C; the difference in weight between measurements: up to 0.01 g) and by calculating the difference in raw material weight before and after the drying. The data were recalculated for the absolute dry lyophilisate weight. The prepared apple samples were stored in dark, tightly closed glass vessels.

#### *3.4. Preparation of the Phenolic Compounds*

During the analysis of phenolic compounds, 2.5 g of lyophilizate powder (exact weight) was weighed, added to 30 mL of 70% (*v*/*v*) ethanol, and extracted in a Sonorex Digital 10 P ultrasonic bath (Bandelin Electronic GmbH & Co. KG, Berlin, Germany) at room temperature for 20 min. The obtained extract was filtered through a paper filter, and the residue on the filter was washed with 70% (*v*/*v*) ethanol in a 50-mL flask until the exact volume was reached. The conditions of the extraction were chosen based on the results of the tests for setting the extraction conditions.

#### *3.5. Qualitative and Quantitative Analysis by HPLC–PDA Method*

The qualitative and quantitative HPLC analysis of phenolic compounds was performed with a Waters 2998 PDA detector (Waters, Milford, CT, USA). Chromatographic separations were carried out by using a YMC-Pack ODS-A (5 µm, C18, 250 × 4.6 mm i.d.) column. The column was operated at a constant temperature of 25 ◦C. The volume of the analyzed extract was 10 µL. The flow rate was 1 mL/min. The mobile phase consisted of 2% (*v*/*v*) acetic acid (solvent A) and acetonitrile (solvent B). Gradient variation: 0–30 min 3–15% B, 30–45 min 15–25% B, 45–50 min 25–50% B, and 50–55 min 50–95% B. For the quantitative analysis, the calibration curves were obtained by injecting the known concentrations of different standard compounds. All the identified phenolic compounds were quantified at λ = 200–400 nm wavelength [5].

#### *3.6. Statistical Analysis*

The statistical analysis of the study data was performed by using Microsoft Office Excel 2013 (Microsoft, Redmond, WA, USA) and SPSS 25.0 (SPSS Inc., Chicago, IL, USA) computer software. All the results obtained during the ESC analysis were presented as means of three consecutive test results and standard deviations. To evaluate the variance in the quantitative composition, we calculated the coefficient of variation. Univariate analysis of variance (ANOVA) was applied in order to determine whether the differences between the compared data were statistically significant. The hypothesis about the equality of variances was verified by applying Levine's test. If the variances of independent variables were found to be equal, Tukey's multiple comparison test was used. The differences were regarded as statistically significant at *p* < 0.05. The comparison of the chemical composition between the apple fruit samples of the studied heirloom cultivars was carried out by applying the hierarchical cluster analysis, using the squared Euclidean distance. Principal component analysis was performed as well.

#### **4. Conclusions**

Apple trees of heirloom cultivars are valuable from the genetic aspect in the selection of new fruit tree cultivars. Their fruit are a source of biologically active compounds and can be used in the development and production of new innovative dietary supplements and medicinal cosmetic products. Apple trees of the heirloom cultivars 'Koštele', 'Lietuvos pepinas', 'Paprastasis antaninis', ˙ 'Virginijos rožinis' and 'Sierinka' grown in the Collection of the Apple Tree Genetic Resources at the Institute of Horticulture of the Lithuanian Research Center for Agriculture and Forestry are not suitable for growing in industrial gardens due to their low fruit yield, poor external quality, small size, and susceptibility to disease. In amateur gardens, growing apples of heirloom cultivars is promising due to their higher content of bioactive substances. Our phytochemical studies of heirloom apple cultivars provide valuable scientific knowledge on the variability of the qualitative and quantitative composition of phenolic compounds. The results of our study will enable a wider cultivation of heirloom apple cultivars in gardens and collections and will help consumers to obtain and use apples with a known chemical composition of phenolic compounds, which determine the use of apples in the healthy food chain and the development of innovative food products.

**Author Contributions:** Conceptualization, V.J.; methodology, M.L.; formal analysis, A.B.; investigation, A.B.; resources, D.G., D.K.; data curation, A.B.; writing-original draft preparation, A.B.; writing-review and editing, V.J., M.L., D.K., D.G.; visualization, A.B.; supervision, V.J., M.L.; project administration, V.J., M.L. All authors have read and agreed to the published version of the manuscript.

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

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

### **References**


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## *Article* **Biopharmaceutical Evaluation of Capsules with Lyophilized Apple Powder**

**Aurita Butkeviˇciut¯ e˙ 1,\*, Mindaugas Liaudanskas <sup>1</sup> , Kristina Ramanauskiene˙ <sup>2</sup> and Valdimaras Janulis <sup>1</sup>**

	- LT-50162 Kaunas, Lithuania; Kristina.Ramanauskiene@lsmuni.lt

**Abstract:** Apples are an important source of biologically active compounds. Consequently, we decided to model hard gelatin capsules with lyophilized apple powder by using different excipients and to evaluate the release kinetics of phenolic compounds. The apple slices of "Ligol" cultivar were immediately frozen in a freezer (at −35◦C) with air circulation and were lyophilized with a sublimator at the pressure of 0.01 mbar (condenser temperature, −85◦C). Lyophilized apple powder was used as an active substance filled into hard gelatin capsules. We conducted capsule disintegration and dissolution tests to evaluate the quality of apple lyophilizate-containing capsules of different encapsulating content. Individual phenolic compounds can be arranged in the following descending order according to the amount released from the capsules of different compositions: chlorogenic acid > rutin > avicularin > hyperoside > phloridzin > quercitrin > (−)-epicatechin > isoquercitrin. Chlorogenic acid was the compound that was released in the highest amounts from capsules of different encapsulating content: its released amounts ranged from 68.4 to 640.3 µg/mL. According to the obtained data, when hypromellose content ranged from 29% to 41% of the capsule mass, the capsules disintegrated within less than 30 min, and such amounts of hypromellose did not prolong the release of phenolic compounds. Based on the results of the dissolution test, the capsules can be classified as fast-dissolving preparations, as more than 85% of the active substances were released within 30 min.

**Keywords:** apple; phenolic compounds; dissolution test; HPLC-DAD

#### **1. Introduction**

Apples are among the most consumed fruits in Lithuania and worldwide. According to the data of 2020, 84.63 million tons of apples were grown in the world [1]. The greatest amounts of apples are grown in China (around 40.92 million tons). In the US, around 5.19 million tons are grown per year, and in Poland, the amount of apples grown per year is around 3.20 million tons [1]. Apples are widely used in the food industry in the production of various products and beverages (juices, wines, and ciders), and are also used unprocessed [2,3].

In the healthy food chain, apples are an important source of biologically active compounds [4]. They have been found to contain a complex of phenolic (8.2–360.75 mg/g−<sup>1</sup> ) [5], and triterpenic (0.47–3.75 mg/g−<sup>1</sup> ) [6] compounds, glucose (1.96–75.91 mg/g−<sup>1</sup> ), fructose (15.52–342.14 mg/g−<sup>1</sup> ), sucrose (11.81–43.79 mg/g−<sup>1</sup> ), xylitol (1.01–13.79 mg/g−<sup>1</sup> ), pectins (4.05–19.72 mg/g−<sup>1</sup> ), organic acids (malic acid (38.96–107.28 mg/g−<sup>1</sup> ), citric acid (1.84– 4.12 mg/g−<sup>1</sup> ), maleic acid (0.17–0.23 mg/g−<sup>1</sup> ), pyruvic acid (0.14–0.34 mg/g−<sup>1</sup> ), shikimic acid (0.04–0.17 mg/g−<sup>1</sup> )) [7,8], vitamins (21.08 mg/g−<sup>1</sup> ) [9], macroelements (K (1.07– 1.12 mg/g−<sup>1</sup> ), P (0.074–0.11 mg/g−<sup>1</sup> ), Mg (0.05–0.08 mg/g−<sup>1</sup> ), Ca (0.04–0.06 mg/g−<sup>1</sup> ), Na (0.007–0.04 mg/g−<sup>1</sup> )), microelements (Fe (0.001–0.003 mg/g−<sup>1</sup> ), Zn (0.0004–0.019 mg/g−<sup>1</sup> ),

**Citation:** Butkeviˇciut¯ e, A.; ˙ Liaudanskas, M.; Ramanauskiene, K.; ˙ Janulis, V. Biopharmaceutical Evaluation of Capsules with Lyophilized Apple Powder. *Molecules* **2021**, *26*, 1095. https://doi.org/ 10.3390/molecules26041095

Academic Editors: Jan Oszmianski and Sabina Lachowicz Received: 13 January 2021 Accepted: 17 February 2021 Published: 19 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Mn (0.0003–0.0004 mg/g−<sup>1</sup> ), Cu (0.0003–0.0005 mg/g−<sup>1</sup> ) [10], and fiber (3700– 4500 mg/g−<sup>1</sup> ) [5].

Biologically active substances in apples affect the biological systems of the human body. They have an antioxidant effect, neutralizing harmful reactive oxygen and nitrogen species that cause structural damage to the body's molecules, which, in turn, is directly linked to the development and progression of numerous diseases (cardiovascular and neurodegenerative diseases, cancer, diabetes, etc.) [11,12]. The biologically active compounds accumulated in apples are potentially valuable for the prevention of various diseases [13,14]. During the recent period, in order to preserve the chemical composition of high-quality fruit, lyophilization is increasingly used in the food industry. Lyophilized apples with a chemical composition identical to that of fresh apples can be used in the functionalization of food supplements and various pharmaceutical dosage forms.

One important step in modeling a solid pharmaceutical dosage form is the selection of the category of the pharmaceutical form. Hard capsules are a convenient and widely used dosage form in which drugs and excipients content are placed in the capsules. One of the most widely used capsules may not be resistant to and dissolved in gastric juice, so we decided to use this capsules type in the study. In addition, the capsules may be resistant to gastric juice and dissolved in a selected part of the gastrointestinal tract if appropriate excipients are selected. Therefore, one of the most important advantages of capsules is that their use allows for the localization of the gastrointestinal tract site of the release of biologically active compounds from the capsule, thus helping to protect the acid-sensitive biologically active substances from the destructive effects of gastric juice [15].

Another equally important property of hard capsules is that the dissolution kinetics of the active substances can be modified by using different excipients in the modeling of hard capsules. In our study, the active substance in capsules is a lyophilized apple powder rich in phenolic compounds. Excipients are used in the manufacturing process of encapsulated mixtures in order to increase the bulk of the encapsulated substances, to reduce their adhesion, to improve flowability, and/or to promote disintegration or penetrability to water, thus differentially modifying the release of the active ingredients from the capsule. The use of excipients can slow down the release of the capsule contents, thus ensuring a longer and more sustained effect of the biologically active substances in the capsule [15,16]. It is important to select suitable excipients that would ensure adequate dissolution kinetics of the medicinal substance. Microcrystalline cellulose, starch, and D (+)-glucose were used as fillers in the encapsulating mixture. Hypromellose was selected as a drug release modifier that would prolong the release of the active ingredient. Silicon dioxide was selected as the tackifier of the encapsulated mixture.

About 50% of the currently biopharmaceuticals are lyophilized, representing the most common formulation strategy [17]. Lyophilization or freeze drying is a process in which water is frozen, followed by its removal from the sample, initially by sublimation (primary drying) and then by desorption (secondary drying). Freeze-drying is a process of drying in which water is sublimed from the product after it is frozen [17,18]. Although on the pharmacy there are quite a number of medicines, and in particular food supplements, which use various plant extracts as the active ingredients in hard capsules, the use of lyophilized fruits and botanical raw materials in the production of hard capsules is still a rare phenomenon. Lyophilized fruits (apples, pears, plums, peaches), berries (blueberries, cranberries, strawberries), and vegetables (carrots, beets) are used in the food industry and can be added to various types of cocktails, cereals, drinks, meals, juices, yogurts, ice cream; freeze-dried fruit powder can also be added to food supplements thus increasing the content of biologically active compounds [19–22]. Dehydrated, freeze-dried fruit, berries, and vegetables promote long-term storage of freeze-dried food or food supplements. Lyophilization preserves most of the biologically active compounds stored in the fruit, while other methods often destroy thermolabile compounds. Lyophilization does not involve the use of chemical substances, which is crucial for providing the consumers with

safe, effective, and environmentally friendly products or food supplements with the highest content of biologically active compounds. The aim of our study was to model hard gelatin capsules with lyophilized apple powder by using different excipients and to evaluate the release kinetics of phenolic com-

with safe, effective, and environmentally friendly products or food supplements with the

The aim of our study was to model hard gelatin capsules with lyophilized apple powder by using different excipients and to evaluate the release kinetics of phenolic compounds. pounds.

#### **2. Results and Discussion 2. Results and Discussion**

#### *2.1. Qualitative and Quantitative Analysis of Phenolic Compounds of Apple Lyophilisate 2.1. Qualitative and Quantitative Analysis of Phenolic Compounds of Apple Lyophilisate*

*Molecules* **2021**, *26*, x FOR PEER REVIEW 3 of 15

highest content of biologically active compounds.

During the first stage of the study, the quantitative composition of the lyophilized apple powder was analyzed. Ethanol extracts of the lyophilized apple powder were analyzed by applying high performance liquid chromatography (HPLC). The obtained results allowed for an accurate assessment of the qualitative and quantitative composition of individual phenolic compounds in the studied apple lyophilisate powder. Different groups of phenolic compounds were identified and quantified in the analyzed apple lyophilisate: quercetin glycosides (rutin, hyperoside, isoquercitrin, reynoutrin, avicularin, and quercitrin), flavan-3-ols (procyanidin B1, procyanidin B2, procyanidin C1, (+)-catechin, and (−)-epicatechin), dihydrochalcones (phloridzin), and phenolic acids (chlorogenic acid). The data obtained by Jakobek et al. corroborate to the results of our research [23]. The chromatogram of the tested apple lyophilisate ethanol extract is shown in Figure 1. During the first stage of the study, the quantitative composition of the lyophilized apple powder was analyzed. Ethanol extracts of the lyophilized apple powder were analyzed by applying high performance liquid chromatography (HPLC). The obtained results allowed for an accurate assessment of the qualitative and quantitative composition of individual phenolic compounds in the studied apple lyophilisate powder. Different groups of phenolic compounds were identified and quantified in the analyzed apple lyophilisate: quercetin glycosides (rutin, hyperoside, isoquercitrin, reynoutrin, avicularin, and quercitrin), flavan-3-ols (procyanidin B1, procyanidin B2, procyanidin C1, (+)-catechin, and (−)-epicatechin), dihydrochalcones (phloridzin), and phenolic acids (chlorogenic acid). The data obtained by Jakobek et al. corroborate to the results of our research [23]. The chromatogram of the tested apple lyophilisate ethanol extract is shown in Figure 1.

**Figure 1.** Chromatogram of the ethanol extract of the apple lyophilisate. Analytes determined at λ = 280 nm wavelength: 1—procyanidin B1; 2—(+)-catechin; 3—chlorogenic acid; 4—procyanidin B2; 5—(−)-epicatechin; 6—procyanidin C1; at λ = 360 nm wavelength: 7—rutin; 8—hyperoside; 9—isoquercitrin; 10—reynoutrin; 11—avicularin; 12—quercitrin; at λ = 280 nm wavelength: 13—phloridzin. **Figure 1.** Chromatogram of the ethanol extract of the apple lyophilisate. Analytes determined at λ = 280 nm wavelength: 1—procyanidin B1; 2—(+)-catechin; 3—chlorogenic acid; 4—procyanidin B2; 5—(−)-epicatechin; 6—procyanidin C1; at λ = 360 nm wavelength: 7—rutin; 8—hyperoside; 9—isoquercitrin; 10—reynoutrin; 11—avicularin; 12—quercitrin; at λ = 280 nm wavelength: 13—phloridzin.

The sum of the identified and quantified individual phenolic compounds found in the apple lyophilisate of the "Ligol" cultivar was 1.94 ± 0.05 mg/g. Studies of the fruit samples of apple cultivars grown in the orchards of the Marche region of Italy showed that the total amount of phenolic compounds ranged from 0.82 to 3.60 mg/g and confirm the results of our research [5]. Chlorogenic acid predominated among all the identified phenolic compounds. Its content was 0.67 ± 0.10 mg/g, which accounted for 34.5% of the total content of all the detected phenolic compounds (Figure 2). The sum of the identified and quantified individual phenolic compounds found inthe apple lyophilisate of the "Ligol" cultivar was 1.94 <sup>±</sup> 0.05 mg/g. Studies of the fruit samples of apple cultivars grown in the orchards of the Marche region of Italy showedthat the total amount of phenolic compounds ranged from 0.82 to 3.60 mg/g and confirmthe results of our research [5]. Chlorogenic acid predominated among all the identified phenolic compounds. Its content was 0.67 ± 0.10 mg/g, which accounted for 34.5% of the total content of all the detected phenolic compounds (Figure 2).

**Figure 2.** The amount of individual flavan-3-ols, phloridzin, and chlorogenic acid in ethanol extracts obtained from the apple fruit of the "Ligol" cultivar grown in Lithuania. **Figure 2.** The amount of individual flavan-3-ols, phloridzin, and chlorogenic acid in ethanol extracts obtained from the apple fruit of the "Ligol" cultivar grown in Lithuania.

The results of this study confirmed those of the previous studies reporting that chlorogenic acid is one of the most predominant components in apples [24,25]. The amount of chlorogenic acid in apple samples grown in Italy was found to vary from 0.12 to 0.63 mg/g [26]. In our study, a higher amount of chlorogenic acid compared with the amount described by Italian scientists was determined. Chlorogenic acid has properties important for human health, such as antioxidant activity [27,28], anti-inflammatory activity [29,30], the reduction in the risk of type 2 diabetes [31], the improvement of cardiovascular function [32,33], and the inhibition of the processes of carcinogenesis [34–36]. The results of this study confirmed those of the previous studies reporting that chlorogenic acid is one of the most predominant components in apples [24,25]. The amount of chlorogenic acid in apple samples grown in Italy was found to vary from 0.12 to 0.63 mg/g [26]. In our study, a higher amount of chlorogenic acid compared with the amount described by Italian scientists was determined. Chlorogenic acid has properties important for human health, such as antioxidant activity [27,28], anti-inflammatory activity [29,30], the reduction in the risk of type 2 diabetes [31], the improvement of cardiovascular function [32,33], and the inhibition of the processes of carcinogenesis [34–36].

Another group of flavan-3-ol compounds with diverse biological activity identified in the apple lyophilisate consisted of monomeric compounds ((+)-catechin and (−)-epicatechin) and oligomeric compounds (procyanidin B1, procyanidin B2, and procyanidin C1). The total amount of compounds in flavan-3-ol group was 0.82 ± 0.03 mg/g, which accounted for 42.3% of the total amount of the identified phenolic compounds (Figure 2). The flavan-3-ol content in fruit samples of apple cultivars grown in Croatian orchards was found to vary from 0.02 to 0.69 mg/g [23]. We established the higher amount of total flavan-3-ols compared with the amount determined by Croatian scientists. The variability in the quantitative composition of the individual compounds of the flavan-3-ol group in the apple lyophilisate is shown in Figure 2. The predominant compounds of the flavan-3-ol group in the apple lyophilisate was procyanidins. The highest amount of procyanidin B2 and procyanidin C1 were determined as 0.30 ± 0.08 mg/g and 0.23 ± 0.06 mg/g, respectively (Figure 2). The amount of procyanidin B2 and procyanidin C1 in fruit samples of apple cultivars grown in Polish orchards ranged from 0.07 to 2.00 mg/g and 0.0006 to 0.97 mg/g, accordingly [37]. Our study results confirmed Polish research results. Procyanidins are important for the human body as they exhibit antioxidant, anticancer, anti-inflammatory, platelet aggregation-reducing, and cholesterol-reducing effects [38–40]. Monomeric flavan-3-ols, which together with procyanidins may be responsible for the cholesterol-lowering [41] and vasodilating [42] effect of the apples, also inhibit sulfotransferases, and thus can regulate the biological activity of hydroxysteroids and can act as natural chemopre-Another group of flavan-3-ol compounds with diverse biological activity identified in the apple lyophilisate consisted of monomeric compounds ((+)-catechin and (−) epicatechin) and oligomeric compounds (procyanidin B1, procyanidin B2, and procyanidin C1). The total amount of compounds in flavan-3-ol group was 0.82 ± 0.03 mg/g, which accounted for 42.3% of the total amount of the identified phenolic compounds (Figure 2). The flavan-3-ol content in fruit samples of apple cultivars grown in Croatian orchards was found to vary from 0.02 to 0.69 mg/g [23]. We established the higher amount of total flavan-3-ols compared with the amount determined by Croatian scientists. The variability in the quantitative composition of the individual compounds of the flavan-3-ol group in the apple lyophilisate is shown in Figure 2. The predominant compounds of the flavan-3-ol group in the apple lyophilisate was procyanidins. The highest amount of procyanidin B2 and procyanidin C1 were determined as 0.30 ± 0.08 mg/g and 0.23 ± 0.06 mg/g, respectively (Figure 2). The amount of procyanidin B2 and procyanidin C1 in fruit samples of apple cultivars grown in Polish orchards ranged from 0.07 to 2.00 mg/g and 0.0006 to 0.97 mg/g, accordingly [37]. Our study results confirmed Polish research results. Procyanidins are important for the human body as they exhibit antioxidant, anticancer, anti-inflammatory, platelet aggregation-reducing, and cholesterol-reducing effects [38–40]. Monomeric flavan-3-ols, which together with procyanidins may be responsible for the cholesterol-lowering [41] and vasodilating [42] effect of the apples, also inhibit sulfotransferases, and thus can regulate the biological activity of hydroxysteroids and can act as natural chemopreventive agents [43].

ventive agents [43]. Phloridzin, a compound of the dihydrochalcone group, was detected in the apple lyophilisate as well. Its quantitative content in the sample was 0.08 ± 0.005 mg/g, which Phloridzin, a compound of the dihydrochalcone group, was detected in the apple lyophilisate as well. Its quantitative content in the sample was 0.08 ± 0.005 mg/g, which accounted for 4.1% of the total amount of phenolic compounds detected in the apple

lyophilisate (Figure 2). The amount of phloridzin in fruit samples of apple cultivars grown in orchards in the Garfagnana region of Italy ranged from 0.01 to 0.05 mg/g [44]. The results obtained in these studies confirm the results obtained in our research. Phloridzin exhibits important antidiabetic activity [45,46], and therefore apples, as botanical raw materials accumulating this compound, can be potentially useful for the prevention of diabetes mellitus [14]. ophilisate (Figure 2). The amount of phloridzin in fruit samples of apple cultivars grown in orchards in the Garfagnana region of Italy ranged from 0.01 to 0.05 mg/g [44]. The results obtained in these studies confirm the results obtained in our research. Phloridzin exhibits important antidiabetic activity [45,46], and therefore apples, as botanical raw materials accumulating this compound, can be potentially useful for the prevention of diabetes mellitus [14].

accounted for 4.1% of the total amount of phenolic compounds detected in the apple ly-

The qualitative and quantitative composition of quercetin glycosides is shown in Figure 3. The total amount of quercetin glycosides was 0.37 ± 0.12 mg/g, which accounted for 19.1% of the total amount of phenolic compounds detected in the apple lyophilisate. The qualitative and quantitative composition of quercetin glycosides is shown in Figure 3. The total amount of quercetin glycosides was 0.37 ± 0.12 mg/g, which accounted for 19.1% of the total amount of phenolic compounds detected in the apple lyophilisate.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 5 of 15

**Figure 3.** Concentration of individual quercetin glycosides in ethanol extracts obtained from the fruit of the "Ligol" apple cultivar grown in Lithuania. **Figure 3.** Concentration of individual quercetin glycosides in ethanol extracts obtained from the fruit of the "Ligol" apple cultivar grown in Lithuania.

Studies of fruit samples of apple cultivars grown in Croatian orchards showed that quercetin glycosides levels ranged from 0.20 to 1.22 mg/g [23]. The results of our study are corroborated by research data obtained by Croatian researchers. Hyperoside predominated among all the identified quercetin glycosides. Its content was 0.12 ± 0.06 mg/g, which accounted for 6.2% of the total content of all the detected phenolic compounds (Figure 3). The amount of hyperoside in fruit samples of apple cultivars grown in Italian orchards ranged from 0.0003 to 0.002 mg/g [47]. The apple lyophilisate contained higher amounts of hyperoside compared to these found in fruit samples of apple cultivars grown in Italian orchards. The amount of quercitrin was 0.09 ± 0.04 mg/g and these results confirmed Italian study results that report that the amount of quercitrin found in apple samples ranged from 0.005 to 0.043 mg/g [47]. Studies of fruit samples of apple cultivars grown in Croatian orchards showed that quercetin glycosides levels ranged from 0.20 to 1.22 mg/g [23]. The results of our study are corroborated by research data obtained by Croatian researchers. Hyperoside predominated among all the identified quercetin glycosides. Its content was 0.12 ± 0.06 mg/g, which accounted for 6.2% of the total content of all the detected phenolic compounds (Figure 3). The amount of hyperoside in fruit samples of apple cultivars grown in Italian orchards ranged from 0.0003 to 0.002 mg/g [47]. The apple lyophilisate contained higher amounts of hyperoside compared to these found in fruit samples of apple cultivars grown in Italian orchards. The amount of quercitrin was 0.09 ± 0.04 mg/g and these results confirmed Italian study results that report that the amount of quercitrin found in apple samples ranged from 0.005 to 0.043 mg/g [47].

All the quercetin glycosides identified and quantified in the ethanol extract of the apple lyophilisate can be ranked in the following ascending order by their content: rutin < isoquercitrin < reynoutrin < avicularin < quercitrin < hyperoside. Hyperoside was a predominant component among quercetin glycosides in the ethanol extracts of the fruit samples of apple cultivars selected for this study. Rutin was the minor component among all the quercetin derivatives. These results are consistent with those of the previously published studies, which reported hyperoside to be one of the predominant compounds All the quercetin glycosides identified and quantified in the ethanol extract of the apple lyophilisate can be ranked in the following ascending order by their content: rutin < isoquercitrin < reynoutrin < avicularin < quercitrin < hyperoside. Hyperoside was a predominant component among quercetin glycosides in the ethanol extracts of the fruit samples of apple cultivars selected for this study. Rutin was the minor component among all the quercetin derivatives. These results are consistent with those of the previously published studies, which reported hyperoside to be one of the predominant compounds [3,48].

[3,48].

#### *2.2. Biopharmaceutical Evaluation of Hard Gelatin Capsules*

Following the analysis of the composition of biologically active compounds in the apple lyophilisate, the selected amount of the active substance was 0.100 g of lyophilized apple powder per capsule. During the next stage of the research, the selection of excipients was performed. Microcrystalline cellulose, starch, and glucose were chosen as fillers to increase the mass of the encapsulated content when modeling apple lyophilisate-containing capsules (Table 1).


**Table 1.** Compositions of the apple lyophilisate-containing capsules.

CC = capsule compositions; AL = apple lyophilisate; SDX = silicon dioxide; MCC = microcrystalline cellulose; HPMC = hydroxypropyl methylcellulose; HEC = hydroxyethyl cellulose; GL = glucose; ST = starch; TCM = theoretical capsule mass; FQ = filling quality; MM = mean mass.

> Microcrystalline cellulose, one of the most commonly used excipients in the manufacture of solid dosage forms, has excellent compressibility and disintegration-enhancing properties [49]. Glucose is well soluble in water and has good sensory properties, but is hygroscopic [50]. Hypromellose was included in the composition of the capsules to prolong their disintegration time and to extend their therapeutic efficacy [51]. All the compositions of the apple lyophilisate-containing capsules are presented in Table 1.

> The quality of the manufactured capsules was evaluated according to the following parameters: uniformity of the capsule mass, capsule disintegration time, and the amount of the active substances released from the capsule (the dissolution test). Data presented in Table 1 shows that the indices of the uniformity of the capsule mass were similar. Thus, it can be stated that the selected excipients and their amounts ensured accurate dosage of the encapsulated mixture into the capsules. During the next stage of the study, capsule disintegration and dissolution tests were performed. Based on the results of these tests, the bioavailability of the modeled capsules can be predicted. The results of these tests are presented in Figure 4 and Table 2.

> Disintegration time is an important indicator in assessing the quality of capsules. The release of the drug is known to begin only after the disintegration of the capsule. The excipients in the encapsulated mixture must not interfere with the solubility and disintegration of the capsule. According to the test results presented in Figure 4, the capsules of the compositions N1–N9 disintegrated within less than 15 min, the capsules of the composition N10 disintegrated within 20 min, while the capsules of the composition N11 did not disintegrate after more than 60 min (Figure 4). The European Pharmacopoeia states that non-modified capsules must disintegrate within 30 min, and thus it can be stated that capsules N1–N10 meet the requirements of the European Pharmacopoeia [52]. The results of our study were confirmed by the results described by other scientists. Vyas et al. showed that lyophilized *Vasa Swaras* capsules undergo disintegration in more than 4.23 min [53]. Esmaeili et al. confirmed our study results, that hard gelatin capsules from *Pinus eldarica* bark extract disintegrate between 9.4 and 20.0 min [54]. There was no statistically significant difference (*p* > 0.05) between the disintegration time of the N1–N9 capsules compared to the disintegration time of the other tested capsules. The results of the testing showed

that capsules disintegrated more slowly when hypromellose was used as a filler (Figure 4). A statistically significant difference (*p* < 0.05) was found between the disintegration time of the N10 capsules and the disintegration time of the other tested capsules. In capsules of this composition, hypromellose made up about 71.4% of the capsule mass. This confirms the literature data indicating that hypromellose has disintegration-prolonging properties [51]. *Molecules* **2021**, *26*, x FOR PEER REVIEW 7 of 15

**Figure 4.** Results of the capsule disintegration test. The means followed by different letters are significantly different at *p* < 0.05. **Figure 4.** Results of the capsule disintegration test. The means followed by different letters are significantly different at *p* < 0.05.

Disintegration time is an important indicator in assessing the quality of capsules. The release of the drug is known to begin only after the disintegration of the capsule. The **Table 2.** Results of the in vitro dissolution test after 30 min. The means followed by different letters in the columns are significantly different at *p* < 0.05.


(Figure 4). A statistically significant difference (*p* < 0.05) was found between the disinte-CC = capsule compositions.

In capsules of this composition, hypromellose made up about 71.4% of the capsule mass. This confirms the literature data indicating that hypromellose has disintegration-prolonging properties [51]. Dissolution test is one of the most important tests used for capsule quality assessment. The dissolution test evaluates the time taken for a defined amount or part of a drug to be released from a dosage form into a solution. During the test, it is important to choose the right dissolution medium. For this reason, the next stage of the study focused on the Dissolution test is one of the most important tests used for capsule quality assessment. The dissolution test evaluates the time taken for a defined amount or part of a drug to be released from a dosage form into a solution. During the test, it is important to choose the right dissolution medium. For this reason, the next stage of the study focused on the selection of a suitable solvent for the active substances. Table 2 presents test results showing that the maximum amount of the active ingredients was released from the capsule when a 1:1 mixture of ethanol and water was used as the solvent.

gration time of the N10 capsules and the disintegration time of the other tested capsules.

selection of a suitable solvent for the active substances. Table 2 presents test results showing that the maximum amount of the active ingredients was released from the capsule when a 1:1 mixture of ethanol and water was used as the solvent. According to the amount of the active substance released from the capsules of the compositions N1–N11, individual phenolic compounds can be arranged in the following descending order: chlorogenic acid > rutin > avicularin > hyperoside > phloridzin > quercitrin > (−)-epicatechin > isoquercitrin (Table 2). Based on the results of the study, chlorogenic acid was the compound that was released in the highest amounts from capsules N1–N11, its released amount ranged from 68.4 to 640.3 µg/mL (Table 2).

The results of the testing showed that the selected excipients did not affect the active ingredients because all the capsule formulations released the compounds that were detected in the apple lyophilisate. The amounts of excipients affected the dissolution kinetics of the modeled capsules. The results showed in Table 3 reveals that unequal amounts of the phenolic compounds were released after 30 min.



There was no statistically significant difference (*p* > 0.05) between the amount of phenolic compounds released from the N1–N7 capsules after 30 min (Table 3). Thus, it can be argued that the amount of hypromellose used in capsules N1–N7 did not prolong the release of the active substance [55]. The results of the testing revealed that the amount of hypromellose used in capsule production did not prolong the release of the active substance when its amount in the capsules ranged from 29% to 41%. The selected excipients with different properties such as glucose, microcrystalline cellulose and starch did not affect the release of the active ingredients, and no statistically significant (*p* > 0.05) difference was found between the amount of the active compounds released from capsules N3, N4, and N5.

The results of the investigation exposed that the higher amount of hypromellose ranging from 50% to 83% in capsules did prolong the release of the active substance. The results show that after 30–60 min the lowest amount of the phenolic compounds is released from capsule N11 containing 0.500 g of the hypromellose. Of studied N8–N11 composition capsules was released than 85% of active compounds after 75 min (Table 4).

There was no statistically significant difference determined between the amount of released active compounds of capsules N8–N11 after 75 and 90 min (*p* > 0.05). The testing results confirmed the literature data indicating that the dissolution test is an informative tool for assessing the quality of solid dosage forms, helping to evaluate how the dosage form releases the active ingredients [56]. The results of the study showed that the selected excipients were suitable for the modeling of capsules with lyophilized apple powder. The results of this study proved that hypromellose is an appropriate excipient for prolonging the release of an active substance. Active compounds release and disintegration rate depends on the quantity of hypromellose in a capsule. The corresponding amount of hypromellose in capsule N1–N7 neither prolonged the disintegration of the capsules nor affected the dissolution rate of the active compounds. The results of the study showed that the use of hypromellose as a filler in higher amounts (N8–N11) prolongs the release of the active compounds.


**Table 4.** Results of the dissolution test. Released content of the individual phenolic compounds after 60, 75, 90 min. The means followed by different letters in the columns are significantly different at *p* < 0.05.

> The literature has demonstrated concerning data related to herbal medicinal product dissolution tests. A study performed with phytomedicines based on *Ginkgo biloba* extract capsules and tablets found marked differences in dissolution behavior were established, with values of 99% and 33% dissolution, on average, after 15 min and 60 min, respectively [57]. The dissolution profile of *Senna* sp. was less than 10% sennoside release from capsules containing dry extract, in a period of 60 min, in contrast to lyophilized *Senna* sp. extract, which attained around 90% dissolution after the same period. Data on the dissolution test of *Passiflora* sp. showed that capsules containing the crude extract presented 50% dissolution while lyophilized extract and other standardized extract reached around 100% for the same period [58].

> The modeled capsules are suitable for internal use as a food supplement that contains all the components of the chemical composition of the phenolic compounds found in lyophilized apples, as these components have a wide range of biological effects on the human body. Given the fact that the bioavailability of phenolic compounds is not particularly high, compounds with a lower molecular weight are more readily absorbed in the gastrointestinal tract [59]. According to scientific literature, various groups of phenolic compounds are absorbed at a rate of 0.3–43%, and the metabolite content circulating in the plasma can be low [60]. Chlorogenic acid absorption is approximately 33%, and the majority of chlorogenic acid will reach the large intestine, while (+)-catechin and (−)-epicatechin are both absorbed by small intestinal epithelial cells [11]. The route of administration, the release of a dosage form, and absorption are known to affect bioavailability [61]. The bioavailability of flavonoids depends on their physicochemical properties. Flavonoids with complex structures and larger molecular weights, bioavailability may be even lower [62,63]. The absorption of flavonoids in the small intestine is limited owing to their molecular weight and hydrophilicity of their glycosides. It is therefore important to select suitable excipients that do not limit the therapeutic efficacy of the active compounds [64].

> Since the active compounds in the apple lyophilisate were not highly soluble, we tried to model capsules whose excipients would not impair their solubility. When modeling capsules that contain active substances of botanical origin, it is expedient to avoid possible interactions between the lyophilized apple powder and the excipients, since dissolution, base structure, molecular size, and interaction with other components are the major physiochemical properties that lower the effectiveness of phenolic compounds [59]. According to the scientific literature, lyophilisate of *Vasa Swaras* capsules improved the stability and oral bioavailability of vasicine as well as reduced its conversion to vasicinone. The pharma-

cokinetic profiles of the various formulations inferred that the highest bioavailability of vasicine was obtained from hard gelatin capsules of lyophilized *Vasa Swaras* [53].

Considering the increasing consumption of herbal medicines, food supplements, there is a need for studies to ensure quality from raw material to finished product. Quality control is essential for the safety of functional food or nutrition supplements. Thus, biopharmaceutical studies of capsules with apple lyophilisate are of great importance since they can contribute toward ensuring future research for the planning and manufacture of innovative nutrition supplements.

#### **3. Materials and Methods**

#### *3.1. Plant Materials*

The study included "Ligol" apple cultivar (a winter cultivar bred in Poland). The apple trees were grown in the experimental orchard (block 2, row 4, trees 21–40) of the Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai, Lithuania (55◦600 N, 23◦480 E). The altitude of Babtai town is 57 m above sea level. Trees were trained as a slender spindle. Pest and disease management was carried out according to the rules of integrated plant protection. The experimental orchard was not irrigated. Tree fertilization was performed based on the results of soil and leaf analysis. Nitrogen was applied before flowering at the rate of 80 kg ha−<sup>1</sup> , and potassium was applied after the harvest at the rate of 90 kg ha−<sup>1</sup> . Soil conditions of the experimental orchard were the following: clay loam, pH—7.3, humus—2.8%, P2O5—255 mg kg−<sup>1</sup> , and K2O—230 mg kg−<sup>1</sup> . The apples harvested in September 2019 were immediately lyophilized and used for the study.

#### *3.2. Chemicals and Solvents*

All solvents, reagents, and standards used were of analytical grade. Acetonitrile, acetic acid, D(+)-glucose monohydrate, hypromellose, AerosilTM 200, microcrystalline cellulose, silicon dioxide and starch were obtained from Sigma-Aldrich GmbH (Buchs, Switzerland), and ethanol was obtained from Stumbras AB (Kaunas, Lithuania). Hyperoside, rutin, quercitrin, phloridzin, procyanidin B1, procyanidin B2, and chlorogenic acid standards were purchased from Extrasynthese (Genay, France), reynoutrin, (+)-catechin and (−)-epicatechin were purchased from Sigma-Aldrich GmbH (Buchs, Switzerland), and avicularin, procyanidin C1, and isoquercitrin were purchased from Chromadex (Santa Ana, USA). In this study, we used deionized water produced by the Milli-Q® (Millipore, Bedford, MA, USA) water purification system.

#### *3.3. Preparation of Apple Lyophilisate*

The apples were cut into slices of equal size (up to 1 cm in thickness), and the stalks and the seeds were removed. The apple slices were immediately frozen in a freezer (at −35 ◦C) with air circulation. Apple samples were lyophilized with a ZIRBUS sublimator 3 × 4 × 5/20 (ZIRBUS technology, Bad Grund, Germany) at the pressure of 0.01 mbar (condenser temperature, −85 ◦C). The lyophilized apple slices were ground to fine powder (particle size about 100 µm) by using a knife mill Grindomix GM 200 (Retsch, Haan, Germany).

#### *3.4. Preparation of Phenolic Extracts*

During the analysis of phenolic compounds, 2.5 g of lyophilizate powder (exact weight) was weighed, added to 30 mL of 70% (*v*/*v*) ethanol, and extracted in a Sonorex Digital 10 P ultrasonic bath (Bandelin Electronic GmbH & Co. KG, Berlin, Germany) at room temperature for 20 min. The obtained extract was filtered through a paper filter, and the residue on the filter was washed with 70% (*v*/*v*) ethanol in a 50 mL flask until the extract volume was reached. The conditions of the extraction were chosen based on the results of the tests for setting the extraction conditions [65].

#### *3.5. Qualitative and Quantitative Analysis by HPLC-PDA Method*

The qualitative and quantitative HPLC analysis of phenolic compounds was performed with a Waters 2998 PDA detector (Waters, Milford, USA). Chromatographic separations were carried out by using a YMC-Pack ODS-A (5 µm, C18, 250 × 4.6 mm i.d.) column. The column was operated at a constant temperature of 25 ◦C. The volume of the analyzed extract was 10 µL. The flow rate was 1 mL/min. The mobile phase consisted of 2% (*v*/*v*) acetic acid (solvent A) and acetonitrile (solvent B). Gradient variation: 0–30 min 3–15% B, 30–45 min 15–25% B, 45–50 min 25–50% B, and 50–55 min 50–95% B. For the quantitative analysis, the calibration curves were obtained by injecting the known concentrations of different standard compounds. All the identified phenolic compounds were quantified at λ = 200–400 nm wavelength [66,67].

#### *3.6. Encapsulation Process*

Compositions of capsules fillings are given in Table 1. Powder was prepared by simple mixing of the mixture of apple lyophilizate with excipient. Filled capsules were prepared using the manual capsule filling machine (Capsuline, Davie, FL, USA).

#### 3.6.1. Test of the Uniformity of Mass of Single-Dose Preparations

The tested capsules were weighed, and the mean weight of 1 capsule was determined [68]. One capsule was weighed, apple lyophilizate and excipients mixture were poured out, and then the capsule shell was weighed. Subsequently, the mass of the content i.e., the difference between the weight of the capsule and the weight of the shell—was calculated. This procedure was applied to each modeled capsule. The allowed deviation for capsules weighing not more than 250 mg was 7.5%.

#### 3.6.2. Capsule Disintegration Test

The capsule disintegration time was determined based on the methodology outlined in Ph. Eur. 2.9.1. [69]. The device C-MAG HS7 (IKA®-Werke GmbH & Co, Staufen, Germany) was used to determine the disintegration time. The disintegration medium was 0.1 M hydrochloric acid solution, temperature 37 ± 0.50 ◦C, observed for 30 min.

#### 3.6.3. Capsule Dissolution Test

The capsule dissolution test was performed using a Sotax AT 7smart dissolution tester (SOTAX AG, Allschwil, Switzerland). The acceptor medium was an ethanol-water mixture at the ratio of 1:1, the temperature being 37.0 ± 0.5 ◦C. The volume of the medium was 250 mL. The samples were taken after 15, 30, 60, 75 and 90 min. The sample volume was 10 mL. The analysis of the active compounds was performed by applying HPLC.

#### *3.7. Statistical Analysis*

The statistical analysis of the study data was performed by using Microsoft Office Excel 2013 (Microsoft, Redmond, WA, USA) and SPSS 25.0 (SPSS Inc., Chicago, IL, USA) computer software. All the results obtained during the HPLC analysis were presented as means of three consecutive test results and standard deviations. To evaluate the variance in the quantitative composition, we calculated the coefficient of variation. Univariate analysis of variance (ANOVA) was applied in order to determine whether the differences between the compared data were statistically significant. The hypothesis about the equality of variances was verified by applying Levine's test. If the variances of independent variables were found to be equal, Tukey's multiple comparison test was used. The differences were regarded as statistically significant at *p* < 0.05.

#### **4. Conclusions**

Excipients and their amounts may affect the dissolution kinetics of the phenolic compounds. Hypromellose prolonged the disintegration time of the modeled capsules when its amount reached 50–83% of the capsule weight. The selected fillers did not affect the kinetics of the release of the phenolic compounds from the capsules. Based on the results of the dissolution test, the capsules can be classified as fast-dissolving preparations since more than 85% of the active substance was released within 30 min.

According to the amount released from the capsules of different encapsulating content, individual phenolic compounds can be arranged in the following descending order: chlorogenic acid > rutin > avicularin > hyperoside > phloridzin > quercitrin > (−)-epicatechin > isoquercitrin. Chlorogenic acid was the compound that was released in the highest amounts from capsules of different encapsulating content: its released amounts ranged from 68.4 to 640.3 µg/mL.

The results of the solubility and disintegration tests proved that the capsules of the proposed composition are appropriate for internal use. The proposed product could serve as a basis for the development of food supplements with lyophilized apple powder.

**Author Contributions:** Conceptualization, V.J. and K.R.; methodology, K.R. and M.L.; formal analysis, A.B. and M.L.; investigation, A.B.; resources, K.R. and M.L.; data curation, A.B.; writing—original draft preparation, A.B., M.L. and K.R. writing—review and editing, V.J.; visualization, A.B.; supervision, V.J. and K.R. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All datasets generated for this study are included in the article.

**Acknowledgments:** The authors wish to thank the LSMU Science Foundation for the support of this study.

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

**Sample Availability:** The samples for this paper's experiment are available from the authors.

#### **References**


## *Article* **Hydrogel Emulsion with Encapsulated Safflower Oil Enriched with Açai Extract as a Novel Fat Substitute in Beef Burgers Subjected to Storage in Cold Conditions**

**Monika Hanula 1,\*, Arkadiusz Szpicer <sup>1</sup> , Elzbieta G ˙ órska-Horczyczak <sup>1</sup> , Gohar Khachatryan <sup>2</sup> , Grzegorz Pogorzelski <sup>1</sup> , Ewelina Pogorzelska-Nowicka <sup>1</sup> and Andrzej Poltorak <sup>1</sup>**


**Citation:** Hanula, M.; Szpicer, A.; Górska-Horczyczak, E.; Khachatryan, G.; Pogorzelski, G.; Pogorzelska-Nowicka, E.; Poltorak, A. Hydrogel Emulsion with Encapsulated Safflower Oil Enriched with Açai Extract as a Novel Fat Substitute in Beef Burgers Subjected to Storage in Cold Conditions. *Molecules* **2022**, *27*, 2397. https://doi.org/10.3390/ molecules27082397

Academic Editors: Sabina Lachowicz-Wi´sniewska, Jan Oszmianski and Francesco Cacciola

Received: 9 March 2022 Accepted: 4 April 2022 Published: 7 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** This study evaluates the effects of using a fat substitute in beef burgers composed of a hydrogel emulsion enriched with encapsulated safflower oil and açai extract. The influences of the fat substitute on the chemical (TBARS, fatty acids, and volatile compounds profile) and physical (weight loss, cooking loss, water-holding capacity, color, and texture analyses) characteristics of the burgers were analyzed after 0, 4 and 8 days of storage at 4 ± 1 ◦C. The obtained results were compared with control groups (20 g of tallow or 8 g of safflower oil). The fat substitute used improved burger parameters such as chewiness, hardness and the a\* color parameter remained unchanged over storage time. The addition of açai extract slowed the oxidation rate of polyunsaturated fatty acids and reduced the changes in the volatile compounds profile during the storage of burgers. The utilization of a fat substitute enriched the burgers with polyunsaturated fatty acids and lowered the atherogenic index (0.49 raw, 0.58 grilled burger) and the thrombogenicity index (0.8 raw, 1.09 grilled burger), while it increased the hypocholesterolemic/hypercholesterolemic ratio (2.59 raw, 2.09 grilled burger) of consumed meat. Thus, the application of the presented fat substitute in the form of a hydrogel enriched with açai berry extract extended the shelf life of the final product and contributed to the creation of a healthier meat product that met the nutritional recommendations.

**Keywords:** konjac; linseed flour; fat substitute; volatile compounds; lipid oxidation; encapsulation

#### **1. Introduction**

The current literature suggests that the development of many human diseases stems from a variety of factors, in which diet type is prominent [1]. Over the last 40 years, the prevalence of obesity has doubled, resulting in ischemic heart disease, strokes, and numerous other diseases. Consumers and international health organizations (the World Health Organization and the European Food Safety Authority) have demanded changes in food quality and content in order to improve the level of human health. Meat products belong to a category of food that is a rich source of saturated and trans fatty acids that can increase the risk of cardiovascular diseases. Due to the evolution of human lifestyles, greater demands for easily accessible and fast-food preparation are observed, for example, beef burgers. Recent studies have focused on meeting these demands through the preparation of burgers with potential health benefits that consider consumers' needs and the nutritional recommendations of global organizations. This has been achieved through a combination of functional ingredients or by using fat substitutes [1–3].

The main challenge of fat substitutes is finding an oil that improves the nutritional profile without affecting consumer acceptability of the final product. To overcome such drawbacks of fat substitutes, many strategies have been proposed such as oil encapsulation and immobilization in oleogels or hydrogels [1]. The conversion of liquid vegetable oils into a solid gel has been the subject of many scientific studies. However, this technique, due to high production cost and the destructive effect of organogelators at high temperatures on the fatty acid profile of the oils, is problematic in the food industry. Unlike oleogels, emulsion hydrogel creation is cheap, simple and requires lower temperatures. Hence, this approach is suitable for heat-sensitive or bioactive compounds that undergo oxidative degradation. Moreover, emulsion hydrogels contain ≤ 50% oil. Thus, they improve the fatty acid profile and also effectively reduce the total fat and caloric content in modified meat products. Furthermore, this method allows for the incorporation of both hydrophilic and hydrophobic functional components into the hydrogel matrix [1]. Researchers have used various emulsifiers, biocomposites, and polysaccharides to create emulsion hydrogels that may be used as fat substitutes. Konjac flour, which is a low-calorie ingredient, has been proposed as a promising biocomposite, as it has been successfully used in dry sausages as a fat substitute in pork [4], salcichón enriched with n-3 [5], and pork liver pates [6].

Current trends in the production of functional foods include the designs of products with lower fat content or with additional health benefits using bioactive compounds and dietary fiber. Açai berries are a popular bioactive additive, due to their antioxidant, antilipidemic, anti-inflammatory, and antiproliferative activities; açai berries have been classified as a "superfood" [7,8]. In contrast, dietary fibre is one of the most common functional ingredients in foods. The incorporation of fiber into meat can increase the daily intake of dietary fiber with food, which is recommended to be >25 g/day. Clinical and epidemiological studies have shown that dietary fiber can increase the feeling of satiety. Moreover, fiber has been shown to reduce hyperlipidaemia, total cholesterol, and the risk of cancer occurrence, as well as to improve glucose tolerance and gastrointestinal health [9]. Flaxseed flour is another ingredient that possesses health-promoting properties, which contains numerous functional compounds. Flaxseed has a favorable profile of fatty acids (polyunsaturated fatty acids) and is a rich source of dietary fiber, protein, and antioxidants (lignans). Moreover, flaxseed is gluten free and can be used to enrich the diet of people suffering from celiac disease [10].

Therefore, the aim of this study was to develop a healthier burger recipe by using a hydrogel emulsion and to study its effect on chemical and physical parameters of raw and grilled meat during storage. To the best of our knowledge, according to the literature, this type of low-fat burger formulation has not yet been tested.

#### **2. Results and Discussion**

#### *2.1. Effect of Encapsulated Safflower Oil Concentration on the Physical Characteristics of Emulsion Hydrogel*

#### 2.1.1. Texture and Rheological Analysis

The values of the hydrogel texture parameters with safflower oil concentrations of 29–48% and a control without oil (0%) are shown in Table 1. The values for the firmness parameter ranged from 5.65 (N) to 11.91 (N). The encapsulated oil content (29%, 33%, and 38%) in the hydrogel emulsion resulted in a statistically significant increase in firmness; a similar trend was observed for the lubricity parameter, where the highest value was 11.22 (N × n). The viscosity and adhesion parameters decreased with increasing oil concentration up to 38%. In contrast, the addition of 42%, 45%, and 48% oil increased these parameters (*p* < 0.05). Hence, the obtained analysis showed that oil concentration influenced the hydrogel texture parameters [11,12].


**Table 1.** Effect of oil concentration on the hydrogel texture parameters.

A–E—Mean values between variants in the same column indicated by different letters indicate a statistically significant difference.

The analysis of the rheological parameters of the hydrogel with encapsulated safflower oil are shown in Table 2. The analyzed samples all behaved as non-Newtonian, pseudoplastic liquids. Based on the Ostwald–de Waele model fitted to the upper flow curves and n values obtained, it was determined that the addition of oil to the hydrogel emulsion promoted a statistically significant increase in pseudoplasticity. The addition of oil increased the shear stress, which was characterized by a higher shear resistance. This was confirmed by a statistically significant increase in the consistency coefficient (K) value. The highest K value was recorded for the sample with 38% oil (139.42 (Pa×s n )). A further increase in oil concentration of the hydrogel decreased the K value. A similar trend was observed for the thixotropy parameter. The report by Cano et al. [13] showed that an increase in the lipid phase content in the studied emulsion increased the K parameter.

**Table 2.** Parameters of the Ostwald–de Waele rheological model and area of the hysteresis loop of the hydrogel with encapsulated safflower oil.


A–F—Mean values between variants in the same column indicated by different letters indicate a statistically significant difference.

#### 2.1.2. SEM and FT-IR Analysis

Figure 1 shows the FT-IR spectra of the analyzed hydrogels with encapsulated safflower oil. The absorbance band characteristics of the oil were recorded for 45% and 0% oil content samples. The spectra showed peaks corresponding to the −CH<sup>3</sup> groups in the range of 1350–1150 cm−<sup>1</sup> . The C−O bond stretching vibrations belonging to ester groups consisted of two coupled asymmetric vibrations, i.e., C−C(=O)-O and O−C−C, which were observed between 1300 and 1000 cm−<sup>1</sup> . The bands corresponding to C−C(=O) −O vibrations of saturated esters were found between 1240 and 1163 cm−<sup>1</sup> , whereas unsaturated esters were observed at lower wave numbers. The vibrational bands of the O−C−C bonds from esters of primary alcohols were found at 1064−1031 cm−<sup>1</sup> and from secondary alcohols at 1100 cm−<sup>1</sup> , both types of esters were present in triacylglycerol molecules [14]. The low-intensity band at 1390 cm−<sup>1</sup> was related to C−H combination vibrations. The bands at 1726 cm−<sup>1</sup> and 1760 cm−<sup>1</sup> corresponded to C−H stretching vibrations of the methyl, methylene and ethylene groups. The band at 1725 cm−<sup>1</sup> corresponded to oleic acid, while saturated and trans-unsaturated triacylglycerols exhibited absorption bands at 1725 cm−<sup>1</sup> and 1760 cm−<sup>1</sup> . The band at 2145 cm−<sup>1</sup> was related to C−C and C−H stretching

*Molecules* **2022**, *27*, x FOR PEER REVIEW 4 of 26

vibrations, and the following vibrations at 2952, 2921, and 2855 cm−<sup>1</sup> corresponded to the valence −C−H vibrations from −CH<sup>3</sup> and −CH<sup>2</sup> groups of triglycerides, respectively [15]. The results indicated that the encapsulation process had no effect on the oil's structure. following vibrations at 2952, 2921, and 2855 cm−1 corresponded to the valence −C−H vibrations from −CH3 and −CH2 groups of triglycerides, respectively [15]. The results indicated that the encapsulation process had no effect on the oil's structure.

cm−1. The band at 2145 cm−1 was related to C−C and C−H stretching vibrations, and the

**Figure 1.** FT-IR analysis of hydrogels with encapsulated safflower oil. **Figure 1.** FT-IR analysis of hydrogels with encapsulated safflower oil.

The images of scanning electron microscope SEM are shown in Figure 2 and show that nanocapsules were successfully obtained in a polysaccharide matrix. The size and shape of the capsules varied depending on the oil concentration. The results indicated that, in the case of 29% and 33% oil content samples, the capsule envelope was multilayered (Figure 2A,B). The size of the capsules decreased with increasing oil concentration (Figure 2C,D). The capsules in the hydrogel emulsion were present throughout the matrix The images of scanning electron microscope SEM are shown in Figure 2 and show that nanocapsules were successfully obtained in a polysaccharide matrix. The size and shape of the capsules varied depending on the oil concentration. The results indicated that, in the case of 29% and 33% oil content samples, the capsule envelope was multilayered (Figure 2A,B). The size of the capsules decreased with increasing oil concentration (Figure 2C,D). The capsules in the hydrogel emulsion were present throughout the matrix and their distribution was uniform.

and their distribution was uniform. Despite slight statistical differences in the tested variants, the hydrogels with concentrations ranging from 29% to 45% were characterized with similar texture parameters. A 48% oil content was too high and differed significantly from other concentrations. Furthermore, the success of hydrogel applications in food depends largely on the ability of the oleogelator to form a network that traps liquid oil [16]. Therefore, the Despite slight statistical differences in the tested variants, the hydrogels with concentrations ranging from 29% to 45% were characterized with similar texture parameters. A 48% oil content was too high and differed significantly from other concentrations. Furthermore, the success of hydrogel applications in food depends largely on the ability of the oleogelator to form a network that traps liquid oil [16]. Therefore, the hydrogel with 45% concentration encapsulated oil was selected for further study as a fat substitute.

#### hydrogel with 45% concentration encapsulated oil was selected for further study as a fat 2.1.3. Biochemical Analysis of Oil and Açai Extract

substitute. The results of the TPC analysis and antioxidant activity of oil and açai extract are shown in Table 3. The concentration of the extract used in the main study was determined based on the study conducted by Mokhtar et al. [17]. Safflower oil containing polyphenols accounted for 0.27 mg gallic acid/g of oil, whereas the antioxidant activity was 0.09 mg ascorbic acid/g of oil according to the ABTS analysis and 0.32 mg ascorbic acid/g of oil according to the FRAP analysis. Nimrouzi et al. [18] reported similar results.

The fatty acid profile analysis showed that safflower oil was characterized by a high PUFA content, especially n-6. This result was also confirmed by a study conducted by Rutkowska et al. [19].

**Figure 2.** SEM analysis of hydrogels with encapsulated oil: (**A**,**B**) Concentration of 29%; (**C**,**D**) concentration in the range of 45%. **Figure 2.** SEM analysis of hydrogels with encapsulated oil: (**A**,**B**) Concentration of 29%; (**C**,**D**) concentration in the range of 45%.


2.1.3. Biochemical Analysis of Oil and Açai Extract **Table 3.** Characteristics of açai extract and safflower oil.

ABTS (mg ascorbic acid/g of sample0 0.09 ± 0.003 50.54 ± 0.296 FRAP (mg ascorbic acid/g of TPC—total phenolic compounds; ABTS—2,20 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid; FRAP—ferric reducing antioxidant power; SFA—saturated fatty acid; MUFA—monounsaturated fatty acid; PUFA—polyunsaturated fatty acid.

Fatty acid profile (%)

#### sample) 0.32 ± 0.004 38.05 ± 1.268 *2.2. Main Study*

#### SFA 10.88 ± 1.36 2.2.1. TBARS Analysis

MUFA 9.74 ± 1.13 PUFA 79.19 ± 0.95 ∑n-6 78.91 ± 0.98 ∑n-3 0.15 ± 0.02 A TBARS analysis is a commonly used indicator of oxidative lipid rancidity in meat, which quantifies the amount of secondary oxidation products. The analysis of the lipid peroxidation content in the studied burgers after 0, 4, and 8 days of storage is shown in Figure 3. The results indicated that, depending on the day of storage and variant, the lipid peroxidation content ranged from 1.14 to 6.92 mg MA/100 g sample. At Day 0, depending

on the variant used, the lipid oxidation values ranged from 1.14 (CO) to 5.2 mg MA/100 g sample (GE), hence, the application of a fat substitute in the form of a hydrogel with encapsulated oil and flaxseed flour increased the TBARS values as compared with the control variants (CT and CO). This result was probably due to the presence of flaxseed flour which contained high PUFA levels [20]. The defatted flaxseed flour used in the experiment contained 10 g of fat per 100 g of flour. The manufacturing process of the flaxseed flour could have influenced the generation of free radicals, aldehydes, and ketones upon exposure of PUFAs to light, heat, and oxygen. A study performed by Hautrive et al. [9] revealed that the addition of defatted flaxseed flour elevated TBARS values as compared with a control variant. Furthermore, our results showed that between 0 and 4 days of storage, the highest increase in lipid oxidation (1.85–2.08) was observed in CT and CO. Therefore, using a fat substitute in the form of gel (konjac flour and sodium alginate) with encapsulated oil and flaxseed flour (G, GT, GE, and GET) exerted a positive effect on the reduction in the peroxidation rate. Thus, structuring the added fat significantly reduced the TBARS value (*p* < 0.05, Figure 3) as compared with the product containing animal or vegetable fat [21]. Additionally, our research showed that the MA value increased with storage time, regardless of variant type. As a result, lipid oxidation contributed to the development of rancidity while reducing the quality of meat products during storage [22]. The threshold value that dictates the loss of sensory quality of food is >1.0 mg malondialdehyde/kg burger [23]. In our case, despite the observed increase in fat oxidation, all values obtained were below the threshold value. sample (GE), hence, the application of a fat substitute in the form of a hydrogel with encapsulated oil and flaxseed flour increased the TBARS values as compared with the control variants (CT and CO). This result was probably due to the presence of flaxseed flour which contained high PUFA levels [20]. The defatted flaxseed flour used in the experiment contained 10 g of fat per 100 g of flour. The manufacturing process of the flaxseed flour could have influenced the generation of free radicals, aldehydes, and ketones upon exposure of PUFAs to light, heat, and oxygen. A study performed by Hautrive et al. [9] revealed that the addition of defatted flaxseed flour elevated TBARS values as compared with a control variant. Furthermore, our results showed that between 0 and 4 days of storage, the highest increase in lipid oxidation (1.85–2.08) was observed in CT and CO. Therefore, using a fat substitute in the form of gel (konjac flour and sodium alginate) with encapsulated oil and flaxseed flour (G, GT, GE, and GET) exerted a positive effect on the reduction in the peroxidation rate. Thus, structuring the added fat significantly reduced the TBARS value (*p* < 0.05, Figure 3) as compared with the product containing animal or vegetable fat [21]. Additionally, our research showed that the MA value increased with storage time, regardless of variant type. As a result, lipid oxidation contributed to the development of rancidity while reducing the quality of meat products during storage [22]. The threshold value that dictates the loss of sensory quality of food is >1.0 mg malondialdehyde/kg burger [23]. In our case, despite the observed increase in fat oxidation, all values obtained were below the threshold value.

TPC—total phenolic compounds; ABTS—2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid; FRAP—ferric reducing antioxidant power; SFA—saturated fatty acid; MUFA—monounsaturated

The fatty acid profile analysis showed that safflower oil was characterized by a high PUFA content, especially n-6. This result was also confirmed by a study conducted by

A TBARS analysis is a commonly used indicator of oxidative lipid rancidity in meat, which quantifies the amount of secondary oxidation products. The analysis of the lipid peroxidation content in the studied burgers after 0, 4, and 8 days of storage is shown in Figure 3. The results indicated that, depending on the day of storage and variant, the lipid peroxidation content ranged from 1.14 to 6.92 mg MA/100 g sample. At Day 0, depending on the variant used, the lipid oxidation values ranged from 1.14 (CO) to 5.2 mg MA/100 g

*Molecules* **2022**, *27*, x FOR PEER REVIEW 6 of 26

fatty acid; PUFA—polyunsaturated fatty acid.

Rutkowska et al. [19].

2.2.1. TBARS Analysis

*2.2. Main Study*

**Figure 3.** Analysis of lipid oxidation (TBARS) in burgers with a fat substitute after 0, 4, and 8 days of storage. G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with aҫai **Figure 3.** Analysis of lipid oxidation (TBARS) in burgers with a fat substitute after 0, 4, and 8 days of storage. G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with açai extract; GET—encapsulated oil with açai extract + tallow; CT—control with tallow; CO—control with oil.

#### 2.2.2. Color and pH Analysis

The pH parameter of meat can affect certain characteristics such as color, flavor, aroma, tenderness, and nutritional value. An analysis of the meat pH parameter was carried out on raw burgers after each storage time. Table 4 lists the obtained pH values, which, depending on the variant and storage time, range from 5.56 to 5.81. The addition of a hydrogel and flaxseed flour contributed to the observed increase in pH on Day 0 as compared with the controls (CT and CO). This effect was possibly due to the addition of fiber in the form of flaxseed flour. Hautrive et al. [9] and Sánchez-Zapata et al. [24] also reported higher pH values in modified fiber burgers as compared with control variants. Our study demonstrated that the addition of tallow to the studied meat increased the pH values of the burgers more than the addition of safflower oil (5.64, respectively, 5.56 *p* < 0.05). The observed increase on Day 4 of storage, especially in the G, GT, GE, and GET variants, may have been due to the accumulation of volatile bases such as trimethylamine and ammonia, which formed during protein hydrolysis and amino acid degradation by endogenous enzymes or microorganisms [25].


**Table 4.** Effect of fat substitute in the form of a hydrogel with encapsulated safflower oil, açai extract, and linseed flour on color parameters, browning index (BI), and pH in raw and grilled burgers at 0, 4, and 8 days of storage.

A–E Mean values between variants on the same storage day with different letters indicate a significant difference. a–c Mean values of the same variants between storage day with different letters indicate a significant difference. G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with açai extract; GET—encapsulated oil with açai extract + tallow; CT—control with tallow; CO—control with oil. L\*—lightness; a\*—redness; b\*—yellowness; BI—browning index.

The effects of hydrogel with encapsulated safflower oil and flaxseed flour on the color parameters of grilled and raw burgers after 0, 4, and 8 days of storage are shown in Table 4. The color parameter of meat influences the willingness of consumers to buy a product due to a growing appreciation for bright red products. The color parameter was significantly affected by storage time and formulation ingredients. The values of the L\* parameter in raw burgers increased with increasing storage time regardless of the variant type. A similar trend

was observed by Carvalho et al. [26]. However, as compared with the L\* color parameter on Day 4, the control variants (CT and CO) had lower values (*p* < 0.05) than the experimental variants (G, GT, GE, and GET). This trend was also observed for a\* and b\* parameters on Days 4 and 8. Furthermore, the addition of açai extract (GE and GET) had no significant effect on the color parameter regardless of storage time. The fat substitute with microencapsulated oil and flaxseed flour contributed to an improved color parameter of raw burgers after Days 4 and 8 of storage. The analysis of the browning index showed that for both raw and grilled meat, the BI was higher in the G, GT, GE, and GET groups. Probably this effect was caused by the addition of flaxseed flour, which was characterized by a brown color. The color analysis performed on the grilled burgers after each storage day showed no differences among the variants. This was also confirmed by Summo et al. [27] and Gök et al. [28]. However, in the case of the study reported by Lucas-González et al. [29], an increase in the a\* color parameter and a decrease in the L\* color parameter in grilled burgers with a fat substitute (chestnut flour, emulsion gel, and chia oil) were noted. Grilling changed the color of the meat due to heat-induced denaturation of myoglobin. Currently, the role of fat type used for grilled burger formulation is not fully understood, but it should have less of an effect on the color than other parameters such as storage conditions or pH [27].

#### 2.2.3. TPA Analysis

Texture changes (chewiness, springiness, and hardness) that occur in meat products as a result of replacing animal fat with vegetable fats are becoming an interesting area of research, especially because texture is importance in sensory attributes. The TPA analysis of the grilled burgers is shown in Table 5. The texture parameter values, regardless of variant used and storage time, were significantly (*p* < 0.05) lower (5.3–15.0 (N)) than those of the control samples (24.8–90.9 (N0). This was also observed by Afshari et al. [30], where a fat substitute was applied as an emulsion of soy protein, inulin, β-glucan, canola oil, and olive oil. Similarly, Lucas-González et al. [29] showed that as the degree of fat substitute (chestnut flour emulsion gel and chia oil in pork burgers) increased, texture parameters decreased as compared with control samples. However, Moghtadaei et al. [31] applied a hydrogel (sesame oil and ethyl cellulose) as a fat substitute in a burger and observed the opposite effect as compared with our results. According to the literature, there are many conflicting reports regarding the effect of hydrogel type on the texture parameters of grilled burgers. These differences may occur due to the method of vegetable oil incorporation into the product, its concentration, and the type of hydrogel used [32]. The grilled burgers (G, GT, GE, and GET) had lower hardness values than that of the CT and CO samples. As expected, the grilling process increased hardness of the control samples due to protein denaturation, water loss, and fat loss [33]. In general, the hardness of meat products is related to the size of the fat molecules therein, allowing the formation of an interfacial protein film around the fat globules by salt-soluble proteins (actin, myosin). Thus, an exponential relationship exists between the size of fat globules and the amount of interfacial protein layer [34,35]. In the case of the studied hydrogel, the fat was encapsulated in a polymer blend, whereas in the control samples, an interfacial protein film likely formed and denaturation occurred because of grilling. The variation in hardness between the control samples was probably due to the amount of interfacial protein layer formed, the type of fat used (beef tallow in tissue form, liquid vegetable oil), and its thermal stability. The higher thermal stability of the hydrogel contributed to a greater decrease in hardness of the product after grilling as compared with beef fat or safflower oil. In addition, the values of grilled burger parameters such as springiness and cohesiveness decreased with increasing storage time, whereas the values of chewiness and firmness parameters increased. This effect was related to natural leakage in burgers, which consequently influenced texture parameters [36]. From the consumer's point of view, reductions in hardness or chewiness parameters are considered to be a favorable characteristics due to their association with enhanced meat quality of the burger [29].


**Table 5.** Effect of a hydrogel emulsion with capsulated safflower oil, açai extract, and linseed flour as a fat substitute, on texture parameters (TPA) of grilled burgers at 0, 4, and 8 days of storage.

A–C Mean values between variants on the same storage day with different letters indicate a significant difference. a–c Mean values of the same variants between storage day with different letters indicate a significant difference. G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with açai extract; GET—encapsulated oil with açai extract + tallow; CT—control with tallow; CO—control with oil.

#### 2.2.4. WHC Analysis, Weight Loss, and Cooking Loss Analysis

The food industry demands the appropriate technological properties of a burger related to mass retention after storage and grilling, and therefore, retention of intrinsic water is of key importance. Adequate water retention in meat products not only reduces mass loss, but also causes accumulation of liquid in the package and consequently changes the texture, color, and ultimately, consumer acceptance of the meat product [37]. Figure 4 shows the analysis of water retention in raw and grilled burgers after 0, 4, and 8 days of storage. The values for the variants G, GT, GE, and GET, regardless of storage time, were in the range of 93.5–98.8%. In contrast, the values of the control samples were 31.8–43.2% depending on the period of storage. Our study showed that a fat substitute with flaxseed flour (G, GT, GE, and GET) improved the WHC about 54–60% as compared with those of the control samples, depending on the storage period and variant. A similar trend was observed by Zinina et al. [38], who observed an increased WHC parameter when flaxseed flour was used. In contrast, a study by Sharefiabadi et al. [39] revealed that the addition of flaxseed flour and coconut flour did not improve the WHC parameter in chicken pasties. The difference in the aforementioned studies may have been the type of fat substitute added to the meat products.

The analysis of mass loss conducted in this study showed that mass loss increased with storage time (*p* < 0.05) in the burgers with a fat substitute as well as the control variants. Nevertheless, the G, GT, GE, and GET variants had much lower mass loss (0.5–0.6%) than that of the CT and CO variants (1.3–2.6%). The replacement of fat with a hydrogel (konjac flour and sodium alginate) enriched with encapsulated oil with flaxseed flour helped to reduce mass changes during storage. The significant difference (*p* < 0.05) observed between the tallow and liquid oil controls was possibly due to the form of added fat [40]. Similar trends were observed for grilled samples after 0, 4, and 8 days of storage, where the fat substitute based on konjac flour and sodium alginate enriched with encapsulated safflower oil showed statistically significant (*p* < 0.05) reduction in the percentage mass loss of the burger. A study by Salcedo-Sandaval et al. [41] showed that konjac gel with added oils (olive oil, flaxseed oil, and fish oil) showed lower mass loss after grilling and baking as

compared with control variants. The limiting role in mass changes during grilling was also supported by Moghtadaei et al. [35]. The analysis showed that the addition of tallow (GT and GET) increased the percentage mass loss of the grilled burgers, which could be due to changes in the consistency of the tallow under the influence of the grilling process. *Molecules* **2022**, *27*, x FOR PEER REVIEW 11 of 26

**Figure 4.** Effect of a fat substitute on water retention (WHC) (**A**); weight loss (**B**); and cooking loss (**C**), after 0, 4, and 8 days of storage. G—encapsulated oil; GT—encapsulated oil + tallow; GE encapsulated oil with aҫai extract; GET—encapsulated oil with aҫai extract + tallow; CT—control with tallow; CO—control with oil. **Figure 4.** Effect of a fat substitute on water retention (WHC) (**A**); weight loss (**B**); and cooking loss (**C**), after 0, 4, and 8 days of storage. G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with açai extract; GET—encapsulated oil with açai extract + tallow; CT—control with tallow; CO—control with oil.

#### 2.2.5. Analysis of the Volatile Compounds Profile 2.2.5. Analysis of the Volatile Compounds Profile

Fat is a precursor to many aromatic compounds, and it also acts as a solvent for these compounds. Changes in the amount and composition of fat can affect the rate and type of Fat is a precursor to many aromatic compounds, and it also acts as a solvent for these compounds. Changes in the amount and composition of fat can affect the rate and type

processes that occur during burger storage [1]. The analysis of changes to the profile of volatile compounds in raw and grilled burgers after 0, 4, and 8 days of storage is shown

between G and GT, between G and GE, between GET and CT, and between GET and CO variants on Day 0. The data revealed that the presence of the extract affected the aroma

of processes that occur during burger storage [1]. The analysis of changes to the profile of volatile compounds in raw and grilled burgers after 0, 4, and 8 days of storage is shown in Figure 5. The obtained results displayed differences in the volatile compounds profile between G and GT, between G and GE, between GET and CT, and between GET and CO variants on Day 0. The data revealed that the presence of the extract affected the aroma profile of the raw burger by reducing the changes in compound growth (Figure 5A), thus, slowing down changes that naturally occur in the burger during storage. The alterations in the volatile compounds profile of burgers with a fat substitute after 4 and 8 days of storage were comparable. The opposite was observed for burgers with tallow and oil, where changes increased with storage time, and the process of changes progressed fastest between 4 and 8 days of storage. In contrast, for grilled burgers (Figure 5B), the variants used showed no difference in the volatile compounds profile regardless of storage time, with only a slight variation at Day 0 in GET. However, this may stem from the presence of both extract and tallow. Fluctuations in volatile compounds profile for the CT variant changed dramatically at Day 8, which was possibly due to the progressive oxidative processes of high tallow content (20%), especially after 4 days of storage. In contrast, the CT variant with safflower oil displayed linear changes, which indicated gradual changes that progressed with storage time.

Table 6 shows the component analysis of the volatile compounds profile of raw burgers with a fat substitute (G, GT, GE, and GET) and control variants (CT and CO) after 0, 4, and 8 days of storage. The analysis revealed that the number of compounds, especially corresponding to aldehydes, alcohols, esters, and ketones, increased with storage time. The addition of oils with increased amounts of unsaturated fatty acids promotes oxidative processes in meat and consequently increases the content of volatile compounds formed by lipid oxidation [26]. Volatile compounds such as alcohols, aldehydes, and ketones are mainly responsible for reducing the sensory quality of meat products [42]. Wantanabe et al. [43] confirmed our findings, where the number of volatile compounds related to alcohols increased with storage time. In contrast, alcohols such as 1−hexanol, 1−pentanol, and 1−octen−3−ol have been considered to be key indicators of lipid oxidation in meat [26]. The component analysis of the volatile compounds profile in grilled burgers was dominated by aldehyde and alcohol group bearing compounds. Emerging compounds such as benzaldehyde, 3−methylbutanal, 2−methylpropanal, pyrazine, and dimethyl sulphide are typical compounds that arise from the Maillard reaction, which is induced by a thermal process [44,45]. The obtained results showed that, apart from lipid degradation and the Maillard reaction, the applied product components were the main factors that influenced the formation of volatile substance characteristics for a particular variant, for example, compounds such as benzaneacetaldehyde, 1−propanol−2 methyl, and pyrazine for variants G, GT, GE, and GET; pentan−2−ol for GE and GET; 2−furanmethanol for CO; and dimethyl sulphide for CT.

progressed with storage time.

**Figure 5.** Changes in the volatile compounds profile of raw burgers (**A**); grilled burgers (**B**), after 0, 4, and 8 days of storage (4 ± 1 °C). G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with aҫai extract; GET—encapsulated oil with aҫai extract + tallow; CT—control with tallow; CO—control with oil. **Figure 5.** Changes in the volatile compounds profile of raw burgers (**A**); grilled burgers (**B**), after 0, 4, and 8 days of storage (4 ± 1 ◦C). G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with açai extract; GET—encapsulated oil with açai extract + tallow; CT—control with tallow; CO—control with oil.

profile of the raw burger by reducing the changes in compound growth (Figure 5A), thus, slowing down changes that naturally occur in the burger during storage. The alterations in the volatile compounds profile of burgers with a fat substitute after 4 and 8 days of storage were comparable. The opposite was observed for burgers with tallow and oil, where changes increased with storage time, and the process of changes progressed fastest between 4 and 8 days of storage. In contrast, for grilled burgers (Figure 5B), the variants used showed no difference in the volatile compounds profile regardless of storage time, with only a slight variation at Day 0 in GET. However, this may stem from the presence of both extract and tallow. Fluctuations in volatile compounds profile for the CT variant changed dramatically at Day 8, which was possibly due to the progressive oxidative processes of high tallow content (20%), especially after 4 days of storage. In contrast, the CT variant with safflower oil displayed linear changes, which indicated gradual changes that



**Table 6.** *Cont.*


193

with oil.

#### 2.2.6. Analysis of Fatty Acid Profile and Nutritional Indexes AI, TI, and h/H

The analysis of the fatty acid profile and nutritional indexes of raw and grilled burgers after 0, 4, and 8 days of storage are shown in Table 7. The fatty acid profile was influenced by the type of formulation used. The analysis of saturated fatty acids (SFA) and unsaturated fatty acids (MUFA and PUFA) in raw burgers showed that SFA increased and MUFA and PUFA decreased with storage time, regardless of the variant used. Our results were consistent with the theory that as the number of double bonds in a fatty acid increased, the susceptibility to oxidation increased [26]. As predicted, the addition of safflower oil significantly increased the PUFA content of the raw burgers from 1% (CT) to 24% (GET). The analysis of PUFAs and MUFAs in reformulated raw burgers showed that the addition of açai extract (GE) contributed to lower oxidation levels. Carvalho et al. [26] and Rutkowska et al. [46] confirmed a positive effect of extracts (pitanga leaves, guarana seeds, and chokeberry fruit) on reducing the rate of oxidative changes in fat. Safflower oil used in the experiment was characterized by high PUFA content (79%), with an n − 6/n − 3 ratio of 526. In contrast, the applied reformulation of the burger with cassava oil with the addition of flaxseed flour reduced the n − 6/n − 3 ratio by as much as 10-fold (50 for CO and 5 for GE), while maintaining high PUFA values. According to dietary recommendations, this ratio should be 5-4:1 [19]. For other nutritional indexes, it is recommended that meat products should have the lowest possible AI and TI values, while the h/H ratio should be as high as possible [47]. In our study, independent of storage time, the burger variants with the fat substitute used had the lowest AI and TI values as compared with CT. Furthermore, hydrogel emulsion with flaxseed flour significantly increased the h/H ratio from 1.3 (CT) to 2.5 (GE). Considering the indexes of healthiness that were analyzed, the best results were obtained for raw burgers with the addition of açai extract (GE) in relation to hydrogel without extract (G). The correlation analysis among SFA, MUFA, PUFA, and CLA and storage day, showed that SFA had a very high positive correlation (0.736, *p* < 0.05). In contrast, the correlation analysis of MUFA and CLA showed moderately high negative correlations (−0.359 and −0.488, *p* < 0.05, respectively). In contrast, the PUFA analysis showed no correlation with storage time. The correlations obtained indicated that changes occurred during storage related to the fat oxidation process as a result of which, polyunsaturated acids were converted to a saturated form [26]. The analysis of the fatty acid profile of grilled burgers showed that the use of reformulated burgers contributed to an increase in PUFA content as compared with CT and CO. In addition, usage of hydrogels contributed to lowering n − 6/n − 3 acid ratios, from 24 (CO) to 5 (GE), which was extremely important to maintain proper nutritional standards. Furthermore, the use of reformulation resulted in improved health values of the grilled burger by lowering the Al and Tl values and increasing the h/H ratio.


**Table 7.** Effects of a fat substitute (hydrogel emulsion with encapsulated oil and acai extract) on the fatty acid profile of raw and grilled burgers at 0, 4, and 8 days of storage in cold conditions.




**Table 7.** *Cont.*

A–E Mean values between variants on the same storage day with different letters indicate a significant difference. SFA—saturated fatty acid; MUFA—monounsaturated fatty acid; PUFA—polyunsaturated fatty acid; CLA—conjugated linoleic acid; Al—atherogenic index; Tl—thrombogenicity index; h/H—hypocholesterolemic/hypercholesterolemic ratio; G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with açai extract; GET—encapsulated oil with açai extract + tallow; CT—control with tallow, CO—control with oil.

2.2.7. Analysis Correlation Coefficients among Color Parameters (L\*, a\*, b\*, BI), Textural (Springiness, Chewiness, Cohesiveness, and Hardness), Cooking Loss, Weight Loss, Fatty Acid Profile (SFA, MUFA, and PUFA), WHC, pH, and TBARS in Raw and Grilled Burgers

The correlation analysis among fatty acid profiles (SFA, MUFA, and PUFA), color parameters (L\*, a\*, b\*, and BI), WHC, pH, weight loss, and TBARS in raw burgers is presented in Table 8. The analysis showed a high negative correlation between PUFA and SFA and between PUFA and MUFA (−0.580 and −0.790, *p* < 0.05, respectively). These correlation are probably related to the change in the number of bonds in fatty acids associated with oxidation processes. In addition, the amount of SFA in the raw burger is negatively correlated with the color parameter a\* and BI; this may indicate an oxidative process during which the color of the fat changes. The analysis of the burger brightness parameter (L\*) showed positive correlations with b\*, WHC, pH, TBARS (0.727, 0.539, 0.497, and 0.848, *p* < 0.05, respectively). Moreover, the correlation analysis showed high positive correlations of the parameter b\* with BI, WHC, pH, and the TBARS analyses (0.751, 0.928, 0.880, and 0.765, *p* < 0.05, respectively). The correlation analysis among fatty acid profiles (SFA, MUFA, and PUFA), color parameters (L\*, a\*, b\*, and BI), WHC, pH, weight loss, and TBARS in raw burgers is presented in Table 8. The analysis showed a high negative correlation between PUFA and SFA and between PUFA and MUFA (−0.580 and −0.790, *p* < 0.05, respectively). This correlation is probably related to the change in the number of bonds in fatty acids associated with oxidation processes. In addition, the amount of SFA in the raw burger was negatively correlated with the color parameter a\* and BI; this may indicate an oxidative process during which the color of the fat changes. The analysis of the burger brightness parameter (L\*) showed positive correlations with b\*, WHC, pH, and TBARS (0.727, 0.539, 0.497, and 0.848, *p* < 0.05, respectively). Moreover, the correlation analysis showed high positive correlations of the parameter b\* with BI, WHC, pH, and the TBARS analyses (0.751, 0.928, 0.880, and 0.765, *p* < 0.05, respectively).

**Table 8.** Analysis of correlations among fatty acid profile (SFA, MUFA, and PUFA), color parameters (L\*, a\*, b\*, and BI) and WHC, pH, weight and cooking loss, TBARS, and TPA parameters (springiness, chewiness, cohesiveness, and hardness) with raw and/or grilled burgers.


#### **3. Materials and Methods**

#### *3.1. Preliminary Studies*

Preliminary studies were conducted to verify the maximum amount of oil absorbed by the base hydrogel without affecting the gel structure. The base for gel formulation consisted of konjac flour (green essence) and sodium alginate (Agnex 1999). Preparation of the gel with encapsulated safflower oil (gold press) was conducted in a multistep process. Gelation involved dissolving 2% sodium alginate and konjac flour in H2O at 60 ◦C with constant stirring until a clear emulsion was obtained. An emulsion was obtained of the mixture of safflower oil and water at a ratio of 2:1. Next, homogenization of the emulsion in base gel was performed using an immersion homogenizer (IKA T18 digital, Ultra Turrax, Germany). The concentrations of encapsulated oil in the emulsion hydrogels were 0% (control), 29%, 33%, 38%, 42%, 45%, and 48%. The biocomposites obtained through this approach were used for further studies: SEM image analysis, FT-IR spectroscopy analysis, texture, and rheology analysis, from which the optimal hydrogel was selected for further analysis and application (see Results and Discussion section).

#### *3.2. Beef Burgers Formation and Packaging*

The beef (neck muscles, Zakłady Mi˛esne Wierzejki, Poland) without excess fat and connective tissue was minced. Then, the beef and beef fat (tallow) were ground separately using a meat grinder with an 8 mm hole diameter plate (PI-22-TU-T, Edesa, Greece). Table 9 shows the composition of the prepared burgers with encapsulated oil without/with addition of açai extract (superfoods, PL-EKO-07) in hydrogel and defatted flaxseed flour (LenVitol, Oleofarm, Wrocław, Poland) (variants G, GT, GE, and GET). An emulsion hydrogel containing 45% encapsulated oil was prepared according to the recipe presented in the preliminary studies. The control variant was a burger with tallow (CT) as the conventional source of fat in meat products and oil (CO) as an alternative source of fat. All ingredients, according to the specified proportions, were mixed and burgers (120 g ± 1) of 10 cm diameter were formed. Then, the burgers were placed on 137 × 187 × 50 mm trays made from polyethylene terephthalate (PET) with an absorbent pad (absorbency 1700 mL/m<sup>2</sup> ). The trays were then sealed with 35 µm thick PSF films (PSF35ZAC, PolTechPack, Olstzn, Poland). Burgers were packed in modified atmosphere (80% O<sup>2</sup> and 20% CO2) and stored for 0, 4, and 8 days at a temperature of 4 ± 1 ◦C. Color, pH, fatty acid profile, thiobarbituric acid reactive substances (TBARS), weight loss, water-holding capacity (WHC), and volatile compounds profile analyses were performed on the raw burgers. After the specified storage time (0, 4, and 8 days), the burgers were grilled on an electric grill (silex), heated to 190 ◦C (bottom plate) and 210 ◦C (top plate) until 75 ◦C at the geometric center of each burger (measured with a thermocouple), and then cooled to 25 ◦C. The prepared burgers were used for the following analyses: color, fatty acid profile, cooking loss, volatile compounds and texture profile analysis (TPA) test. The experimental set-up covered 3 biological replicates (162 samples).


**Table 9.** Beef burger composition.

G—encapsulated oil; GT—encapsulated oil + tallow; GE—encapsulated oil with açai extract; GET—encapsulated oil with açai extract + tallow; CT—control with tallow; CO—control with oil.

#### *3.3. Rheology Analysis*

Melt flow curves were measured using a MARS III rheometer (MTMC—MARS Temperature Module) in CR mode with a coaxial cylinder measuring system (CC25 DIN Ti) with a 5.3 mm gap, wherein the measurements were determined at 5 ◦C. The shear rate was increased from 0 to 80 s−<sup>1</sup> , for 5 min, and then decreased from 80 to 0 s−<sup>1</sup> , for 5 min. The Ostwald–de Waele rheological analysis (Steffe 1996) was used to describe the melt flow curves. Thixotropy and hysteresis loop areas were calculated according to the method described by Sikora et al. [48].

#### *3.4. Analysis of the Textural Parameters of Hydrogel Emulsion*

Analysis of the hydrogel emulsion and burger texture textural parameters of the hydrogel were analyzed using a texture analyzer (TA.XTplusC Texture Analyzer) by penetration using Perspex 45◦ TTC Spreadability Rig Cone Sensors (Stable Micro System, Ltd., Goldaming, UK), according to method described by Ö˘gütcü and Yilmaz [12] with some modifications. Initially, the temperature of the sample was maintained at 5 ◦C for 24 h, then, penetration was carried out to a depth of 23 mm, at a speed of 3 mm/s. From the data obtained, the following parameters were determined using exponent connect software: firmness (N), lubricity (N × s), viscosity (Pa s) and stickiness (N × s).

The texture profile analysis (TPA) of the grilled beef burgers after 0, 4, and 8 days of storage at 4 ± 1 ◦C was conducted in a double compression cycle using an Instron 5965 universal testing machine (Instron, USA) with a 500 (N) load cell connected to the Bluehill®2 software, following the methodology described by Afshari et al. [13]. Samples of 2.45 cm in diameter and 2.50 cm height, cut out from the center of the burger, were subjected to double compression with 3 s relaxation time until their initial height was reduced by 50%. The analysis was performed at a constant head travel speed of 200 mm·min−<sup>1</sup> , at a temperature of 4 ± 1 ◦C. Six measurements were made for each group. Texture parameters, such as cohesiveness (−) (the ratio of the area under the curve from the second compression to the area under the curve from the first compression), hardness (N0 (the maximum force of the first compression), elasticity (−), and chewiness (N) (hardness × cohesiveness × elasticity) were calculated using methodology described by Półtorak et al. [49].

#### *3.5. Scanning Electron Microscope (SEM) Analysis*

The morphology of the prepared oil-encapsulated gels was examined using a JEOL JSM-7500F high-resolution scanning electron microscope.

#### *3.6. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis*

The FT-IR spectra of the biocomposites were recorded in the range of 4000–700 cm−<sup>1</sup> using a MATTSON 3000 FT-IR spectrophotometer (Madison, Wisconsin, USA). The instrument was equipped with a 30SPEC 30 Degree Reflectance adapter and a MIRacle ATR

accessory (PIKE Technologies Inc., Madison, WI, USA). The FT-IR spectra were performed on dried gels (films were obtained).

#### *3.7. Water-Holding Capacity (WHC) Analysis*

The WHC of raw burgers after 0, 4 and 8 days of storage was analyzed according to the method reported by Grau and Hamm [50]. Three hundred milligrams of the sample was placed on Whatman No. 1 filter paper between two glass plates under a weight of 2000 g for 5 min. In order to take press stain images, a Kaiser system (Germany) equipped with the OImaging MicroPublisher 5.0 RTV software (Canada) was used. Meat and fluid areas were evaluated using the Image-Pro Plus software (v.7.0). The WHC values were calculated using the following formula:

$$\text{WHC}\% = \text{(Am)} / \text{Ap} \times 100 \tag{1}$$

Am—meat areas; Ap—fluid areas.

#### *3.8. Color and pH Analysis*

The color evaluation was performed on raw and grilled burgers after 0, 4, and 8 days of storage. Measurements were obtained at 5 different locations on the burgers' surfaces. The analysis was performed in a CIE L\*a\*b\* system using a Minolta CR-400 chromameter with a CA-A98 attachment (Konica Minolta Inc., Tokyo, Japan) and D65 illuminant. The browning index (BI) was calculated using L\*, a\*, and b\* values according to the following formula [51]:

$$\text{BI} = 100 \,\text{(x} - 0.31) / 0.17 \,\tag{2}$$

where x = (a\* + 1.75 L\*)/(5.645 L\* + a\* − 3.012 b\*).

The pH was measured using a digital pH meter (FiveEasy F20, Mettler Toledo, Warsaw, Poland).

#### *3.9. Weight Loss and Cooking Loss during Storage Analysis*

The technological properties of the tested burgers were determined in 3 biological replicates using 2 samples for each variant. Raw burgers were weighed on Day 0 and after each day of storage to determine mass loss during storage. Samples were also weighed after grilling and cooling the burgers to room temperature (25 ◦C). Mass loss after both storage (F) and grilling (G) were calculated according to the following equations:

$$\text{weight loss\%} = (\text{BR0} - \text{BRx}) / \text{BR0} \times 100 \tag{3}$$

$$\text{cooking loss\%} = (\text{BRx} - \text{BGx}) / \text{BRx} \times 100\tag{4}$$

BR0—raw burger weight on day 0; BRx—raw burger weight after each day of storage; BGx—grilled burger weight after each day of storage

#### *3.10. Analysis of Total Phenolic Content (TPC) and Antioxidant Activity of the 2,2-Azinobis(3-ethylbenzothiazoline-6-suslfonic Acid (ABTS) and Ferric Reducing Antioxidant Power (FRAP)*

#### 3.10.1. Extraction Process

Safflower oil extraction for the analysis of TPC and antioxidant activity was prepared according to the procedure described by Ablay et al. [52]. Briefly, 5 g of oil was shaken in 5 mL of n-hexane for 5 min. Then, 5 mL of MeOH/H2O (80:20, *v*/*v*) was added, centrifuged, and the resulting extract was stored at 4 ◦C. TPC and antioxidant activity of the açai extract (superfoods, PL-EKO-07) was analyzed after extraction according to the study reported by Hanula et al. [8] with minor modifications. First, 1 g of lyophilized extract was shaken in 25 mL of H2O for 1 h. Then, the extraction process was carried out using ultrasound for 5 min. The obtained extract was centrifuged and stored at 4 ◦C for further analysis.

#### 3.10.2. TPC Analysis

The TPC analysis of the studied oil and açai extract was carried out using the method described by Singleton and Rossi [53] with some modifications. First, 0.1 mL of extract, 0.5 mL of Folin–Ciocalteu, 2.9 mL of H2O, and 1.5 mL of 7% Na2CO<sup>3</sup> were mixed and incubated for 40 min in the dark. The absorbance was measured at 765 nm using a UV-Vis spectrophotometer. The obtained results were expressed as mg gallic acid/g sample.

#### 3.10.3. ABTS and FRAP Analyses

The ABTS and FRAP analyses were performed according to the methods described by Belwal et al. [54] with slight modifications. The ABTS analysis was conducted mixing 0.1 mL of extract with 2.9 mL of ABTS and incubation for 30 min. The FRAP analysis was based on mixing 2.9 mL of FRAP solution (20 mM ferric chloride in H2O, 10 mM 2,4,6-tri(2 pyridyl)-s-triazine in 40 mM hydrochloric acid, and 300 mM sodium acetate buffer at a 1:1:10 ratio) with 0.1 mL of the extract and further incubation in the dark for 15 min. The ABTS and FRAP concentrations were measured at 520 nm and 593 nm, respectively, using a UV-Vis spectrophotometer. The results were expressed as mg ascorbic acid/g sample.

#### *3.11. Thiobarbituric Acid Reactive Substances (TBARS) Analysis*

The analysis of lipid oxidation was evaluated by TBARS changes and was performed according to the procedure described by Brodowska et al. [55]. 1,1,3,3-Tetramethoxypropane was used to prepare the standard curve. The absorbance of the resulting color complex was measured using a UV-Vis spectrophotometer (UV-1800, Shimadzu Corp., 115 VAC, Tokyo, Japan). The TBARS values were calculated in mg of malondialdehyde (MDA) per 100 g sample.

#### *3.12. Fatty Acid Profile Analysis, Thrombogenicity Index (Tl), Atherogenic Index (AI), and Hypocholesterolemic/Hypercholesterolemic (h/H) Ratio Analysis*

The fatty acid profile analysis was performed for safflower oil in each burger variant (raw and grilled) after 0, 4, and 8 days of storage. Lipids were directly methylated as described by Wojtasik-Kalinowska et al. [56] and Heck et al. [3] with slight modifications. The fatty acid methyl ester composition was analyzed using gas chromatography (Shimadzu GC-2010) with a flame ionization detector (FID) equipped with a Zebron ZB-FAME column (GC Cap. Column, 60 mL × 0.25 mm ID × 0.2 µm df). The initial column temperature was 100 ◦C held for 3 min, which was increased to 240 ◦C at a rate of 2.5 ◦C/min, and held for 10 min. The detector was maintained at 260 ◦C. In order to identify the FAME composition in burgers or oils, FAME Mix-37 standard (Supelco, TraceCERT®, EC:200-838-9, SKU: CRM47885) was used. The obtained results were presented as a fatty acid profile. In addition, TI and AI were determined according to the method described by Ulbricht and Southgate [57] and the hypocholesterolemic/hypercholesterolemic (h/H) ratio was calculated according to Fernandez et al. [58] as follows:

$$\text{TI} = (\text{C14:0} + \text{C16:0} + 18\text{d}) / ((0.5 \times \Sigma \text{MUFA}) + (0.5 \times \Sigma \text{n} - 6) + (3 \times \Sigma \text{n} - 3) + (\Sigma \text{n} - 3) / (\Sigma \text{n} - 6))\tag{5}$$

$$\text{AI} = \text{(C12:0} + \text{(4} \times \text{C14:0}) + \text{16:0}) / \text{((\Sigma PUFA n - 3) + (\Sigma PUFA n - 6) + (\Sigma MUFA))} \tag{6}$$

$$\text{h/H} = \text{(C18:1n9 + ΣPUFA)/(C14:0 + C16:0)}\tag{7}$$

#### *3.13. Analysis of the Volatile Compounds Profile*

The analysis of the volatile compounds profile was performed using a Heracles II enose (Alpha MOS Co., Toulouse, France)based on ultrafast gas chromatograph with a flame ionization detector and a retention index counting application using the AroChemBase library (AlphaSoft software, Alpha MOS Co., Toulouse, France). The gas chromatograph was equipped with two capillary columns of different polarities, i.e., DB-5 and DB-1701, with 10 m × 0.18 mm ID x 0.4 µm film thickness. The analysis was conducted according to the methodology reported by Górska-Horczyczak et al. [59]. Calibration was performed on a standard mixture of C6-C16 alkanes (Restek, ANCHEM Plus, Warsaw, Poland).

#### *3.14. Statistical Analysis*

The results were analyzed using the Statistica software version 13.3 (StatSoft, Tulsa, OK, USA). The normality of data distribution was verified using the Shapiro–Wilk test. Factorial analysis of variance (ANOVA) was performed in the case of the TBARS analysis. The results of hydrogel texture and rheology as well as color of meat were analyzed with one-way ANOVA. Texture parameters of the burgers were subjected to the Kruskal–Wallis ANOVA, followed by multiple comparisons of mean ranks. Moreover, the strengths of the relationships among fatty acid profile (SFA, MUFA, and PUFA), texture parameters (springiness, chewiness, cohesiveness, and hardness), cooking loss, weight loss, WHC, pH, and color parameters (L\*, a\*, b\*, and BI) methods were determined using Pearson's correlation coefficients. For all analyses, 95% confidence intervals were established. The AlfaSoft package with statistical quality control was used in order to perform a comparative analysis and to evaluate the chromatographic fingerprints of the volatile compounds.

#### **4. Conclusions**

In conclusion, an emulsion hydrogel formulated based on konjac flour and sodium alginate with encapsulated oil was used as a functional ingredient in beef burgers enriched with flaxseed flour to develop a healthier alternative. Each variant of the fat substitute containing extract contributed to a reduction in the changes of volatile compounds profile rate and preserved a\* color parameter. In addition, the fat substitute reduced the hardness and chewiness parameters of the grilled burgers. According to the obtained results, the applied fat substitute promoted a reduction in the nutritional AI and TI indexes, as well as increased the amount of the PUFAs and h/H ratio. Thus, a hydrogel emulsion enriched with encapsulated oil and açai extract along with the addition of flaxseed flour in a burger recipe instead of beef fat results in a healthier alternative that is rich in fiber.

**Author Contributions:** M.H., conceptualization, formal analysis, investigation, methodology, supervision, writing—original draft, visualization, writing—review and editing; A.S., formal analysis, investigation, methodology; E.G.-H., formal analysis, methodology; G.K., formal analysis, methodology; G.P., formal analysis; E.P.-N., formal analysis, investigation, methodology, supervision, writing review and editing; A.P.; funding acquisition, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research reported in this manuscript has been financed by the Polish Ministry of Science and Higher Education within the fund from the Institute of Human Nutrition Sciences, Warsaw, University of Life Sciences (WULS), for scientific research.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Available from the authors.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Sample Availability:** Samples of the compounds are available from the authors.

#### **References**


## *Article* **Influence of Agronomic Practice on Total Phenols, Carotenoids, Chlorophylls Content, and Biological Activities in Dry Herbs Water Macerates**

**Kalina Sikorska-Zimny 1,2,\* , Paweł Lisiecki 2,3 , Weronika Gonciarz <sup>4</sup> , Magdalena Szemraj <sup>3</sup> , Maja Ambroziak 2,†, Olga Suska 2,†, Oliwia Turkot 2,†, Małgorzata Stanowska 2,†, Krzysztof P. Rutkowski <sup>1</sup> , Magdalena Chmiela <sup>4</sup> and Wojciech Mielicki 2,5**


**Abstract:** Oregano (*Origanum vulgare* L.) and thyme (*Thymus vulgaris* L.) have long been known for their organoleptic properties. Both plants are widely used in cuisine worldwide in fresh and dried form and as a pharmaceutical raw material. The study aimed to assess if the type of cultivation influenced chosen chemical parameters (total polyphenols by Folin-Ciocalteu method; carotenoids and chlorophyll content by Lichtenthaler method), antimicrobial activity (with chosen reference microbial strains) and shaped cytotoxicity (with L929 mouse fibroblasts cell line) in water macerates of dry oregano and thyme. Polyphenols content and antimicrobial activity were higher in water macerates obtained from conventional cultivation (independently from herb species), unlike the pigments in a higher amount in macerates from organic herbs cultivation. Among all tested macerates stronger antimicrobial properties (effective in inhibiting the growth of *Pseudomonas aeruginosa, Bacillus cereus* and *Salmonella enteritidis*) and higher cytotoxicity (abilities to diminish the growth of L929 fibroblasts cytotoxicity) characterized the conventionally cultivated thyme macerate.

**Keywords:** thyme; oregano; dry herbs; polyphenols; chlorophyll; carotenoids; microbial; cytotoxicity

## **1. Introduction**

Oregano (*Origanum vulgare* L., also known as wild marjoram), and thyme (*Thymus vulgaris* L.) are popular and widely used in cuisines and the cosmetic industry. Oregano and thyme are native species of the Mediterranean region [1,2] although they became popular herbs in the Baltic Sea region as well [3–5]. Due to their chemical composition, both herbs are willingly used in the cuisines and medicine/herbal medicine and cosmetics industries.

The properties of the oregano are connected not only with its flavor and scent, but this plant also supports the digestive system (secretion of gastric juices and bile), respiratory system (helps with catarrh of the upper respiratory tract), has a relaxing and expectorant effect, but also has antifungal and bactericidal properties [1,3]. Some of these pro-health

**Citation:** Sikorska-Zimny, K.; Lisiecki, P.; Gonciarz, W.; Szemraj, M.; Ambroziak, M.; Suska, O.; Turkot, O.; Stanowska, M.; Rutkowski, K.P.; Chmiela, M.; et al. Influence of Agronomic Practice on Total Phenols, Carotenoids, Chlorophylls Content, and Biological Activities in Dry Herbs Water Macerates. *Molecules* **2021**, *26*, 1047. https://doi.org/10.3390/ molecules26041047

Academic Editors: Jan Oszmianski and Sabina Lachowicz Received: 26 January 2021 Accepted: 12 February 2021 Published: 17 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

properties are the result of the antioxidant effect of oregano [6]. This effect is related to the presence of numbers of compounds such as phenolic acids, flavonoids, and essential oils like sabinene, carvacrol, geranial, terpineol, linalool or cymene [1]. The concentration of these compounds is shaped not only by the variety and the environmental conditions of the plant during growth (greater exposure to sun, rain, the season of picking the plant) but also by the type of cultivation (organic, conventional), the geographical position but also by the method of oil extraction [6]. The beneficial antioxidant effect is a derivative of the (variable) composition, and therefore these properties will also be influenced by the factors mentioned. Antimicrobial activity of oregano is connected with compounds like carvacrol, thymol, γ-terpinene, and *p*-cymene [1], Faleiro et al. estimated the concentration as 33% for thymol, 26% for γ-terpinene and 11% for *p*-cymene [7].

Thyme in the cuisine is used as a spice for meat, potato, and mushroom dishes. The healing properties of thyme focus on the respiratory system's effect; hence thyme is used in cough syrups, expectorants, and soothing throat irritations, in the form of ointments, drops used in the case of upper respiratory tract catarrh [8]. Thyme is also used in dental prophylaxis as a toothpaste component and liquids for teeth cleaning and oral care (also in endodontics, during disinfecting root canals) [9]. Thyme, similar to oregano, is used against digestive problems and has antibacterial, antifungal, and antiviral properties [6]. These effects are, inter alia, a derivative of the presence of thymol and carvacrol in thyme. These antioxidant compounds (phenolic derivatives) are present in thyme in large amounts: about 18–80% and 1–20% (for thymol and carvacrol, respectively) [10,11]. Two of these phenolic compounds (carvacrol and thymol) have strong antimicrobial activity against both bacteria and fungi, therefore, are considered to be useful as future fighting agents against drugresistant microbial [12]. Detailed examination pointed on carvacrol with high anti-*Listeria monocytogenes* properties [13], experiments conducted by Rota et al. showed that carvacrol and thymol had a high inhibitory effect against the Gram-positive *L. monocytogenes* and *S. aureus,* the Gram-negative *S. enteritidis, E. coli O157:H7, Yersinia enterocolitica,* and *Shigella flexneri* [14].

Different factors have been demonstrated to influence the content of the biologically active compounds, such as technological process, cultivation method, climatic conditions or variety. Among them technological processes such as drying increase the herbs' availability on the market throughout the year, thereby not tied to climatic and cultivation conditions [15]. It enables transport over long distances, thus increasing the availability of these herbs on the market. Moreover, drying, by limiting the water content, helps to inhibit the growth of microorganisms on the product [16]. It needs to be remembered that the drying process changes the chemical composition of plants [15].

Other factors that influence the content of the biologically active compounds in plants are cultivation method, climatic conditions, variety, therefor content of, e.g., polyphenols may differ in published articles [17].

As mentioned before, oregano and thyme possess antioxidant properties connected with the presence of polyphenols compounds like phenolics: acids, diterpenes; flavonoids and volatile oils [18]. These broad group of secondary metabolites of plants are present in flowers, fruits, leaves, stems, roots, and tubers. The antioxidant properties of polyphenols are connected with the ability to scavenge free radicals, counteract lipid peroxidation, and reduced enzymes' activity [17]. Polyphenols are sensitive for high temperatures; therefore, the drying process reduces their plant tissue content [19]. Consumers' important factors during shopping are the color of dried spices, preferably intensive green (most similar to fresh plant) and less popular discolored in grey shades [20]. The color of a dried green plant is related to the content of chlorophyll and carotenoids. These pigments decompose during the technological procedures of drying herbs; therefore, except for visual evaluation of herbs color, the chemical analysis of chlorophylls and carotenoid content is useful.

The cultivation method influences all the features mentioned above. They compare organic and conventional method points on differentials in the usage of synthetic substances during and before plant growth. Organic cultivation method prohibits the use of synthetic plant protection products and fertilizers, which are substituted with pheromone traps, plant extracts and sticky boards (as a substitute of synthetic plant protection products) or manure (of different origin) and compost (as a substitute of synthetic fertilizers) [21,22]. Organically cultivated herbs are expected to be richer in bioactive compound due to elicitation phenomenon: "this process induces a defense response in a plant and leads to several biochemical processes resulting from which numerous groups of secondary metabolites are produced" [23].

The possibility to use both spices in cuisines is connected with their usage in the water solution. Therefore, important is to conduct an experiment with water macerates (of oregano and thyme) and examine their chemical and biological properties to evaluate the pro-healthy values.

The authors focused on determining the content of chosen compounds in water macerates of oregano and thyme, obtained from organic and conventional cultivation, its cytotoxicity, and the assessment of their impact on microorganisms' development.

#### **2. Results**

#### *2.1. Chemical Composition*

#### 2.1.1. Polyphenols

The highest content of total polyphenols was determined in conventionally cultivated thyme (0.42 mg GAE/mL sample) and the lowest in "organic" oregano (0.20 mg GAE/mL sample). Total polyphenols content in water macerates for both herbs were higher in conventional cultivations of oregano and thyme; significant differences were obtained in "organic" oregano comparing to other samples. There were no significant differences among the thyme samples (Table 1).

**Table 1.** The chosen chemical compounds are determined in water macerates of dry oregano and thyme cultivated in conventional (CONV) and organically (ORG).


According to Duncan's test, the means in columns followed by the same letters are not significantly different at *p* = 0.05.

#### 2.1.2. Chlorophyll and Carotenoids

Oregano had a higher content of chlorophyll (a and b) than thyme. The highest chlorophyll a and b content was determined in "organic" oregano (7.81 µg/mL and 12.23 µg/mL of macerates, for chlorophyll a and b, respectively), the lowest in "conventional" thyme (2.96 µg/mL and 4.56 µg/mL of macerates, for chlorophyll a and b, respectively). Chlorophylls (a and b) were significantly higher in organically cultivated herbs (for both species).

Carotenoids were in higher amount in macerates of organically cultivated herbs (2.07 and 1.67 µg/mL for oregano and thyme, respectively) than a conventional one (1.03 and 0.76 µg/mL for oregano and thyme, respectively).

The determined results (higher content of pigments in organic herbs) were not noticeable during the preparation (Figures 1 and 2) of macerates, where colors of organic and conventional cultivated, dry plants were similar.

and conventional cultivated, dry plants were similar.

and conventional cultivated, dry plants were similar.

*Molecules* **2021**, *26*, x 4 of 14

The determined results (higher content of pigments in organic herbs) were not noticeable during the preparation (Figures 1 and 2) of macerates, where colors of organic

The determined results (higher content of pigments in organic herbs) were not noticeable during the preparation (Figures 1 and 2) of macerates, where colors of organic

**Figure 1.** Comparison of colors of dry oregano from organic and conventional cultivation. **Figure 1.** Comparison of colors of dry oregano from organic and conventional cultivation. **Figure 1.** Comparison of colors of dry oregano from organic and conventional cultivation.

**Figure 2.** Comparison of colors of dry thyme from organic and conventional cultivation. **Figure 2.** Comparison of colors of dry thyme from organic and conventional cultivation. **Figure 2.** Comparison of colors of dry thyme from organic and conventional cultivation.

#### *2.2. Antimicrobial Activity 2.2. Antimicrobial Activity 2.2. Antimicrobial Activity*

The effects of the water macerates of both herbs on tested standard microbial are presented in Table 2. Four strains of Gram-negative bacteria and five strains of Grampositive bacteria were used in the research. The experiment was repeated triplicate, and the results were consistent. The effects of the water macerates of both herbs on tested standard microbial are presented in Table 2. Four strains of Gram-negative bacteria and five strains of Grampositive bacteria were used in the research. The experiment was repeated triplicate, and the results were consistent. The effects of the water macerates of both herbs on tested standard microbial are presented in Table 2. Four strains of Gram-negative bacteria and five strains of Grampositive bacteria were used in the research. The experiment was repeated triplicate, and the results were consistent.

The macerates of both herbs obtained from organic cultivation did not exhibit antibacterial activity against standard strains of Gram-negative bacteria MIC/MBC > 50 mg/mL (Table 2). Oregano macerate obtained from conventional cultivation exhibited weak activity against *E. coli* and *Salmonella* Enteritidis (MIC/MBC = 50 mg/mL) and moderate activity against *P. aeruginosa* with MIC/MBC= 12.5 mg/mL. Thyme macerate received from conventional cultivation had been effective in inhibiting the growth of *S*. Enteritidis (MIC/MBC = 12.5 mg/mL). The highest sensitivity to the conventional thyme macerate showed the tested strains of *P. aeruginosa* (MIC/MBC = 0.626 mg/mL). Both macerates obtained from organic herbs were less active in inhibiting the growth The macerates of both herbs obtained from organic cultivation did not exhibit antibacterial activity against standard strains of Gram-negative bacteria MIC/MBC > 50 mg/mL (Table 2). Oregano macerate obtained from conventional cultivation exhibited weak activity against *E. coli* and *Salmonella* Enteritidis (MIC/MBC = 50 mg/mL) and moderate activity against *P. aeruginosa* with MIC/MBC= 12.5 mg/mL. Thyme macerate received from conventional cultivation had been effective in inhibiting the growth of *S*. Enteritidis (MIC/MBC = 12.5 mg/mL). The highest sensitivity to the conventional thyme macerate showed the tested strains of *P. aeruginosa* (MIC/MBC = 0.626 mg/mL). The macerates of both herbs obtained from organic cultivation did not exhibit antibacterial activity against standard strains of Gram-negative bacteria MIC/MBC > 50 mg/mL (Table 2). Oregano macerate obtained from conventional cultivation exhibited weak activity against *E. coli* and *Salmonella* Enteritidis (MIC/MBC = 50 mg/mL) and moderate activity against *P. aeruginosa* with MIC/MBC= 12.5 mg/mL. Thyme macerate received from conventional cultivation had been effective in inhibiting the growth of *S. enteritidis* (MIC/MBC = 12.5 mg/mL). The highest sensitivity to the conventional thyme macerate showed the tested strains of *P. aeruginosa* (MIC/MBC = 0.626 mg/mL).

of all the tested strains of Gram-positive bacteria, excluding *L. monocytogenes*, compare to

Both macerates obtained from organic herbs were less active in inhibiting the growth of all the tested strains of Gram-positive bacteria, excluding *L. monocytogenes*, compare to


**Table 2.** Determined values of MIC and MBC for tested water macerates in relation to standard microorganisms.

\* Gentamicin—broad-spectrum antibiotic, fluconazole—antifungal chemotherapeutic; ˆ not detection; nt—the activity of fluconazole (against bacteria) and gentamicin (against fungi) have not been tested.

> Both macerates obtained from organic herbs were less active in inhibiting the growth of all the tested strains of Gram-positive bacteria, excluding *L. monocytogenes*, compare to conventional macerates (Table 2). The bactericidal effect of thyme conventional macerate against *S. aureus* was achieved at the concentration of 12.5 mg/mL, against *E. faecalis* and *B. subtilis* at 25 mg/mL. Thyme organic macerate was active against *B. subtilis* at concentration 25 mg/mL and did not inhibit the growth of *S. aureus* and *E. faecalis* (MIC/MBC > 50 mg/mL). The thyme and oregano macerates obtained from herbs cultivation in organic and conventional condition demonstrated weak activity against *L. monocytogenes* with MIC/MBC values, 50 mg/mL, and 25 mg/mL, respectively.

> The "organic" oregano macerate was not active against *E. faecalis*(MIC/MBC > 50 mg/mL), but oregano conventional macerate was effective at concentration 25 mg/mL. The highest inhibitory effect on the tested strains of *S. aureus* and *B. subtilis* showed the oregano conventional macerate, resulting in MIC/MBC 3.125 mg/mL and 6.25 mg/mL, respectively (Table 2). The oregano organic macerate demonstrated activity against *S. aureus* and *B. subtilis* in higher concentrations with MIC/MBC 6.25 mg/mL and 25 mg/mL, respectively.

> The highest sensitivity of conventional oregano and thyme macerates demonstrated Bacillus cereus' strain, resulting in MIC/MBC 3.125 mg/mL and 6.25 mg/mL, respectively. The thyme and oregano organic macerates showed the same effect at twofold higher concentrations, 6.25 mg/mL, and 12.5 mg/mL, respectively.

> The macerates' antifungal activity was tested against two fungal strains—*C. albicans* (yeast fungus) and *A. brasiliensis* (mould fungus). None of the tested macerates and those obtained from herbs cultivation in organic and conventional conditions did not inhibit the fungal strains' growth ((MIC/MBC > 50 mg/mL).

> The tested macerates' antibacterial activity was lower than the activity of gentamicin or fluconazole used as the antimicrobial reference standard.

> The "conventional" thyme in the highest concentration of 50; 25 mg/mL and 12.5 significantly diminished the growth of L929 fibroblasts (43–71% of dead cells) (Figure 3), while thyme "organic" did not show cytotoxic activity at any of the tested concentrations (Figure 3). Moreover, "organic" thyme significantly increased the ability of L929 cells

*Molecules* **2021**, *26*, x 6 of 14

to reduce MTT within the range 5–50 mg/mL compare to extract isolated from thyme "conventional" (Figure 3). fibroblasts (44%–47% of dead cells) (Figure 3). Furthermore, oregano "organic" significantly increased the ability of L929 cells to reduce MTT within the range 50–2.5 mg/mL compare to extract isolated from oregano "conventional" (Figure 3).

in the highest concentration 50 and 25 mg/mL significantly diminished the growth of L929

**Figure 3.** Cytotoxic effect of plants extracts: (**A**) thyme, (**B**) oregano towards L929 cells. The cytotoxicity was assessed by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] reduction assay. The cell viability was calculated for four experiments, including three repeats for each compound. Complete RMPI-1640 medium (cRPMI) was used as a positive control (PC) of cell viability (100% viable cells) and 0.03% H2O2 as a negative control (NC) of cell viability (100% dead inactive cells). Statistical significance: \* *p* < 0.05; \*untreated cells vs cells treated with tested plants extracts "conventional" extract (light grey bars) vs "organic" extract (dark grey bars). **Figure 3.** Cytotoxic effect of plants extracts: (**A**) thyme, (**B**) oregano towards L929 cells. The cytotoxicity was assessed by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] reduction assay. The cell viability was calculated for four experiments, including three repeats for each compound. Complete RMPI-1640 medium (cRPMI) was used as a positive control (PC) of cell viability (100% viable cells) and 0.03% H2O<sup>2</sup> as a negative control (NC) of cell viability (100% dead inactive cells). Statistical significance: \* *p* < 0.05; \* untreated cells vs cells treated with tested plants extracts "conventional" extract (light grey bars) vs "organic" extract (dark grey bars).

Oregano "conventional" has a stronger inhibitory effect on L929 cell metabolic activity at range concentrations 10–50 mg/mL (40–60% of dead cells), while thyme "organic" in the highest concentration 50 and 25 mg/mL significantly diminished the growth of L929 fibroblasts (44–47% of dead cells) (Figure 3). Furthermore, oregano "organic" significantly increased the ability of L929 cells to reduce MTT within the range 50–2.5 mg/mL compare to extract isolated from oregano "conventional" (Figure 3).

#### **3. Discussion**

According to the authors' best knowledge, there was no conducted analysis of organic and conventional cultivated oregano and thyme water macerates (not infusions). Available in literature are results over chemical analysis of herbs infusion dedicated as beverages (melissa, mint) or macerates based on other solution (methanol, ethanol), mostly used as a solvent in the method of extraction or infusions (pouring with hot water).

In conducted analysis higher polyphenol content was determined in thyme than in oregano that stands according to values obtained by Dragland et al. which determined 45 mmol/100 g and 63.7 mmol/100 g for oregano and thyme respectively in commercial cultivation [24]. Moreover, Vallverdú-Queralt et al. determined total polyphenols in dried oregano and thyme at the level of 2.23 and 3.36 mg GAE/g DW for oregano and thyme, respectively [25]. Jałoszy ´nski determined polyphenols in dry oregano at the level: 63.96–168.87 mg GAE/100 g, where the lower content is similar to obtained in presented work (calculate on DM herbs) [19]. Taghipour et al. determined thyme's polyphenols at a level of 30 mg GAE/g DM that is similar to values obtained in presented work [26]. It also needs to be highlighted that phenolic losses during drying of herbs can reach 50% (dependently of temperature and drying method). Generally, the compound degrades at a higher level with higher temperature applied [19].

Although determined higher polyphenols content in macerates for conventional cultivation stands against the work of Matłok et al., who determined the much higher bioactive potential of dry oregano cultivated with organic fertilizers comparing to conventional cultivation [27]. However, Lv et al. compared peppermint and cinnamon cultivated organically and conventionally and determined in both herbs higher total phenolic content in conventionally cultivated herbs [28].

Still, there is no clear explanation of higher polyphenol content in a specific type of plant cultivation (sometimes higher in organic, sometimes in conventional). Some authors divide polyphenols (into soluble and hydrolysable), where soluble are more stable during processing (cooking) [29], although there are still no data available about polyphenols profiles in dry herbs, together with water macerates. Moreover, the water content of fresh plant might decrease polyphenols content, since the drying process of high-water content must undergo a more extended drying time procedure, or higher temperature must be applied. In any case, both factors are reducing polyphenols content in the final product.

Chlorophylls play an important role in the photosynthetic membranes, where they are present usually in a ratio of 3:1 [30], although this content can be shaped by the growth conditions and environmental factors [31,32]. Chlorophyll a and b differ in a side chain's composition, which for chlorophyll a its methyl group and chlorophyll b its aldehyde group.

Matłok et al. pointed on a strict correlation between chlorophylls content and carotenoids in foliar plants due to general biosynthetic pathways in the plants' chloroplasts [27]. Therefore, the higher content of pigments involved in photosynthesis is related to higher carotenoid content (beta carotene) [21].

In the experiment over water macerates of herbs from organic and conventional cultivation, higher content of chlorophylls and carotenoids were determined in organic cultivation, which is opposite to Hallmann et al. [21]. Although Onofrei et al. pointed on a higher content of pigments presents in organically cultivated thyme and oregano [33]. It needs to be added that Hallmann et al. refer to lower content only of beta-carotene, and in the conducted experiment, the authors were determining the total carotenoids

content [21]. Simultaneously Halmann et al. [21] pointed to the fact of a higher content of lutein and zeaxanthin (belonging to carotenoids) in organically cultivated herbs comparing to conventional one and in case of thyme major carotenoid is zeaxanthin [34].

Onofrei et al. explained the higher content of pigments in herbs cultivated with organic fertilizers by the possible improvement in the availability of nutrients caused by these fertilizers, and thus enhancing the metabolic pathways (e.g., photosynthesis) synthesis of several plant secondary compounds [33]. According to Skubij and Dzida, over the influence of organic fertilizers on the plants' chemical compounds [35]. The other factor influencing carotenoid content is UV radiation that also increases antioxidant content [21,27].

Manukyan determined higher content of chlorophyll a and b in thyme at a level of 0.03–0.052 mg/g and 0.13–0.21 mg/g, (for chlorophyll a and b, respectively) [34]. Tzima et al. obtained the lower results: 88.6 mg/100 g DM for chlorophyll a, and 24.00 mg/100 g DM for chlorophyll b [36].

Kulbat-Warycha et al. determined similar to presented in work content of chlorophyll a in oregano but lower chlorophyll b and higher of total carotenoids (0.009 g/L; 0.002 g/L and 0.004 g/L FW for chlorophyll a, b, and carotenoids, respectively) [37].

Antimicrobial activity of water macerates was higher in conventional cultivated herbs. The obtained results go along with higher polyphenols content determined in these macerates. The highest antimicrobial activity (of "conventional" thyme) was determined against *Bacillus cereus*, which is especially important in dry herbs. These products may have a high number of bacteria and molds, which in favorable conditions, can quickly develop [38]. Research of Fogele et al. and Boer et al. pointed on a high level of *Bacillus cereus* determined in dry spices [38,39]. Boer et al. determined antimicrobial activity of 2% water macerates and pointed of weak or non-antimicrobial abilities of most of examined herbs solutions (rosemary, basil, ginger, sage) [38]. Other investigation showed that 20% cold water oregano extract was effective against the strains of *P. aeruginasa* and *K. pneumoniae* [40]. Our study demonstrated the highest antibacterial effect of thyme water macerate obtained from conventional cultivation against the strains of *P. aeruginosa*. This opportunistic human pathogen was found to contaminate herbal plants and spices caused by the unsafe collection, drying, preparation or storage [41].

Quadir et al. examined antimicrobial activity of chosen herbal extracts, but they have not confirmed any activity of thyme extract against *B. subtilis*, explanation to this fact may be connected with used solution concentrate (1:10) [42]. Our investigation shows that "conventional" oregano macerate exhibited moderate activity against *B. subtilis*.

Our study demonstrated that water macerates of oregano obtained from the conventional method of cultivation had been effective in inhibiting the growth of *S. aureus*. Other studies also pointed to a high activity oregano water extract against this microorganism [43]. *S. aureus* is most commonly isolated from dry herbs [41].

Research is connecting herbs' antimicrobial ability with the presence of phenols that can precipitate proteins (or react with cells by sulfhydryl groups of proteins causing unavailability of the substrate) and inhibit microorganisms' enzymes [44,45].

Plant extracts including thyme and oregano have been used in the traditional medicine for the treatment of several respiratory diseases like asthma and bronchitis [46] as well as other pathologic processes, thanks to several properties such as antiseptic, antispasmodic, antitussive, antimicrobial, antifungal, antioxidative, and antiviral [47,48]. Our study showed that extract isolated from thyme "organic" does not show cytotoxic activity according to ISO 10993-5 in all concentrations used in this study (1–50 mg/mL) whereas the activity of extract isolated from thyme "conventional" was below the cytotoxicity norm in the range 12.5–50 mg/mL. Similar results were observed in the study by de Oliveira et al. [49]. They showed that the *T. vulgaris* extract at 25, 50 and 100 mg/mL provided cell viability above 50% to RAW 264.7, FMM-1, MCF−7 and HeLa cell line [49]. New plant extracts are also tested for antioxidant activity in conjunction with the modulation of the inflammatory response. For example, Loizzo et al. examined different oregano's oils varieties inhibition of NO production in the murine monocytic macrophage cell line

RAW 264.7, obtaining an IC50 value of 66.4 µg/mL and >200 µg/mL for *O. ehrenbergii* and *O. syriacum*, respectively [50]. The antioxidant activity of our preparations with the use of gastric epithelial cells is planned to be tested in the context of minimizing the risk of epithelial damage and the development of an inflammatory reaction. Plant preparations with a cytotoxic effect against cancer cells are extremely valuable. Jamali et al. show that thymol induced toxicity, apoptosis, and cell cycle arrest in MDA-MB231 BC cells [51]. Our study shows that oregano extracts have strong cytotoxic activity against L929 fibroblasts. Oregano extracts: ethyl acetate and ethanol extracts from leaves also have reported the cytotoxicity activity against human breast cancer cells (MCF7) [52]. On the other hand, carvacrol, the major component of oregano, showed antimutagenic activity, which seems to be mainly linked to the induction of mitochondrial dysfunction [53,54].

#### **4. Materials and Methods**

#### *4.1. Macerates Preparation*

Dried oregano and thyme were bought in a local shop. Both types of herbs (organic and conventional cultivated) were obtained from the same manufacturer. Thyme and oregano (conventional and organic, separately) were soaked with distilled water (1:5) and left for five days for the maceration process. The extracts were filtered through a cellulose filter (fine pore, 0.45 µm) and then subjected to further analysis.

#### *4.2. Chemical Analysis*

#### 4.2.1. The Content of Polyphenols

The Folin-Ciocalteu assay was carried out according to the method described by Singleton and Rossi (1965) [55]. The results were expressed as mg/mL of the sample as gallic acid. Measurements were conducted in six replicates; absorbance was measured at 750 nm against the blank sample.

#### 4.2.2. The Content of Chlorophylls and Carotenoids

Carotenoids and chlorophyll a and b were determined according to Lichtenthaler (1983) spectrophotometric method, with the extraction of 80% acetone and absorbance measured at 470, 646, 663 nm [30].

#### *4.3. Microbial Analysis*

#### 4.3.1. Microbial Strains and Culture Conditions

Reference microbial strains were obtained from the American Type Culture Collection (ATCC), including *Staphylococcus aureus* ATCC 25923, *Enterococcus faecalis* ATCC 29212, *Bacillus subtilis* ATCC 6635, *Escherichia coli* ATCC 25922, *Pseudomonas aeruginosa* ATCC 27833 and *Shigella flexneri* ATCC 12022. The *Listeria monocytogenes* PCM 2191 and *Bacillus cereus* PCM 1948 strains were taken from the Polish Collection of Microorganisms (PCM). One bacterial strain *Salmonella* Enteritidis ZMF 279 was derived from the collection of the Department of Pharmaceutical Microbiology and Diagnostic Microbiology, Medical University of Lodz. Two fungal strains were also used: *Candida albicans* ATCC 10231 and *Aspergillus brasiliensis* ATCC 16404. All tested microorganisms were stored at –80 ◦C in 15% glycerol stocks. Before the investigation, the bacterial strains were transferred to Mueller-Hinton agar medium (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA) and cultured overnight at 37 ◦C. Fungal strains were transferred on Sabouraud agar medium (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA) and cultured for two days at 30 ◦C.

#### 4.3.2. Antimicrobial Assay

Before the investigation, the extracts were concentrated to 100 mg/mL by lyophilization. The antimicrobial activity of extracts was assessed according to their minimum inhibitory concentrations (MIC), and minimum bactericidal/fungicidal concentrations (MBC) expressed in mg/mL. According to the European Committee on Antimicrobial Susceptibility recommendations, antibacterial and antifungal activities were determined

using the broth microdilution method [56]. The Mueller-Hinton broth (pH~7.2) (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA) was used for bacteria. Liquid medium RPMI-1640 (w/o red phenol, pH~7.2) (Sigma-Aldrich, Darmstad, Germany) was used for the fungal strains. Two-fold series dilutions of extract in the growth medium were performed in 96-well sterile microtiter plates (Kartell Labware, Noviglio, Italy) in concentrations ranging from 50 to 0.09 mg/mL. The MIC values were defined as the lowest extract concentrations with no bacterial growth after the incubation. The MBCs were determined by seeding 5 µL from all clear MIC wells onto Mueller-Hinton agar plates (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA) (bacterial strains) or Sabouraud agar medium (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA) (fungal strains). MBC was defined as the lowest concentration that killed 99.9% of the final inocula after 24 h incubation at 37 ◦C (bacterial strains) or 48 h at 30 ◦C (fungal strains). The antimicrobial tests were performed in triplicate. Gentamicin and fluconazole were used as an antimicrobial reference standard.

#### *4.4. Cytotoxicity Studies*

#### 4.4.1. In Vitro Cell Culture

According to the ISO (International Organization for Standardization, 2009) norm 10993-5 (Biological evaluation of medical devices—Part 5: Tests for in vitro cytotoxicity), testing of cytotoxicity was performed using L929 mouse fibroblasts (LGC Standards, Middlesex, UK). The cells were maintained under standard conditions (37 ◦C, 5% CO2) in compelled culture medium (cRPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics: 100 U/mL penicillin and 100 µg/mL streptomycin) Sigma-Aldrich (Darmstad, Germany) as previously described [57]. Before being used in the cytotoxicity assay, the cells' viability was assessed by excluding trypan blue dye and was in the range of 93–95%.

#### 4.4.2. Measurements of Cellular Metabolic Activity and Global Growth Inhibition

The metabolic activity of the L929 cells was tested after application of water extracts obtained from dried plants. Cells in culture medium were seeded in 96-well plates (2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well) for 24 h at 37 ◦C, 5% CO2. The tested extracts were diluted in cRPMI-1640 medium in concentrations of 50; 25; 12.5; 10; 5; 2.5 and 1 mg/mL, added to the cells (100 µL/well), and incubated under standard conditions for 24 h. Following incubation, the cell monolayers were carefully screened using light microscopy, as recommended by ISO norm 10993-5, to evaluate cell morphology. Cell metabolic activity was estimated by measuring the ability of cells to reduce MTT [(3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide)], which is one of the tests recommended by the Food and Drug Administration (FDA) and ISO as previously described [57].

#### *4.5. Statistical Analysis*

Statistical analysis over chemical parameters was performed with Duncan's test (*p* = 0.05). Obtained results are presented results are shown as the mean value of six replications. The statistical significance of the cytotoxicity results was determined by Kruskal-Wallis test (*p* < 0.05). Data are presented as mean values ± SD. For statistical analysis, the STATISTICA 12 and 13 PL software was used (Stat Soft, Kraków, Poland).

#### **5. Conclusions**

Many commonly used antibiotics are becoming useless due to the increasing resistance of pathogens. For this reason, there is an urgent need to search for new antimicrobial, antiinflammatory and pro-regenerative drugs with high biocompatibility and targeted activity. Plant biocomponents with well-characterized properties and good biocompatibility are good candidates for developing new drugs, medicinal food, or dietary supplements. A significant impact on such formulations expected therapeutic value is the plant's breeding conditions, limiting the content of potentially toxic compounds, or determining an active biological substance's content.

Water macerates of oregano and thyme are a valuable solution. Dependently, they can be a good source of polyphenols and have good antimicrobial abilities (conventional) and are rich in pigments (organic). It is worth pointing that "conventional" thyme macerate exhibited strong activity against the strains of *P. aeruginosa*. This opportunistic pathogen alleged to cause gastroenteritis in humans if ingested in large numbers. Can be isolated from soil and water and is commonly associated with spoilage of food such as eggs, cured meats, fish, and milk and cosmetic.

Thyme "conventional" macerates diminished the growth of L929 fibroblasts cytotoxicity, and "organic" oregano increased the ability of L929 cells to reduce MTT. Cultivation type had shaped influence on the ability of L929 cells to reduce MTT—both organic herbs macerates had significant higher abilities to increase this property; however, conventional cultivated herbs (water macerates) had higher antimicrobial activity and higher polyphenols content. Extracts, isolated from ecological plant cultures, may be used in higher concentrations in formulations for medical use due to their lower cytotoxic activity. This allows achieving a higher concentration of biologically active substances while maintaining biological safety, and faster achievement of the therapeutic and cosmetic effect.

**Author Contributions:** Conceptualization: K.S.-Z.; methodology: K.S.-Z., P.L., W.G., M.C., M.S. (Magdalena Szemraj); validation: K.S.-Z., W.G.; chemical analysis: K.S.-Z., M.A., O.S., O.T., M.S. (Małgorzata Stanowska); microbial analysis and description: P.L., M.S. (Magdalena Szemraj); cytotoxicity analysis and description: W.G., M.C.; writing—original draft preparation: K.S.-Z.; review and editing: W.G., P.L., M.C., K.P.R., W.M.; supervision: K.S.-Z.; funding acquisition: K.S.-Z., K.P.R., W.M., P.L., M.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** Microbial analysis were partially supported financially by the Department of Pharmaceutical Microbiology and Microbiological Diagnostics (503/3- 012-03/503-31-001), Faculty of Pharmacy, Medical University of Lodz, Poland.

**Institutional Review Board Statement:** Faculty of Biology and Environmental Protection University of Lodz, Poland, fulfills the statutory conditions for conducting research on cell lines in accordance with the European Union low.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

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

**Sample Availability:** Not available.

#### **References**


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