#### **1. Introduction**

Sweet potato (*Ipomoea batatas*), an important root tuber crop, provides food and energy for people, especially in Asia and Africa [1]. Sweet potato contains 15–30% and 46–68% starch in its wet and dry root tuber, respectively, among different varieties, and has become the first choice for producing starch due to its short growth cycle, strong environment adaptability, low planting cost, and high yield in the world, especially in developing countries [2,3]. Starch isolated from the sweet potato root tuber has been used to produce noodles and vermicelli. In addition, it is also a good thickening agent in cooking foods and an important raw material in producing syrup, film, lactic acid, ethanol, and other chemicals [4,5]. The applications of starch are influenced by its physicochemical properties including size, components, crystalline structure, and thermal properties [3,6–9].

Sweet potato is planted widely in the world, and has hundreds of varieties or lines due to artificial selection, natural hybrids, and mutations [1,4]. The root tuber of sweet potato has different colored skin and flesh, but starch properties have no significant correlation with skin and flesh color, and are determined by the genotypes of varieties [2,10,11].

**Citation:** Li, Y.; Zhao, L.; Shi, L.; Lin, L.; Cao, Q.; Wei, C. Sizes, Components, Crystalline Structure, and Thermal Properties of Starches from Sweet Potato Varieties Originating from Different Countries. *Molecules* **2022**, *27*, 1905. https:// doi.org/10.3390/molecules27061905

Academic Editor: Mirella Nardini

Received: 2 March 2022 Accepted: 14 March 2022 Published: 15 March 2022

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**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/).

Starches from different sweet potato varieties have been studied for their structures, functional properties, and applications [10,12–14]. For example, Collado et al. [12] investigated the genetic variation in physical properties of sweet potato starches from 44 genotypes adapted to Philippine conditions, and found that wide variation and distinctly different pasting properties exist among genotypes. Zhu and Xie [14] studied the swelling power, water solubility, rheological properties, gelatinization, and retrogradation of starches from seven New Zealand sweet potatoes, and concluded that the starch properties from different varieties exhibit significant diversity due to their different internal unit chain parameters of amylopectin. Kim et al. [13] compared the physicochemical properties of starches from eight Korean sweet potato varieties including purple-, orange-, and white/cream-fleshed tubers. The starches exhibit polygonal and semi-oval shapes with different granule sizes among different varieties, and have A- and CB-type X-ray diffraction patterns. The pasting properties, amylose contents and water binding capacities of starches are closely related to the chain length distribution of amylopectin, and have no relationship with tuber color. Similar results are also reported in starches from eight Chinese sweet potato varieties with light yellow-, orange-, and purple-fleshed tubers. The molecular structure of amylose and amylopectin exhibits differences among different varieties, and is the main influencing factor in determining starch physicochemical properties [10]. Though the above references have reported the differences in starches from different varieties, the varieties originate from the same country or region in the same report. At present, no research reports the starch properties of sweet potato varieties originating from different countries. The growing environment and field management significantly influence the development and properties of starch in plant resources [15–18]. In addition, the measuring and analysis methods of starch properties also affect the property parameters. Therefore, it is necessary to study the characteristics of starches from sweet potato varieties originating from different countries but planted in the same growing conditions.

In this research, 44 sweet potato varieties originating from 15 countries were planted in the same conditions, and their starches were isolated and investigated for granule size, iodine absorption, amylose content, crystalline structure, and thermal properties. The hierarchical cluster analysis was carried out to reveal the differences in sweet potato varieties based on the starch property parameters. The objective of this study was to evaluate the germplasm resources of sweet potato starch. This research offers some references for the utilization of sweet potato germplasm.

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

#### *2.1. Granule Morphology and Size Distribution of Starch*

The granule morphology and size are important properties of starch, affecting functional properties and applications of starch [9]. The morphologies of isolated starches were observed under normal and polarized light (Figure 1). Starch granules had round, polygonal, oval, and semi-oval shapes, and contained small and large granules with typical "Maltese crosses" having the hila in the center of the granules. No significant differences in starch morphology were observed among all sweet potato varieties. Similar morphology has also been reported in sweet potato starch [19,20]. However, the granule size distributions exhibited significant differences among some sweet potato starches (Table 1). The surface- (D[3,2]) and volume-weighted mean diameter (D[4,3]) are usually used to indicate the size of starch, and ranged from 3.866 to 7.681 µm and from 8.013 to 15.296 µm among 44 sweet potato starches, respectively. The starch size in this study agreed with the previous report of sweet potato starch [2,19,20]. The starch size is influenced by plant source, variety genotype, plant physiology, and growing environment [9,21,22].

**Figure 1.** *Cont*.

**Figure 1.** Morphologies of starch granules under normal light and polarized light. Scale bar = 20 μm. **Figure 1.** Morphologies of starch granules under normal light and polarized light. Scale bar = 20 µm.

**Table 1.** Granule size distribution of starches from 44 sweet potato varieties.

 Granule size distribution of starches from 44 sweet potato varieties.

The d(0.2), d(0.5), and d(0.8) are the granule size at which 20%, 50%, and 80% of all the granules by volume are smaller. The D[3,2] and D[4,3] are the surface- and volume-weighted mean diameter, respectively. Sig.: the significance of normal distribution of the 44 samples by Shapiro–Wilk test. Data are means ± standard deviations, *n* = 3. The values with different letters in the same column are significantly different (*p* < 0.05).

#### *2.2. Iodine Absorption and Amylose Content of Starch*

Starch contains amylose and amylopectin. Both amylose and amylopectin influence the absorption of starch and iodine [23]. In this research, the iodine absorption spectra of 44 starches were analyzed (data not shown), and the iodine absorption parameters including the value of optical density (OD) at 550 nm (OD550), 620 nm (OD620), and 680 nm (OD680) were measured. The OD680 is usually defined as blue value (BV) of starch, and displays the iodine binding capacity of starch [24]. The ratio of OD620 to OD550

(OD620/550) can reflect the relative proportion of long chains in starch [23]. The OD680 and OD620/550 ranged from 0.259 to 0.382 and from 1.142 to 1.237 among 44 starches, respectively (Table 2), indicating that amylose and amylopectin were different among these starches.

**Table 2.** Iodine absorption parameters and amylose contents of starches.

AAC: apparent amylose content; TAC: true amylose content; ∆AC: the difference between AAC and TAC (AAC–TAC); Sig.: the significance of normal distribution of the 44 samples by Shapiro–Wilk test. Data are means ± standard deviations, *n* = 3. The values with different letters in the same column are significantly different (*p* < 0.05).

Amylose content is the important structure parameter, determining the applications of starch [25,26]. The amylose content is usually determined with the iodine colorimetry method according to the OD620 of starch–iodine complex. However, the iodine colorimetry usually overestimates the amylose content of starch because of the branch-chains of amylopectin that can bind the iodine. Therefore, the amylose content determined by iodine colorimetry is called the apparent amylose content (AAC) [23]. In this study, the AAC varied from 19.2% to 29.2% (Table 2), and is in the range of sweet potatoes reported by previous references [14,19,27]. The concanavalin A (Con A) precipitate method can accurately determine the amylose content to avoid the overestimation of amylose content. Using this method, starch is completely dissolved into amylose and amylopectin, and separated into two aliquots. The Con A specifically binds and precipitates the amylopectin in an aliquot, and the remaining amylose in the supernatant is hydrolyzed by amylolytic enzymes to glucose, which is measured with glucose oxidase/peroxidase reagent for determining the quality of amylose. The amylose and amylopectin in another aliquot are similarly hydrolyzed to glucose for measuring their quality. The amylose content is the quality percentage of amylose to both amylose and amylopectin. The measured amylose content is not influenced by the purity and moisture of starch, and is usually defined as true amylose content (TAC) [23]. The TAC ranged from 14.2% to 20.2% among 44 sweet potato starches (Table 2). The TAC was significantly lower than AAC in all starches, and the ∆AC (AAC– TAC) was significantly different among different varieties from 4.0% to 11.8%, indicating that varieties with different genotype backgrounds had significantly different amylopectin structure with different iodine binding capacity. The amylose is biosynthesized simply by granule-bound starch synthase I (GBSSI) encoded by the *Waxy* (*Wx*) gene in plant storage tissue, and amylopectin is biosynthesized complexly by soluble starch synthases (SSSs), starch branching enzymes (SBEs), and starch debranching enzymes (DBEs) [28]. There are many *Wx* alleles responsible for amylose synthesis from low to high in different varieties of the same species [29]. The SSSs, SBEs, and DBEs have many isoforms, and their quality and activities determine the amylopectin structure [30]. In this research, sweet potato varieties originate from different countries and areas. Their different genotype backgrounds with different qualities and activities of GBSSI, SSSs, SBEs, and DBEs led to different amylose contents and amylopectin structures among 44 sweet potato varieties.

The hierarchical cluster analysis based on OD680, OD620/550, AAC, and TAC was carried out to investigate the differences and similarities between sweet potato varieties (Figure 2). The 44 sweet potato varieties were divided into cluster 1 (C1) and cluster 2 (C2). The C2 contained 16 sweet potato varieties with OD680 from 0.259 to 0.319, OD620/550 from 1.167 to 1.237, AAC from 19.2% to 25.3%, and TAC from 15.0% to 20.2%. The C1 was further divided into two subgroups of C1A and C1B. The C1A contained eight sweet potato varieties with OD680 from 0.303 to 0.382, OD620/550 from 1.146 to 1.186, AAC from 25.0% to 29.2%, and TAC from 14.2% to 17.7%, and the C1B contained 20 varieties with OD680 from 0.284 to 0.326, OD620/550 from 1.142 to 1.222, AAC from 21.8% to 28.3%, and TAC from 14.5% to 19.7% (Figure 2; Table 2). The ∆AC ranged from 4.0% (PE04) to 6.6% (US02) in C2, from 6.8% (US06) to 9.6% (NG01) in C1B, and from 10.1% (PH01) to 11.8% (JP06) in C1A (Table 2), indicating that amylopectin structure plays an important role in influencing starch components and iodine absorption. Though some varieties originated from the same area, their genotypes exhibited significant differences. For example, for 15 varieties (JP01-JP15) originating from Japan, there were three, six, and six varieties in C1A, C1B and C2, respectively.

**Figure 2.** Hierarchical cluster analysis of 44 sweet potato varieties based on OD680, OD620/550, AAC, and TAC in Table 2. **Figure 2.** Hierarchical cluster analysis of 44 sweet potato varieties based on OD680, OD620/550, AAC, and TAC in Table 2.

#### *2.3. Crystalline Structure of Starch 2.3. Crystalline Structure of Starch*

The branch-chains of amylopectin can form A- and B-type crystallinity, which can be detected by X-ray diffraction (XRD). The XRD patterns of 44 starches are presented in Figure 3. Starches from plant tissues have A-, B-, and C-type. A- and B-type starch contains only A- and B-type crystallinity, respectively, but C-type starch simultaneously contains both A- and B-type crystallinities. According to the ratio of A- to B-type crystallinities from high to low, C-type starch is usually further classified into CA-, CC-, and CB-type. The CC-type starch has diffraction peaks at 2θ 5.6°, 15°, 17°, and 23°; CA-type starch exhibits significant shoulder peak at 2θ 18°, a characteristic peak of A-type crystallinity; and CBtype starch has significant shoulder peaks at 2θ 22° and 24°, two characteristic peaks of Btype crystallinity [7,31]. In this study, 44 sweet potato varieties had A-, CA-, CC-, and CBtype starches (Figure 3). The JP14 starch was A-type, the PE06 starch was CB-type, the AO01, CB01, CN02, JP01, JP06, JP12, JP13, JP15, ML01, PE01, PE04, PH01, PH02, TH01, TZ01, US03, US06, and US07 starches were CA-type, and the other starches were CC-type (Figure 3). The A-, CA-, CC-, and CB-type starches have been reported in sweet potato varieties [10,13,14,19], but the B-type starch has not been reported in references. Genkina et al. [16,32] reported that the soil temperature determines the crystallinity of sweet potato starch. The root tuber synthesizes A-type starch in soil temperature above 33 °C and Btype starch in soil temperature below 15 °C. In fact, the temperature, moisture, and amylopectin branch-chain length all influence the crystal conformation during starch synthesis. The low temperature, wet condition, and amylopectin long branch-chains tend to form B-type crystallinity, and the high temperature, dry condition, and amylopectin short branch-chains tend to form A-type crystallinity [33]. In this study, all varieties grew in the same environment, and underwent the same soil temperature and moisture variation during starch synthesized and accumulated. The expression and activities of starch synthesis related enzymes were different among the 44 sweet potato varieties because their different genotypes responded to the variation in soil temperature differently, leading to different crystalline structures among the 44 sweet potato starches. The RC of starch was measured, and ranged from 15.9% to 27.3% among 44 starches (Figure 3). The RC of starch is affected by amylose, amylopectin, crystallinity, and granule size [7,24,34]. The branch-chains of amylopectin can form A- and B-type crystallinity, which can be detected by X-ray diffraction (XRD). The XRD patterns of 44 starches are presented in Figure 3. Starches from plant tissues have A-, B-, and C-type. A- and B-type starch contains only A- and B-type crystallinity, respectively, but C-type starch simultaneously contains both A- and B-type crystallinities. According to the ratio of A- to B-type crystallinities from high to low, C-type starch is usually further classified into CA-, CC-, and CB-type. The CC-type starch has diffraction peaks at 2θ 5.6◦ , 15◦ , 17◦ , and 23◦ ; CA-type starch exhibits significant shoulder peak at 2θ 18◦ , a characteristic peak of A-type crystallinity; and CB-type starch has significant shoulder peaks at 2θ 22◦ and 24◦ , two characteristic peaks of B-type crystallinity [7,31]. In this study, 44 sweet potato varieties had A-, CA-, CC-, and CB-type starches (Figure 3). The JP14 starch was A-type, the PE06 starch was CB-type, the AO01, CB01, CN02, JP01, JP06, JP12, JP13, JP15, ML01, PE01, PE04, PH01, PH02, TH01, TZ01, US03, US06, and US07 starches were CA-type, and the other starches were CC-type (Figure 3). The A-, CA-, CC-, and CB-type starches have been reported in sweet potato varieties [10,13,14,19], but the B-type starch has not been reported in references. Genkina et al. [16,32] reported that the soil temperature determines the crystallinity of sweet potato starch. The root tuber synthesizes A-type starch in soil temperature above 33 ◦C and B-type starch in soil temperature below 15 ◦C. In fact, the temperature, moisture, and amylopectin branch-chain length all influence the crystal conformation during starch synthesis. The low temperature, wet condition, and amylopectin long branch-chains tend to form B-type crystallinity, and the high temperature, dry condition, and amylopectin short branch-chains tend to form A-type crystallinity [33]. In this study, all varieties grew in the same environment, and underwent the same soil temperature and moisture variation during starch synthesized and accumulated. The expression and activities of starch synthesis related enzymes were different among the 44 sweet potato varieties because their different genotypes responded to the variation in soil temperature differently, leading to different crystalline structures among the 44 sweet potato starches. The RC of starch was measured, and ranged from 15.9% to 27.3% among 44 starches (Figure 3). The RC of starch is affected by amylose, amylopectin, crystallinity, and granule size [7,24,34].

**Figure 3.** XRD patterns of starches from different sweet potato varieties. The relative crystallinity (RC) is given in the parenthesis after accession ID. **Figure 3.** XRD patterns of starches from different sweet potato varieties. The relative crystallinity (RC) is given in the parenthesis after accession ID.

#### *2.4. Thermal Properties of Starch*

The thermal properties are mainly functional properties and an important index for evaluating starch applications. The differential scanning calorimetric (DSC) thermograms of the 44 starches are shown in Figure 4. Significantly different thermograms were observed among the 44 sweet potato starches. These thermograms were divided into three types of one-peak thermogram (namely, JP14, JP06, ML01, and MY01), two-peak ther-

mogram (namely, JP04, CN03, US04, and PE05), and three-peak thermogram (namely, JP10, AO01, US01, and TZ01) according to their peak patterns (Figure 4). The gelatinization onset (To), peak (Tp), and conclusion temperature (Tc) ranged from 52.2 to 73.8 ◦C, from 59.7 to 80.0 ◦C, and from 75.3 to 86.9 ◦C among the 44 varieties, respectively. The gelatinization temperature range (∆T) varied from 13.1 to 29.2 ◦C, and the gelatinization enthalpy (∆H) ranged from 10.7 to 16.9 J/g (Table 3). The large gelatinization parameter variation among the 44 varieties was mainly due to the significantly different thermal curves. Similar thermal properties have been shown in different sweet potatoes from different references [13,19,35–37]. For example, Osundahunsi et al. [37] reported one-peak thermogram with gelatinization temperature from 67 to 75 ◦C in sweet potato varieties white-skinned TIS-1499 and red-skinned TIB-2. Kim et al. [13] reported the ∆T from 13.0 to 36.7 ◦C among eight sweet potato varieties. Lee and Lee [36] reported the ∆T from 24.0 to 28.0 ◦C among three sweet potato varieties. Duan et al. [35] reported a two-peak DSC curve in sweet potato variety Jishu 25. Genkina et al. [32] fitted the two-peak DSC curve of sweet potato starch into two gelatinization peaks with the low gelatinization temperature peak for B-type crystallinity and the high gelatinization temperature peak for A-type crystallinity. Guo et al. [38] fitted one-, two-, and three-peak DSC curves of different sweet potato starches into three gelatinization peaks, and the low, middle, and high gelatinization temperature peaks are the gelatinization of B-, C-, and A-type starch granules, respectively, meaning that sweet potato root tuber contains A-, B-, and C-type starch. In the present study, the different proportions of A-, B-, and C-type starch in sweet potatoes led to the different patterns of DSC curves among the 44 sweet potato starches.

The hierarchical cluster analysis based on Tp, Tc, ∆T, and ∆H was carried out to investigate the differences and similarities between sweet potato varieties (Figure 5). The JP14 was divided into one cluster (C2), and exhibited significant differences to the other sweet potato varieties in cluster 1 (C1) because JP14 starch had a typical one-peak DSC pattern with the highest gelatinization temperature and the most narrow ∆T among the 44 sweet potato starches. The cluster 1 contained two subclusters of C1A and C1B. The C1A had 21 sweet potato varieties with Tp, Tc, ∆T, and ∆H from 59.7 to 77.1 ◦C, from 75.3 to 86.7 ◦C, from 22.0 to 29.2 ◦C, and from 10.7 to 15.0 J/g, respectively. The C1B had 22 sweet potato varieties with Tp, Tc, ∆T, and ∆H from 66.8 to 77.5 ◦C, from 78.8 to 85.4 ◦C, from 18.0 to 23.9 ◦C, and from 13.2 to 16.9 J/g, respectively. It is noteworthy that some varieties from the same country, such as the JP01-JP15 varieties originating from Japan, were distributed into different groups, indicating that varieties from the same country had significantly different thermal properties.

#### *2.5. Relationships of Starch Properties and Cluster Analysis of Sweet Potato Varieties*

The relationships between starch properties were analyzed (Table 4). Among the 44 sweet potato starches, the granule size (D[4,3]) had no significant relationship with amylose content, RC, and thermal properties except the ∆T. The OD680 was significantly positively correlated to AAC, TAC, and ∆H, the OD620/550 was negatively correlated to AAC, TAC, Tp, Tc, and ∆H, and the AAC, TAC, and RC were positively correlated to Tp, Tc, and ∆H. Amylose and amylopectin are important components in starch, especially amylose, its content significantly affects the physicochemical properties and applications of starch [23,25,26]. Although granule size also influences the starch thermal properties [9,34], the thermal properties of starch were mainly affected by amylose content (AAC, TAC), amylopectin structure (OD620/550), and crystal structure (RC) in sweet potato (Table 4).

The hierarchical cluster analysis based on D[4,3], OD680, OD620/550, AAC, TAC, RC, Tp, Tc, ∆T, and ∆H was further carried out to investigate the differences and similarities between sweet potato varieties (Figure 6). The JP14 was divided into cluster 2 (C2), and the other sweet potato varieties were divided into cluster 1 (C1), indicating that JP14 had significantly different starch properties, especially for its thermal properties. The result also agreed with their crystalline structure that only JP14 had A-type starch. In cluster 2,

the 43 sweet potato varieties were divided into different groups, showing that they had different starch properties due to different genotype backgrounds. *Molecules* **2022**, *27*, x FOR PEER REVIEW 11 of 19

**Figure 4.** DSC thermograms of starches from different sweet potato varieties. **Figure 4.** DSC thermograms of starches from different sweet potato varieties.

AO01 57.4 ± 0.6 fgh 71.6 ± 0.1 klm 82.7 ± 0.6 jklmn 25.3 ± 1.2 klmnop 13.6 ± 0.1 bcdefghijk AR01 59.8 ± 0.6 jklm 71.8 ± 0.4 lmn 82.9 ± 0.0 jklmno 23.2 ± 0.6 ghijklm 14.3 ± 0.0 fghijklm CB01 56.7 ± 0.5 def 76.5 ± 0.4 vw 84.5 ± 0.3 nopqr 27.9 ± 0.2 qr 14.2 ± 0.3 efghijklm CG01 57.3 ± 0.3 efg 70.5 ± 0.6 ij 84.2 ± 1.0 mnopqr 26.9 ± 0.7 opqr 13.8 ± 0.0 cdefghijkl CN01 64.2 ± 0.2 qr 75.3 ± 0.3 tu 85.4 ± 0.4 pqrst 21.3 ± 0.2 cdefgh 16.9 ± 0.5 o

**Table 3.** Thermal property parameters of starches.

**Accession ID To (°C) Tp (°C) Tc (°C) ΔT (°C) ΔH (J/g)**

**Table 3.** Thermal property parameters of starches.

To, Tp and Tc: onset, peak, and conclusion temperature of gelatinization, respectively; ∆T and ∆H: gelatinization temperature range and enthalpy, respectively; Sig.: the significance of normal distribution of the 44 samples by Shapiro–Wilk test. Data are means ± standard deviations, *n* = 3. The values with different letters in the same column are significantly different (*p* < 0.05).

**Figure 5.** Hierarchical cluster analysis of the 44 sweet potato varieties based on Tp, Tc, ΔT, and ΔH in Table 3. **Figure 5.** Hierarchical cluster analysis of the 44 sweet potato varieties based on Tp, Tc, ∆T, and ∆H in Table 3.

*2.5. Relationships of Starch Properties and Cluster Analysis of Sweet Potato Varieties* **Table 4.** Correlation coefficients between starch property parameters.

ble 4). **Table 4.** Correlation coefficients between starch property parameters. The analysis is based on property parameters with the significance of normal distribution over 0.05. The RC is relative crystallinity, and its significance of normal distribution is 0.067. The other abbreviations and their normal distributions are listed in Tables 1–3. \* and \*\* indicate the significance at the *p* < 0.05 and *p* < 0.01 levels, respectively. *Molecules* **2022**, *27*, x FOR PEER REVIEW 14 of 19

The hierarchical cluster analysis based on D[4,3], OD680, OD620/550, AAC, TAC, RC, Tp, Tc, ΔT, and ΔH was further carried out to investigate the differences and similarities between sweet potato varieties (Figure 6). The JP14 was divided into cluster 2 (C2), and **Figure 6.** Hierarchical cluster analysis of the 44 sweet potato varieties based on D[4,3], OD680, OD620/550, AAC, TAC, RC, Tp, Tc, ΔT, and ΔH in Table 4. **Figure 6.** Hierarchical cluster analysis of the 44 sweet potato varieties based on D[4,3], OD680, OD620/550, AAC, TAC, RC, Tp, Tc, ∆T, and ∆H in Table 4.

the other sweet potato varieties were divided into cluster 1 (C1), indicating that JP14 had significantly different starch properties, especially for its thermal properties. The result also agreed with their crystalline structure that only JP14 had A-type starch. In cluster 2, the 43 sweet potato varieties were divided into different groups, showing that they had different starch properties due to different genotype backgrounds. Though the 44 sweet potato varieties were divided into different clusters according to their starch properties (Figure 6), it is noteworthy that the growing conditions significantly affect starch properties in sweet potato, especially for temperature [16] and fertilizer treatment [21]. Recent study also shows that starch physicochemical properties are Though the 44 sweet potato varieties were divided into different clusters according to their starch properties (Figure 6), it is noteworthy that the growing conditions significantly affect starch properties in sweet potato, especially for temperature [16] and fertilizer treatment [21]. Recent study also shows that starch physicochemical properties are affected

affected significantly by varieties, growing locations, and their interaction in sweet potato [39]. In this study, the sweet potato varieties originating from different countries were

different from growth habits of varieties in their original country. Therefore, the starch properties of sweet potato varieties in the present study might be different from those in

The 44 sweet potato varieties were randomly chosen, and their fresh root tubers were provided by Sweetpotato Research Institute, China Agricultural Academy of Sciences. These germplasm resources are all public varieties or landraces, and conserved in National Sweetpotato Genebank in Xuzhou, China. Among the 44 varieties, 15 were from Japan, 7 from United States, 6 from Peru, 4 from China, 2 Philippines, and the others from Angola, Argentina, Cambodia, Congo, Morocco, Mali, Malaysia, Nigeria, Thailand, and Tanzania. Their accession IDs and original areas are listed in Table 5. These sweet potato varieties were planted simultaneously in the farm of Xuzhou Sweetpotato Research Center (32°27′ N, 117°29′ E), Jiangsu Province, China, on April 28, and harvested on 26 October 2020. The soil in the experimental field was yellow fluvo-aquic soil with a sandy texture. The available K, available P, hydrolysable N, total N, and organic matter in 0–20 cm soil

, 19.7 mg kg−<sup>1</sup>

, respectively, and its pH was 7.54. The fertilizer including 75 kg ha−<sup>1</sup> P2O<sup>5</sup> (cal-

cium superphosphate) and 120 kg ha−<sup>1</sup> K2O (potassium chloride) was applied as base fertilizer in the ridge before planting. The seedlings were planted in 5 rows with 90 cm between rows and 20 cm between hills. The temperature and rainfall of growing location

, 94.2 mg kg−<sup>1</sup>

, 1.06 g kg−<sup>1</sup>

, and

their original countries.

*3.1. Plant Materials*

16.2 g kg−<sup>1</sup>

**3. Materials and Methods**

layer before the experiment were 95 mg kg−<sup>1</sup>

during sweet potato growth stage are presented in Table 6.

significantly by varieties, growing locations, and their interaction in sweet potato [39]. In this study, the sweet potato varieties originating from different countries were planted in the same place and cultivated conditions to avoid the effects of growing environment on starch properties. However, the selected growing conditions are significantly different from growth habits of varieties in their original country. Therefore, the starch properties of sweet potato varieties in the present study might be different from those in their original countries.

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

#### *3.1. Plant Materials*

The 44 sweet potato varieties were randomly chosen, and their fresh root tubers were provided by Sweetpotato Research Institute, China Agricultural Academy of Sciences. These germplasm resources are all public varieties or landraces, and conserved in National Sweetpotato Genebank in Xuzhou, China. Among the 44 varieties, 15 were from Japan, 7 from United States, 6 from Peru, 4 from China, 2 Philippines, and the others from Angola, Argentina, Cambodia, Congo, Morocco, Mali, Malaysia, Nigeria, Thailand, and Tanzania. Their accession IDs and original areas are listed in Table 5. These sweet potato varieties were planted simultaneously in the farm of Xuzhou Sweetpotato Research Center (32◦270 N, 117◦290 E), Jiangsu Province, China, on April 28, and harvested on 26 October 2020. The soil in the experimental field was yellow fluvo-aquic soil with a sandy texture. The available K, available P, hydrolysable N, total N, and organic matter in 0–20 cm soil layer before the experiment were 95 mg kg−<sup>1</sup> , 19.7 mg kg−<sup>1</sup> , 94.2 mg kg−<sup>1</sup> , 1.06 g kg−<sup>1</sup> , and 16.2 g kg−<sup>1</sup> , respectively, and its pH was 7.54. The fertilizer including 75 kg ha−<sup>1</sup> P2O<sup>5</sup> (calcium superphosphate) and 120 kg ha−<sup>1</sup> K2O (potassium chloride) was applied as base fertilizer in the ridge before planting. The seedlings were planted in 5 rows with 90 cm between rows and 20 cm between hills. The temperature and rainfall of growing location during sweet potato growth stage are presented in Table 6.

#### *3.2. Isolation of Starch*

Starches from fresh root tubers were prepared following the method of Guo et al. [2]. Briefly, the root tubers were washed cleanly and cut into some pieces. The sample was homogenized in H2O with a home blender. The tissue suspension was filtered through four layers of gauze, and the residue was homogenized and filtered again to release more starch. The filtrate was filtered through 100-, 200-, and 300-mesh sieve, successively, and centrifuged (3000× *g*, 5 min). The starch precipitate was washed 5 times with H2O and 3 times with anhydrous ethanol. Finally, the starch was dried at 40 ◦C for 2 d, and ground into powder through 100-mesh sieve.

#### *3.3. Granule Morphology and Size Analysis of Starch*

Isolated starch in 25% glycerol was viewed and photographed with a polarized light microscope (BX53, Olympus, Tokyo, Japan) under normal and polarized light. Starch size was analyzed with a laser size analyzer (Mastersizer 2000, Malvern, Worcestershire, UK) according to the procedures of Guo et al. [2]. The obscuration of starch–water suspension was about 12%, and the sample was stirred at 2000 rpm during analysis.

#### *3.4. Analysis of Starch–Iodine Absorption*

Starch was dispersed in dimethyl sulfoxide and stained with iodine solution according to the procedures of Man et al. [24]. Briefly, 10 mg starch in 5 mL dimethyl sulfoxide containing 10% 6.0 M urea was heated at 95 ◦C for 1 h. The starch suspension was swirled at intervals of 15 min during heating. The dispersed amylose and amylopectin (1 mL) was colorized for 20 min with iodine solution (1 mL of 0.2% I<sup>2</sup> and 2% KI) in 50 mL volumetric flask diluted with H2O. The sample was scanned from 400 to 900 nm using a spectrophotometer (BioMate 3S, Thermo Scientific, Chino, CA, USA).

**Table 5.** List of sweet potato accessions used in this study.

**Table 6.** The climatic data of Xuzhou city in 2020.

#### *3.5. Measurement of Amylose Content*

The apparent amylose content was determined using the absorption value of starch– iodine complex at 620 nm [24]. The true amylose content was measured using concanavalin A precipitation method through an Amylose/Amylopectin Assay Kit (K-AMYL, Megazyme, Bray, Ireland).

#### *3.6. XRD Analysis of Starch*

Starch was analyzed with an X-ray diffractometer (XRD) (D8, Bruker, Karlsruhe, Germany). Before analysis, the sample was moisturized for 1 week at 25 ◦C in a desiccator containing a saturated solution of NaCl with a relative humidity about 75%. The testing setting contained an X-ray beam at 200 mA and 40 kV and scanning range of diffraction angle from 2θ 3 to 40◦ with a step size of 0.02◦ . The relative crystallinity (RC) was evaluated with the percentage of diffraction peak area to total diffraction area over the diffraction angle 2θ 4 to 30◦ following the method of Wei et al. [40].

#### *3.7. DSC Analysis of Starch*

The 5 mg starch and 15 µL water were mixed and sealed hermetically in an aluminum pan. After equilibrating for 2 h at room temperature, the sample was heated from 25 to 130 ◦C at 10 ◦C/min using a differential scanning calorimeter (DSC 200-F3, Netzsch, Selb, Germany).

#### *3.8. Statistical Analysis*

The statistical differences between data from varieties were detected using one-way analysis of variance (ANOVA) by Tukey's test using the SPSS 19.0 Statistical Software Program. The Pearson correlation analysis and hierarchical cluster analysis were also evaluated using SPSS 19.0 Statistical Software Program. Prior to the analysis, the normal distribution of structural property parameters was assessed using the Shapiro–Wilk test with SPSS 16.0. Only structural property parameters with their significances of normal distributions over 0.05 were used to evaluate the Pearson correlation and hierarchical cluster analysis.

#### **4. Conclusions**

Starches from 44 sweet potato varieties originating from 15 countries were investigated for their sizes, amylose contents, crystalline structures, and thermal properties. The D[4,3], AAC, TAC, and ∆AC (AAC–TAC) ranged from 8.01 to 15.30 µm, from 19.2% to 29.2%, from 14.2% to 20.2%, and from 4.0% to 11.8% among the 44 starches, respectively. Starches had A-, CA-, CC-, and CB-type with RC from 19.5% to 27.3%. One-, two-, and three-peak DSC curves were detected, and the To, Tp, Tc, and ∆T ranged from 52.2 to 73.8 ◦C, from 59.7 to 80.0 ◦C, from 75.3 to 86.9 ◦C, and from 13.1 to 29.2 ◦C among the 44 starches, respectively. Based on starch property parameters, the 44 sweet potato varieties were divided into different groups. This research offers references for the utilization of sweet potato germplasm resources.

**Author Contributions:** C.W. and Q.C. conceived the study and designed the experiments; Y.L., L.Z., L.S. and L.L. performed the experiments; Y.L. and L.Z. wrote the original draft manuscript; and C.W. reviewed and edited the manuscript. All authors discussed the contents of the manuscript and approved the submission. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financially supported by grants from the National Key Research and Development Program of China (2018YFD1000705, 2018YFD1000700), Accurate Identification of Sweetpotato Excellent Germplasm Resources (19211139), and Priority Academic Program Development of Jiangsu Higher Education Institutions.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are available upon request from the corresponding author.

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

**Sample Availability:** Samples of some starches are available from the corresponding author upon reasonable request.

#### **References**

## *Article* **Influence of Gamma Irradiation on Different Phytochemical Constituents of Dried Rose Hip (***Rosa canina* **L.) Fruits**

**Manol Ognyanov 1,\* , Petko Denev <sup>1</sup> , Desislava Teneva <sup>1</sup> , Yordan Georgiev <sup>1</sup> , Sabina Taneva <sup>2</sup> , Iskra Totseva <sup>2</sup> , Mariana Kamenova-Nacheva <sup>3</sup> , Yana Nikolova <sup>3</sup> and Svetlana Momchilova <sup>2</sup>**

**Abstract:** Gamma irradiation is efficiently applied to many foods, but nevertheless there is a distinct lack of information about the changes of macro- and micronutrients (e.g., carbohydrates, lipids, organic acids, and phenolics) in dried rose hip (RH) fruits. Therefore, in this study, for the first time, the effect of gamma irradiation (10 and 25 kGy) on RH constituents is investigated. Different analytical techniques (GC-FID, HPLC-UV, HPSEC-RID, IR-FT, and SEM) are employed to examine this effect. The irradiation treatment (10 kGy) increased the glucose content by 30% and released cellobiose from RH fruits, thus revealing cellulose destruction. The extractability of total uronic acids increased from 51% (control) to 70.5% (25 kGy-irradiated), resulting in a higher pectin yield (10.8% < 12.8% < 13.4%) and molecular heterogeneity. Moreover, de-esterification was not a major effect of the irradiation-induced degradation of pectin. The sample exposure to the highest dose did not change the content of total carotenoids, *β*-carotene, and (un)saturated fatty acids, but it affected the tocopherols levels. Gamma rays had a negligible effect on the phenolic constituents and did not affect ORAC and HORAC antioxidant activity. In conclusion, it can be compromised that the exposition of dried RH is safe and can be successfully applied to decontaminate fruits without affecting their nutritional value and biological activity.

**Keywords:** gamma irradiation; rose hip; seed oil; polysaccharides; carbohydrates; lipids

#### **1. Introduction**

The irradiation of foodstuffs with gamma rays was developed as a technology in the second half of the 20th century, and is nowadays among the most widely used approaches for microbial decontamination and the shelf-life extension of foods [1,2]. It is considered a safe, efficient, environmentally friendly, and energy-efficient method, and is particularly important in industrial food decontamination [3]. Preventing microbial spoilage by irradiating dried foods, such as fruits, herbs, spices, and nuts, at doses of 3–10 kGy is an important alternative to fumigation with microbicidal gases, however, doses above 20 kGy have also been used to reduce the microbial population in dry foods [4,5]. Along with the benefits of gamma ray treatment, adverse effects associated with changes in the chemical composition of irradiated foods can be expected. It is known, for example, that irradiation could have a negative effect on the structure of fatty acids in nuts or to affect the anthocyanin and

**Citation:** Ognyanov, M.; Denev, P.; Teneva, D.; Georgiev, Y.; Taneva, S.; Totseva, I.; Kamenova-Nacheva, M.; Nikolova, Y.; Momchilova, S. Influence of Gamma Irradiation on Different Phytochemical Constituents of Dried Rose Hip (*Rosa canina* L.) Fruits. *Molecules* **2022**, *27*, 1765. https://doi.org/10.3390/ molecules27061765

Academic Editor: Mirella Nardini

Received: 9 February 2022 Accepted: 7 March 2022 Published: 8 March 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/).

polyphenol content of fresh fruits, such as pomegranates [6,7]. Due to conflicting results, scientists are still looking for fresh evidence to accept or reject the hypothesis about the positive or negative influence of irradiation on foods. Moreover, due to a great variety of foods with specific chemical compositions, each food must be examined individually. about the positive or negative influence of irradiation on foods. Moreover, due to a great variety of foods with specific chemical compositions, each food must be examined individually. Rose hip (RH) fruits (*Rosa canina* L.) are mainly used for producing jams, jellies, marmalades, tea, soups, food additives, and functional ingredients [8,9]. Although fresh RH

could have a negative effect on the structure of fatty acids in nuts or to affect the anthocyanin and polyphenol content of fresh fruits, such as pomegranates [6,7]. Due to conflicting results, scientists are still looking for fresh evidence to accept or reject the hypothesis

*Molecules* **2022**, *27*, x FOR PEER REVIEW 2 of 21

Rose hip (RH) fruits (*Rosa canina* L.) are mainly used for producing jams, jellies, marmalades, tea, soups, food additives, and functional ingredients [8,9]. Although fresh RH fruits having the highest nutrient quality are particularly suitable for the production of various functional foods and food supplements, they are most often stored, transported, sold, and processed in a dried form. Some fruits' characteristics appeared interesting enough to require a more detailed study of their changes after irradiation. Many studies, for example, provide empirical evidence to prove that RH is a rich source of vitamins, lipids, phenolic antioxidants, macro- and micro-elements, and polysaccharides (PSs) [8–11]. Moreover, because of their well-known beneficial health properties, the RH fruits are also used as the main ingredient in fruit teas [12]. In spite of this, to the best of our knowledge, studies on the effect of gamma irradiation on the changes in the chemical composition of dried RH have not been found. fruits having the highest nutrient quality are particularly suitable for the production of various functional foods and food supplements, they are most often stored, transported, sold, and processed in a dried form. Some fruits' characteristics appeared interesting enough to require a more detailed study of their changes after irradiation. Many studies, for example, provide empirical evidence to prove that RH is a rich source of vitamins, lipids, phenolic antioxidants, macro- and micro-elements, and polysaccharides (PSs) [8– 11]. Moreover, because of their well-known beneficial health properties, the RH fruits are also used as the main ingredient in fruit teas [12]. In spite of this, to the best of our knowledge, studies on the effect of gamma irradiation on the changes in the chemical composition of dried RH have not been found. Therefore, the present study aims to investigate the effects of gamma irradiation on

Therefore, the present study aims to investigate the effects of gamma irradiation on the changes in the levels of different macro- and micronutrient constituents of dried RH fruits. the changes in the levels of different macro- and micronutrient constituents of dried RH fruits.

#### **2. Results 2. Results**

#### *2.1. Influence of Gamma Irradiation on Sugar and Organic Acid Composition 2.1. Influence of Gamma Irradiation on Sugar and Organic Acid Composition*

The changes in the content of free sugars in dried RH fruits caused by gamma irradiation are shown in Figure 1. It can be seen that glucose (53%) was the major sugar in non-irradiated fruits. Together with fructose (33%), it occupied more than 80% of the total free sugars, whereas galactose and xylose were very minor constituents. Surprisingly, the glucose content increased substantially by 30% (*p* < 0.05) with increasing the irradiation dosage, while the quantity of the other sugars seemed to be mainly unaffected by irradiation. It is interesting to note that this effect was particularly noticeable at a lower dose (10 kGy), and a further increase in the dose (25 kGy) did not have any considerable influence on the quantity of the glucose. Despite this, the irradiation influenced the pattern of sugar accumulation. One of the most interesting observations was that cellobiose was found in 10 kGy- and 25 kGy-irradiated fruits. The presence of cellobiose combined with an additionally liberated glucose amount presented a clear sign that a higher dose could inflict damage on the cellulose fibers that constituted the primary cell walls of plants. Additionally, the quantity of non-reducing sucrose was found to increase when RH fruits were irradiated (≥10 kGy). It was scarcely possible that a part of the starch/cellulose was converted into sucrose and glucose by affecting the carbohydrate-active enzymes. The changes in the content of free sugars in dried RH fruits caused by gamma irradiation are shown in Figure 1. It can be seen that glucose (53%) was the major sugar in nonirradiated fruits. Together with fructose (33%), it occupied more than 80% of the total free sugars, whereas galactose and xylose were very minor constituents. Surprisingly, the glucose content increased substantially by 30% (*p* < 0.05) with increasing the irradiation dosage, while the quantity of the other sugars seemed to be mainly unaffected by irradiation. It is interesting to note that this effect was particularly noticeable at a lower dose (10 kGy), and a further increase in the dose (25 kGy) did not have any considerable influence on the quantity of the glucose. Despite this, the irradiation influenced the pattern of sugar accumulation. One of the most interesting observations was that cellobiose was found in 10 kGy- and 25 kGy-irradiated fruits. The presence of cellobiose combined with an additionally liberated glucose amount presented a clear sign that a higher dose could inflict damage on the cellulose fibers that constituted the primary cell walls of plants. Additionally, the quantity of non-reducing sucrose was found to increase when RH fruits were irradiated (≥10 kGy). It was scarcely possible that a part of the starch/cellulose was converted into sucrose and glucose by affecting the carbohydrate-active enzymes.

**Figure 1.** Sugar composition of non-irradiated, 10 kGy-, and 25 kGy-irradiated RH fruits. Different letters denote statistically different values, *p* < 0.05; dw—dry weight.

Further, the effect of gamma irradiation on the organic acid profile was examined (Table 1). It should be mentioned that organic acids are a very important component of foods, which have a part to play in determining the taste of foods. As can be seen, citric acid, a member of the Krebs cycle, was predominantly present (2768 mg/100 g), because it occupied 55% of the total organic acids. Non-irradiated RH fruits also contained quinic acid (1344 mg/100 g) and ascorbic acid (624 mg/100 g) in smaller amounts, but, nevertheless, they accounted for almost 40% of the total organic acids content. From the data in Table 1, it is apparent that the exposition of fruits to gamma radiation has a minor or no effect on quinic, malic, and ascorbic acid content. It is evident that citric, *α*-ketoglutaric, and succinic acids increase in quantity. The increasing levels of these acids suggested a stimulation of respiration through the citric acid cycle, if we assume the presence of viable cells. However, the latter statement looked highly unlikely because the cell's/tissue's metabolism could be dramatically slowed down by moisture loss during freeze-drying (<10%). Thus, it is not wrong to assume that the cell walls and/or even (non)membrane-bound cell organelles (such as vacuole and mitochondrion) suffered extensive damage by irradiation, thus making small molecules more easily extractable. Further, the level of tartaric acid, a product of the oxidation of sugars, decreased slightly, but it was statistically significant at the highest level of radiation (25 kGy). An important point brought to mind is that organic acids and, above all, tartaric acid, have closely related metabolism to ascorbic acid [13]. It is possible that the level of tartaric acid was reduced in an attempt to compensate for the loss of ascorbic acid whose amount did not change significantly.

**Table 1.** Organic acid composition of non-irradiated, 10 kGy- and 25 kGy-irradiated RH fruits (mg/100 g dw).

\* Different letters within each row indicate statistically different values (*p* < 0.05).

#### *2.2. Influence of Gamma Irradiation on the Lipid Composition of Seed Oil*

The results of the lipid composition of RH seed oil are presented in Table 2. Our experiment indicated that the yield of oil did not change considerably with irradiation (6.3–6.5%). Further, it can be seen that palmitic acid (16:0; 3.7%) and stearic acid (18:0; 2.2%) were the most abundant saturated fatty acids in the oil of non-irradiated RH fruits. Considering unsaturated fatty acid composition, seed oil was characterized by a high degree of unsaturation. Linoleic acid (18:2; 55%) represented the major polyunsaturated fatty acid constituent, followed by *α*-linolenic (18:3; 21%) and oleic acid (16:1; 16%). Despite this fact, it seemed that irradiation did not cause any significant changes in the fatty acid composition (Table 2), even though the sample was exposed to the highest dose (25 kGy). Considering the carotenoids, it was evident that neither the 10 kGy nor 25 kGy doses had any negative influence on *β*-carotene and, in general, on the total carotenoid content. It is worth mentioning that the total carotenoid content did not change significantly not only in seed oil, but also in fruit flesh (about 820 mg/kg) when the irradiation doses increased (Table 2). Our findings were in line with those of an earlier study that demonstrated that a 10 kGy irradiation of RH tea did not cause any change in the carotenoid content [14]. Further, one of the most interesting findings was that there was a noticeable difference in the amounts of *α*- and *γ*-tocopherol as a result of irradiation (*p* < 0.05) (Table 2). It is well known that tocopherols (vitamin E) are powerful antioxidants able to scavenge free

radicals. It may be that the tocopherols were reduced because of their active participation in the neutralization of free radicals.

**Table 2.** Yield and lipid composition of seed oil extracted from non-irradiated, 10 kGy-, and 25 kGy-irradiated RH fruits.

\* in each row, no statistically significant changes are observed for the yield and each fatty acid. For the other components, different letters within each row denote statistically significant differences between the values (*p* < 0.05); \*\* Values in brackets represent the total carotenoid content of RH fruit flesh.

In addition, important parameters, such as the acid value, amounts of conjugated dienes, and trienes, were determined. These parameters are reliable indicators of the degree of lipid oxidation. The results obtained are also summarized in Table 2. Our findings indicated that the conjugated trienes, and especially dienes, were slightly influenced by a higher dose (25 kGy). The results obtained are broadly in line with the expectations, because unsaturated fatty acids comprised 93% of the total fatty acid content. In general, a higher amount of unsaturated fatty acids suggested a higher instability of oil due to oxidation. Therefore, the determination of the induction period of auto-oxidation of RH oil has a more practical significance when the degree of oxidation stability should be evaluated (Figure 2). It represents the time interval during which the antioxidants present in the sample still effectively inhibit the auto-oxidation processes in the oil, and the rate of formation and accumulation of hydroperoxides still does not increase exponentially. As illustrated in Figure 2, it seems that gamma irradiation (<25 kGy) does not significantly change the oxidative stability of RH seed oil because of the presence of antioxidants.

#### *2.3. Influence of Gamma Irradiation on the Phenolic Constituents and Antioxidant Activity*

The content of the total phenolics and in vitro antioxidant activity of non-irradiated, 10 kGy-, and 25 kGy-irradiated RH fruits was investigated. Together with the quantitative data for the phenolic components, they are included in Table 3.

In general, gamma irradiation had a negligible effect on the content of the phenolic compounds and their profile. As it is evident from the results, the total polyphenol content shows a trend towards increase, however without reaching significant differences (*p* < 0.05). The only significant difference was observed in the quercetin content, which increased from 13.9 mg/100 g to 18.9 mg/100 g, and 18.3 mg/100 g for fruits irradiated with 10 kGy and 25 kGy, respectively (*p* < 0.05). On the other hand, the detected amounts of quercetin-3-*O*-glucoside and rutin decreased dose-dependently after irradiation (*p* > 0.05), which explained the increase in quercetin content and could be attributed to the cleavage of

glycosidic bonds from the high-energy electromagnetic radiation [15]. Gamma irradiation showed no effect on the amounts of chlorogenic acid, catechin, and epicatechin in RH fruits. Even though quercetin is a stronger radical scavenger than its glycosides, the antioxidant activity measured by the ORAC and HORAC assays did not change as a result of the treatment. *Molecules* **2022**, *27*, x FOR PEER REVIEW 5 of 21

**Figure 2.** Induction periods of RH seed oils extracted from non-irradiated, 10 kGy-, and 25 kGyirradiated RH fruits. **Figure 2.** Induction periods of RH seed oils extracted from non-irradiated, 10 kGy-, and 25 kGyirradiated RH fruits.

*2.3. Influence of Gamma Irradiation on the Phenolic Constituents and Antioxidant Activity*  The content of the total phenolics and in vitro antioxidant activity of non-irradiated, **Table 3.** Phenolic constituents and in vitro antioxidant activity of non-irradiated, 10 kGy-, and 25 kGy-irradiated RH fruits.

#### \* Different letters within each row denote the statistically significant differences between values (*p*  < 0.05); ORAC—oxygen radical absorbance capacity; TE—Trolox equivalents; HORAC—hydroxyl *2.4. Influence of Gamma Irradiation on Polysaccharide Constituents*

radical averting capacity; and GAE—gallic acid equivalents. In general, gamma irradiation had a negligible effect on the content of the phenolic compounds and their profile. As it is evident from the results, the total polyphenol content shows a trend towards increase, however without reaching significant differences (*p* < 0.05). The only significant difference was observed in the quercetin content, which increased from 13.9 mg/100 g to 18.9 mg/100 g, and 18.3 mg/100 g for fruits irradiated with 10 kGy and 25 kGy, respectively (*p* < 0.05). On the other hand, the detected amounts of We were very interested in knowing more about the potential changes in different PSs because they are the most important plant cell wall constituents. The results of different analyses are summarized in Table 4. From the table, it is evident that the yield of alcoholinsoluble solids (AISs) decreases with increasing the radiation dose (0 > 10 > 25 kGy). Accordingly, this means having a higher proportion of alcohol-soluble small molecules as a result of irradiation (0 < 10 < 25 kGy). These results provide us with the next clear evidence to support our assertion about the damage of plant cell wall constituents during irradiation. These cell wall changes should facilitate the extraction of initially present

quercetin-3-*O*-glucoside and rutin decreased dose-dependently after irradiation (*p* > 0.05), which explained the increase in quercetin content and could be attributed to the cleavage

tion showed no effect on the amounts of chlorogenic acid, catechin, and epicatechin in RH

or additionally formed (as a result of irradiation) low molecular weight alcohol-soluble compounds (such as sugars and oligomers). It is interesting to mention that the yield of PS also increased significantly when RH fruits were exposed to higher doses of radiation: non-irradiated—10.8%, 10 kGy—12.8%, and 25 kGy—13.4%. These results suggest that some cell wall constituents are adversely affected by irradiation, thus directly contributing to the additional release of PS populations. The latter results are very closely related to the finding of a considerable percentage of uronic acids extracted from the fruits as a result of irradiation (Table 4; control—51% and 25 kGy—71%). It is well known that uronic acids are major constituents of pectic PSs and, together with hemicelluloses, mainly comprise the primary cell walls of plants. Table 4 shows that the 10 kGy- and 25 kGyirradiation does not exert any changes in the amount of the total uronic acid in the initial RH fruits (9.5–10.5%). At first sight, it can be interpreted to mean a 'lack of changes in pectin'. The PSs obtained were characterized as typical high-methyl-ester pectins having a uronic acid content in the range between 49 and 53%. Surprisingly, it seemed that the degree of methyl-esterification was not affected by radiation (Table 4; 69–70 mol%), strongly suggesting that de-esterification is not a major effect of the irradiation-induced degradation of pectic PS. However, the degree of acetylation tended to be increased with increasing the irradiation dose. It may be that PS fractions carrying a higher number of acetyl groups were additionally released as a result of irradiation. Further, it could be observed that irradiation exerted a profound influence on cellulose that led to a reduction in its amount (Table 4). This was a key finding, which directly confirmed our above-mentioned statement about cellulose destruction. It could be argued that a minor part of the cellulose skeleton was broken down into cellobiose, low-molecular-weight cello-dextrins, or other oligomers (alcohol soluble), thus causing a reduction in the cellulose level and a rise in the yield of alcohol-soluble solids. Taken together, the above results suggest that a part of the pectin was trapped in the (hemi)cellulose network, and by degrading a part of the cellulose segments, additional pectic PSs were released leading to a rise in the yield of PSs. An interaction between pectin and cellulose was demonstrated in a previous study [16]. In fact, it should not rule out the possibility that the gamma radiation caused a direct degradative action on the pectin, in addition to that of cellulose.

**Table 4.** Yield and chemical characteristics of alcohol-insoluble solids, initial RH fruits, and polysaccharides (*w*/*w*%).

\* Different letters in each row denote the statistically significant difference between values (*p* < 0.05); \*\* moles methanol or acetyl per 100 moles of uronic acids (mol%).

#### 2.4.1. Influence of Gamma Irradiation on Monosaccharide Composition

Table 5 shows the results of the monosaccharide composition analysis of non- and 25 kGy-irradiated PSs fraction. It can be seen that commonly reported pectic monosaccharides constitute the PS fractions. The PS isolated from non-irradiated fruits was composed

mostly of galacturonic acid, glucose, and rhamnose. Other monosaccharides, such as galactose and arabinose, were present in lower amounts. Irradiation did not have a considerable influence on different sugar constituents. It appears that gamma-irradiation contributes only to the reduction in the glucose content, whereas that of fucose is raised, possibly due to changes in the extractability of PSs and plant cell walls.

**Table 5.** Monosaccharide composition of the polysaccharides isolated from non-irradiated and 25 kGy-irradiated RH fruits (mol%).

\* Different letters in each row denote the statistically significant difference between values (*p* < 0.05).

#### 2.4.2. Influence of Gamma Irradiation on Molecular Weight Distribution

The molecular weight distribution pattern of the isolated PSs fraction is shown in Figure 3A. It can be seen that the PS fraction, isolated from non-irradiated fruits, consists mainly of two high molecular weight populations covering the range between 78.8 <sup>×</sup> <sup>10</sup><sup>4</sup> and 21.2 <sup>×</sup> <sup>10</sup><sup>4</sup> g/mol (Figure 3A solid line). Moreover, the main peak is accompanied by a pronounced tailing suggesting that heterogeneity exists within the sample. It ranges between 21.2 <sup>×</sup> <sup>10</sup><sup>4</sup> and 4.73 <sup>×</sup> <sup>10</sup><sup>4</sup> g/mol. What is immediately striking, from a close inspection of Figure 3A, is that irradiation causes the PS molecular weight elution pattern to change visibly. A 10 kGy-irradiation leads to a decrease in the main peak intensity (78.8 <sup>×</sup> <sup>10</sup><sup>4</sup> g/mol), suggesting that part of the high molecular weight populations is converted into lower molecular weight compounds (Figure 3A dash line). As a consequence, the increase in the area representing medium and low molecular weight fractions is found (RT > 7.1 min). Interestingly, this negative effect on the PSs became stronger when the sample was exposed to the highest dose (25 kGy), and thus resulted in an even greater increase in the area representing lower molecular weights. More specifically, it is easy to be observed that, if we integrate the chromatograms (Figure 3A), the high molecular weight populations (RT 5.5–7.0 min) occupied a smaller percentage of the total (100%) peak area, hence a smaller percentage of PSs, when the irradiation dose increased: 0–60%, 10 kGy—48%, 25 kGy—40%. Accordingly, the percentage of lower molecular weight populations (RT 7.0–11.6 min) that comprised the PSs jumped: 0–40%, 10 kGy—52%, 25 kGy—60%.

To receive more information about the structural features of PSs, an *endo*polygalacturonase (*endo*-PG) hydrolysis of the PS fraction was performed. It is a wellknown fact that *endo*-PG can cleave *α*-1,4 glycosidic bonds between two non-esterified galacturonic acid units in the homogalacturonan (HG) region of pectin [17]. Figure 3B illustrates the elution pattern of *endo*-PG-treated samples. It can be observed that the *endo*-PG carried out the conversion of the part of the high molecular weight material into a broad range of lower molecular weight compounds. As a result, the change of the elution profile was observed by comparison with the non-enzyme-treated PS (Figure 3A solid line). This presented a reasonable reason to suppose that the HG region of pectin was adversely affected by gamma irradiation. A surprising finding of this study was that the PSs, isolated from 10 kGy- and 25 kGy-irradiated fruits, were degraded by *endo*-PG more efficiently than those isolated from the non-irradiated control. This led to an additional increase in the

peak area and shift in the retention time reflecting the accumulation of a large number of lower molecular weight compounds (Figure 3B dash and dot line). *Molecules* **2022**, *27*, x FOR PEER REVIEW 9 of 21

**Figure 3.** High-performance size-exclusion chromatography (HPSEC) elution pattern of PSs isolated from non-irradiated, 10 kGy-, and 25 kGy-irradiated RH fruits as: (**A**) initial PSs; (**B**) *endo*-PG-degraded PSs (solid—non-irradiated; dash—10 kGy; dot—25 kGy); and (**C**) Pullulan standards (0.59– 78.8 × 104 g/mol) were used to estimate the molecular weights. **Figure 3.** High-performance size-exclusion chromatography (HPSEC) elution pattern of PSs isolated from non-irradiated, 10 kGy-, and 25 kGy-irradiated RH fruits as: (**A**) initial PSs; (**B**) *endo*-PGdegraded PSs (solid—non-irradiated; dash—10 kGy; dot—25 kGy); and (**C**) Pullulan standards (0.59–78.8 <sup>×</sup> <sup>10</sup><sup>4</sup> g/mol) were used to estimate the molecular weights.

These results could be expressed in numerical data, if we integrate the chromatograms (Figure 3B). It was precisely calculated that the percentage of the total peak area ascribed to high molecular weight compounds (RT 5.5–7.0 min) decreased by 7%: 0–23%, 10 kGy—18%, 25 kGy—16%. Accordingly, the area percentage of lower molecular weight populations (RT 7.0–11.6 min) increased by 7%: 0–77%, 10 kGy—82%, 25 kGy—84%.

This finding can be interpreted in different ways. On the one hand, it is known that *endo*-PG requires a sequence of 2–3 unesterified galacturonic acid units to cleave them [17]. However, keeping in mind that the irradiation did not cause any appreciable effect on the degree of methyl-esterification (Table 5), we can suppose a difference between the esterification pattern of the PSs. On the other hand, it may be that lower molecular weight HG segments were a better substrate for *endo*-PG action.

#### 2.4.3. Fourier Transformed Infrared (FT-IR) Spectroscopy

The FT-IR spectra of the studied PSs are presented in Figure 4. FT-IR can identify the presence of specific functional groups prevailing in the sample. The typical bands for pectic PSs were found [18]. The broadened band that occurs in the region ranging from 3730 to 3100 cm−<sup>1</sup> was assigned to the ν(OH) stretching vibrations of free hydroxyl groups involved in intra- and intermolecular H-bonding. The band at 2940–2960 cm−<sup>1</sup> was attributed to the ν(CH) stretching of CH<sup>2</sup> groups. The presence of a well-separated sharp signal that appears at 1745–1735 cm−<sup>1</sup> was typical for the C=O stretching vibration of methylesterified carbonyl groups of pectic PSs (ν(C=O)COOH). The bands at 1630 and 1438 cm−<sup>1</sup> , together with that at 1440 cm−<sup>1</sup> , were due to the asymmetric νas(O=CO–) and symmetric νs(O=CO–) stretching vibrations of the non- and esterified carboxyl groups, respectively. The signals at 1523–1525 cm−<sup>1</sup> could originate from the aromatic ring vibrations of residual co-extracted phenolics in PSs because their protein content was low (Table 4). The presence of a band at 1238 cm−<sup>1</sup> corresponded to –C–O stretching of the acetyl residue, which was consistent with a higher value of the degree of acetylation (Table 4). In addition, bands at 1145 cm−<sup>1</sup> , 1101 cm−<sup>1</sup> , and 1018 cm−<sup>1</sup> could be interpreted with the glycosidic bond vibrations involving ν(C–OC), –ν(CC)(CO), and –δ(O=C–H) bending in the pyranose ring. The C1-OH vibrations of the monosaccharide ring at 831 cm−<sup>1</sup> showed the presence of *α*-anomeric configuration, which is typical for galacturonic acid in pectins.

It was apparent that the characteristic infrared bands neither disappeared from the spectra nor appeared new ones. At first sight, according to these spectra, there was no reason to suppose that gamma irradiation caused a negative effect on the chemical composition of pectic PS. It is possible for the changes to have been minor, as they could not be seen in the spectra. However, an increase in the band intensities suggested some changes in the amorphous state of PSs, as demonstrated for starch.

#### *2.5. Scanning Electron Microscopy (SEM) of the Fruit Material*

If the gamma irradiation of the fruits caused severe damage to the components of the plant cell walls and tissues, they would be reflected in a change in the surface morphology of the plant material. Therefore, we examined the surface and internal parts of non-irradiated and 25 kGy-irradiated RH fruit material. Figure 5 shows the generated images of the internal and external parts of the fruits. No characteristic changes in the morphology of the surface (exocarp) of the irradiated sample were observed, despite the fact that a very large part of the surface was carefully inspected (Figure 5A,B). However, the examination of the internal part (mesocarp) of the 25 kGy-irradiated fruits revealed that the inner walls became less thick, and the cavities were enlarged (Figure 5C,D). Nevertheless, a more precise histochemical test must be performed to help confirm the latter statement.

meric configuration, which is typical for galacturonic acid in pectins.

2.4.3. Fourier Transformed Infrared (FT-IR) Spectroscopy

The FT-IR spectra of the studied PSs are presented in Figure 4. FT-IR can identify the presence of specific functional groups prevailing in the sample. The typical bands for pectic PSs were found [18]. The broadened band that occurs in the region ranging from 3730 to 3100 cm−1 was assigned to the ν(OH) stretching vibrations of free hydroxyl groups involved in intra- and intermolecular H-bonding. The band at 2940–2960 cm−1 was attributed to the ν(CH) stretching of CH2 groups. The presence of a well-separated sharp signal that appears at 1745–1735 cm−1 was typical for the C=O stretching vibration of methyl-esterified carbonyl groups of pectic PSs (ν(C=O)COOH). The bands at 1630 and 1438 cm−1, together with that at 1440 cm−1, were due to the asymmetric νas(O=CO–) and symmetric νs(O=CO– ) stretching vibrations of the non- and esterified carboxyl groups, respectively. The signals at 1523–1525 cm−1 could originate from the aromatic ring vibrations of residual co-extracted phenolics in PSs because their protein content was low (Table 4). The presence of a band at 1238 cm−1 corresponded to –C–O stretching of the acetyl residue, which was consistent with a higher value of the degree of acetylation (Table 4). In addition, bands at 1145 cm−1, 1101 cm−1, and 1018 cm−1 could be interpreted with the glycosidic bond vibrations involving ν(C–OC), –ν(CC)(CO), and ––δ(O=C–H) bending in the pyranose ring. The C1-OH vibrations of the monosaccharide ring at 831 cm−1 showed the presence of *α*-ano-

**Figure 4.** FT-IR spectra of the PSs isolated from non-irradiated (**A**), 10 kGy- (**B**), and 25 kGy-irradiated (**C**) RH fruits. **Figure 4.** FT-IR spectra of the PSs isolated from non-irradiated (**A**), 10 kGy- (**B**), and 25 kGy-irradiated (**C**) RH fruits. nation of the internal part (mesocarp) of the 25 kGy-irradiated fruits revealed that the inner walls became less thick, and the cavities were enlarged (Figure 5C,D). Nevertheless,

a more precise histochemical test must be performed to help confirm the latter statement.

It was apparent that the characteristic infrared bands neither disappeared from the

**Figure 5.** SEM images of the surface of non-irradiated (**A**) and 25 kGy-irradiated (**B**) RH fruit, and the internal part of non-irradiated (**C**) and 25 kGy-irradiated (**D**) RH fruit (8 kV, 0.1 mm, 500×). **Figure 5.** SEM images of the surface of non-irradiated (**A**) and 25 kGy-irradiated (**B**) RH fruit, and the internal part of non-irradiated (**C**) and 25 kGy-irradiated (**D**) RH fruit (8 kV, 0.1 mm, 500×).

In general, gamma irradiation is a low-cost technology for the sterilization and decontamination of foods. However, together with the positive results, irradiation brings

pends upon the chemical composition (for instance, water, lipids, proteins, and PSs), and thus the changes in the level of constituents have an immediate impact on them. Therefore, in the current study, RH was investigated in detail to determine whether the gammairradiation processing contributed to the changes in the phytochemical constituents of the

Our research findings indicated that the carbohydrate constituents of RH fruits were among the most seriously affected by irradiation. Irradiation contributed to an increase in the reducing sugars content (mainly glucose), which can be ascribed to the structural breakdown of cellulose and, to a lesser extent, starch. A similar effect of gamma radiation on reducing the sugar content was observed in a previous study on the irradiation of dates [19]. According to these authors, irradiation (0.9–1.8 kGy) contributed to a high level of

*3.1. Influence of Gamma Irradiation on Carbohydrate Constituents*

**3. Discussion**

fruits.

#### **3. Discussion**

In general, gamma irradiation is a low-cost technology for the sterilization and decontamination of foods. However, together with the positive results, irradiation brings about a negative impact on the nutritional and functional properties of the foods. This effect is strongly determined by the received irradiation dose [3]. The quality of food depends upon the chemical composition (for instance, water, lipids, proteins, and PSs), and thus the changes in the level of constituents have an immediate impact on them. Therefore, in the current study, RH was investigated in detail to determine whether the gamma-irradiation processing contributed to the changes in the phytochemical constituents of the fruits.

#### *3.1. Influence of Gamma Irradiation on Carbohydrate Constituents*

Our research findings indicated that the carbohydrate constituents of RH fruits were among the most seriously affected by irradiation. Irradiation contributed to an increase in the reducing sugars content (mainly glucose), which can be ascribed to the structural breakdown of cellulose and, to a lesser extent, starch. A similar effect of gamma radiation on reducing the sugar content was observed in a previous study on the irradiation of dates [19]. According to these authors, irradiation (0.9–1.8 kGy) contributed to a high level of glucose, but it could not bring about a significant change in the fructose content. Moreover, scientists highlighted the fact that the decrease in starch content is paralleled by an increase in the glucose amount [19]. Our results on cellulose and glucose content follow a similar pattern. It is useful to recall that cellulose is a glucose-containing polymer organized into stable microfibrils with crystalline and amorphous regions. Cellulose meshes into a cell wall polymer matrix together with pectin, lignin, and hemicellulose. More importantly, cellulose fibers play a crucial role, mainly in maintaining the strong mechanical resistance of plants [20]. Therefore, it can be safely predicted that irradiation (≥10 kGy) leads to a change in the mechanical properties of dried RH fruits. It is interesting to speculate whether the strength of this effect can be increased after the pectic backbone also suffers serious damage as a result of irradiation. Pectin is an amorphous heteropolysaccharide found in the middle lamella, which is composed of the HG, rhamnogalacturonan-I, and rhamnogalacturonan-II regions. HG is the main pectic structural fragment that only consists of an unbranched chain of *α*-1,4-linked galacturonic acid units. These residues can be methyl-esterified and/or acetyl-esterified. Our results strongly suggest that RH pectin also mostly consists of HG (Table 5; ≈50 mol%). For that reason, together with a decrease in the molecular weight before and after *endo*-PG action (Figure 3A,B), we assumed HG to be mainly affected. The depolymerization of HG is a clear sign pointed to contribute to an undesired defect of RH fruits. Further, monosaccharide composition analysis revealed that gamma irradiation contributed only to the reduction in the glucose content, while that of fucose was raised. Glucose, in this regard, is not a typical constituent of pectin. However, this observation could be explained by the decomposition of very closely crosslinked pectin and cellulose complexes. Apart from that, it can be speculated that a higher amount of fucose in 25 kGy-irradiated PS could be due to an increased extractability of fucose-containing rhamnogalacturonan-II segments of pectin.

The degradation of cellulose and pectin by gamma irradiation was investigated in previous studies [21–23]. None of these studies, however, investigated the changes in polymer nature directly in the plant material. In contrast, observations of the effects of radiation were performed on purified dry substances and/or sugar-containing solutions (for pectin). As a sign of degradation, authors mainly used the changes in the viscosities of the solution of the irradiated samples. Concerning cellulose, the authors' findings suggested that crystallinity and the amorphous region were changed due to additional oxidation seen by a decrease in the degree of polymerization and an increase in the absorbance of the carbonyl groups band at 1730–1750 cm−<sup>1</sup> [21,23]. It should be noted that the alteration in cellulose was observed at dosages (100–2000 kGy) far exceeding ours (10 and 25 kGy). As regards the pectin, an increase in the degree of heterogeneity and a decrease in specific viscosity of 1% citrus pectin solution occurred on irradiation at a high dose level (20.56 kGy) [24]. Other

scientists demonstrated by viscosity measurements and jelly tests that, unlike dried pectin, pectin in solution degraded more rapidly by irradiation (10–40 kGy). Interestingly, the jellies were not affected [25]. Our findings concerning the changes in the molecular weights were in accordance with another study that dealt with apple pectin exposed to 0.5–10 kGy of gamma radiation. On the contrary, the degree of esterification was a sharp contrast to our result [26]. However, our result is in agreement with a previous study on the examination of pectin solution exposed to 0.1–1.0 kGy of radiation, where any changes in methanol content were not confirmed [27]. It can be concluded that the differences that can be observed with the other results lie in the way the plant material is prepared (fresh or dried), the amount of carbohydrate constituents, and last but not least the irradiation doses applied.

#### *3.2. Influence of Gamma Irradiation on Organic Acids*

Concerning organic acids, we found evidence to suggest that the exposition of RH fruits to gamma radiation had a negligible effect on the content of quinic, malic, and ascorbic acids. However, most previous studies showed that ascorbic acid was highly susceptible to irradiation. It has been noted that an oxidized dehydroascorbic acid was formed as a direct result of irradiation. For example, the exposition of dried lycium fruit to gamma irradiation (2–14 kGy) had a devastating effect on ascorbic acid at higher doses (14 kGy)—close to the used in the current study [28]. An appreciable loss of ascorbic acid was also found in spice and herb extracts of black pepper, cinnamon (32%), nutmeg (48%), oregano, and sage after being exposed to irradiation (10 kGy) [29]. However, another study indicated that vitamin C content had not been dramatically affected when using a lower dose of radiation (0.15–1 kGy) [30]. The thing to remember is that ascorbic acid is a major water-soluble antioxidant molecule in RH fruits, and therefore the lack of change in its amount was an important finding for the preservation of the quality of treated fruits. Furthermore, it has little information about the changes in the citric and malic acid contents [31]. These authors linked the changes in the citric acid content of fresh potatoes exposed to irradiation (150 Gy) to the storage temperatures and time. A marked increase in citric acid content, for example, was observed at the sixth month of storage, whereas malic acid did not significantly change [31]. It should be noted that great care is needed when interpreting these results, because a large variety of experimental patterns occur.

#### *3.3. Influence of Gamma Irradiation on Lipid Constituents*

Our results reveal that irradiation at doses 10 and 25 kGy have no significant effect on the RH seed fat content, fatty acid composition, and carotenoids amount. Although a slight increase in the acid value, conjugated dienes, and trienes, together with a decrease in the tocopherols were detected, the oxidative stability of oil presented by the induction period of autoxidation was not practically changed. The scientific literature on the topic was not full of examples of irradiated RH fruits and seed oil in particular, and thus to be compared with our results. To date, to our knowledge, only one study, which has not specifically dealt with RH fruits, demonstrated that the carotenoid content of RH tea was not influenced by a dose of up to 10 kGy, probably due to the very low moisture content [14]. Our results generally support this view. It all points to the conclusion that not only plant sources, but also irradiation and post-irradiation storage conditions (such as temperature, light, and oxygen) were all important factors in cases of different lipid compositions.

#### *3.4. Influence of Gamma Irradiation on Phenolic Constituents and In Vitro Antioxidant Activity*

Our study provided evidence to prove that gamma irradiation did not have a dramatic effect on the content of the phenolic components of dried RH fruit. However, it is a known fact that gamma irradiation can affect the number of antioxidant components in plant foods. It could be attributed either to the increased ability of a plant to produce radioprotective antioxidants, or the increased extractability of phenolic antioxidants from the degraded plant matter [32]. The literature contains some observations on the effect of gamma irradiation on the antioxidant activity and polyphenol content of food matrices, but researchers found

conflicting results. For example, the total polyphenol content and antioxidant activity of pomegranate peel powder and dried apricots increased as a result of irradiation, whereas no changes were observed in navel oranges [33–35]. The effect of gamma rays on foodstuffs depends on many factors, such as the radiation dose, moisture content, and the type and amount of endogenous polyphenols [32]. The enhanced antioxidant activity of irradiated plant material was mainly attributed to the increased enzyme activities or to the increased extractability from degraded plant tissues. On the other hand, gamma irradiation could reduce the antioxidant activity, which can be ascribed to the irradiation-derived degradation components or the formation of free radicals [32]. In our study, despite the degradation of dried RH matter, no significant changes in both the polyphenol content and the antioxidant activity of the treated samples were observed. This can be attributed to the lower moisture content of RH, which drastically limited enzymes' activity, to the absence of the oxidative changes in irradiated fruits, and/or the impossibility of polyphenols taking part in the process of inactivating free radicals. This effect was in agreement with the findings of other authors, who observed that the total polyphenols in linden flowers did not alter during irradiation (<10 kGy) [14].

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

#### *4.1. Fruit Material*

The RH fruit material (an orange-red color; reached maturity; 47% dry matter) was obtained from a local producer (Smolyan, the Rhodope Mountains, Bulgaria). The material was enclosed in polyethylene bags and stored frozen (−18 ◦C) before further freeze-drying (<10% moisture content).

#### *4.2. Gamma Irradiation*

Before irradiation, the fruit flesh was separated from the seeds mechanically. The samples were irradiated separately at a radionuclide <sup>60</sup>Co source with 8200 Ci activity. The gamma rays facility has a mobile irradiation chamber with 4.0 L volume and dimensions: 13.5 cm diameter and 22 cm height. During the irradiation, the chamber rotates on its vertical axis. For the study of the absorbed dose, distribution Alanine dosimeters (Kodak BioMax) were used, measured by ESR spectrometer E-scan Bruker, and calibrated in units of absorbed dose in water. At each point, three dosimeters were placed. The chosen absorbed doses were 10 kGy and 25 kGy. The fruit flesh and seeds (control and irradiated samples) were kept in polyethylene bags at room temperature. The irradiation of samples was performed in the National Center of Radiobiology and Radiation Protection at the Ministry of Health (Sofia, Bulgaria). Irradiated fruits and seeds, together with non-irradiated (control) samples were subjected to analyses immediately after irradiation.

#### *4.3. Chemical Characterization of Non-irradiated and Irradiated Fruits*

#### 4.3.1. Preparation of Alcohol-Insoluble Solids

RH fruits (non-, 10 kGy-, and 25 kGy-irradiated) without seeds were used for the preparation of AIS. For that purpose, the initially ground samples were incubated at 50 ◦C for 1 h with 70% (*v*/*v*) aqueous ethanol and then the solids were separated by centrifugation (18.187× *g*, 10 min, 5 ◦C). The same procedure was repeated 3 times. Finally, the residue was washed 2 times with acetone at room temperature and vacuum-dried until there was no change in the mass.

#### 4.3.2. Crude Protein Content

The crude protein content was evaluated by the micro-Kjeldahl method. The determination of nitrogen expressed as the ammonia content of the digested sample was performed by the acetylacetone–formaldehyde colorimetric method using ammonium sulfate as a standard [36]. The results were calculated using 6.25 as a conversion factor.

#### 4.3.3. Total Uronic Acid and Cellulose Content

The uronic acid content of the plant material was estimated as described [37]. As a sample for the analysis served AIS-prepared as described above (Section 4.3.1). In brief, the sample was suspended in 72% (*w*/*w*) H2SO<sup>4</sup> (1 h, 30 ◦C), and after dilution with water to 1 M H2SO4, hydrolysis was achieved through further incubation for 3 h at 100 ◦C. An aliquot of hydrolysate was taken for an automated 3-phenylphenol colorimetric analysis using a continuous flow analyzer Skalar San++ system (Skalar Analytical BV, Breda, the Netherlands). The analysis was conducted according to the instructions of the manufacturer. Absorption was measured at 530 nm and galacturonic acid (12.5–100.0 µg/mL) was used for a calibration curve construction.

The quantitative estimation of cellulose in control and irradiated plant material was performed according to the gravimetric method of Kürschner and Hanak with a modification [38]. Briefly, a sample (0.5 g) was gently boiled (30 min) with 25 mL of acetic acid-HNO<sup>3</sup> reagent (acetic acid: H2O:HNO<sup>3</sup> 8:2:1 *v*/*v*/*v*) in a round-bottom flask fitted with a reflux condenser. After cooling the insoluble residue was filtered through a sintered glass filter (G3) under vacuum, washed with deionized water to neutral pH, then with ethanol (96% *v*/*v*), and finally with an excess of petroleum ether. The obtained residue was dried in a laboratory oven at 50 ◦C to a constant weight. The resulting cellulose was corrected for its ash content.

#### 4.3.4. Total Lipid Content

The total lipid content of the seeds was determined gravimetrically after an exhaustive extraction with *n*-hexane in a Soxhlet extractor (8 h) [39].

#### 4.3.5. Total Carotenoids, β-Carotene, and Tocopherol Content

For the estimation of the total carotenoid content of the fruit flesh, 1 mL methanol, 2 mL chloroform, and 1 mL of 50 mM Tris buffer (pH 7.5, containing 1 M NaCl) were added to 20 mg of the ground sample. The mixture was centrifuged (6000 rpm, 10 min), and after that, the chloroform-containing bottom phase was removed. The upper phase was re-extracted with 2 mL chloroform, and after centrifugation, two chloroform phases were combined, dried under a stream of nitrogen, re-dissolved in a mixture of cyclohexane and dichloromethane (9:1 *v*/*v*), and the absorbance of the solution was then measured at 445 nm (Cecil Series 8000 UV/VIS spectrophotometer, Cecil Instruments Ltd., Cambridge, UK). A calibration curve constructed with *β*-carotene was used for the quantification [40].

The quantities of *β*-carotene and tocopherols were performed by a high-performance liquid chromatography (HPLC) analysis of a hexane solution of oil. The analyses were conducted by Agilent 1100 apparatus equipped with a DAD detector operating at 292, 298, and 450 nm for the monitoring of *α*-, *γ*- tocopherols, and *β*-carotene, respectively, on a Nucleosil 100-5 (250 mm × 4.6 mm, 5 µm) column connected with a pre-column EC 4/3 Nucleosil 100-5 (Macherey-Nagel). The elution of tocopherols was performed with a mobile phase of hexane and tetrahydrofuran (96:4 *v*/*v*), at a flow rate of 1 mL/min [41], whereas *β*-carotene was eluted with a mixture of *n*-hexane and 2-propanol (98:2 *v*/*v*) at 1.0 mL/min. The identification of different analytes was performed by the comparison of retention times with those of individual standards, while the concentration was calculated using calibration curves.

#### 4.3.6. Conjugated Dienes and Trienes Contents

The conjugated dienes and trienes contents of RH seed oils were estimated by measuring the absorbance at 232 nm and 268 nm, respectively, of 1% solution of oil in *iso*-octane (Cecil Series 8000 UV/VIS spectrophotometer) with reference of pure solvent [42].

#### 4.3.7. Acid Value, Peroxide Value, and Induction Period

The acid value of RH seed oils was determined titrimetrically with ethanolic KOH [43]. The peroxide value, expressed as meq O2/kg oil, was determined according to the modified

iodometric method of Yanishlieva et al. [44]. The induction period was evaluated as follows: oxidation of oil (2 g) was carried out at 100 ◦C by blowing a stream of air (50 mL/min). The peroxide value was estimated periodically at different intervals during oxidation and the increase in the value was monitored graphically. The induction period was determined by the method of Le Tutour and Guedon [45].

#### 4.3.8. Fatty Acid Composition

The fatty acid composition was determined by gas chromatography (GC) with a flame ionization detector after transesterification of oil to methyl esters with 1% H2SO<sup>4</sup> in methanol [46]. Prior to the GC analysis, fatty acid methyl esters (FAMEs) were purified by thin-layer chromatography on a silica gel plate with a mobile phase composed of hexaneacetone (100:6 *v*/*v*). GC was conducted on a Shimadzu 17A apparatus coupled to a flame ionization detector using a Simplicity-wax (30 m × 0.32 mm × 0.25 µm, Supelco) column. The separation took place at the following temperature regime: from 170 ◦C, at 2 ◦C/min to 260 ◦C (5 min). The injector and detector temperatures were kept at 260 ◦C and 280 ◦C, respectively. Helium was used as a carrier gas at a flow rate of 0.5 mL/min and a split ratio of 1:50. The peak identification was according to retention times compared to that of a standard mixture of FAMEs.

#### 4.3.9. HPLC Determination of Free Sugars

About 1 g of the ground sample was extracted with a 3% solution of *meta*-phosphoric acid in water (30 mL) for 1 h at 30 ◦C shaking on a thermostatic water bath (NÜVE, Turkey). The residue and extract were separated through centrifugation (6000×*g*, 20 min) and an additional filtration through a PTFE filter (0.45 µm). The filtrate was taken for chromatographic analysis. The separation of sugars was performed on a ZORBAX Carbohydrate (5 µm, 4.6 × 150 mm, Agilent, Santa Clara, CA, USA) and a ZORBAX Reliance Cartridge guard column connected to an Agilent 1220 Infinity HPLC system with a 1260 refractive index detector (RID). The elution was performed at a flow rate of 1.0 mL/min at 25 ◦C with a mobile phase composed of acetonitrile and water (80:20 *v*/*v*). The concentration of sugars in the sample was deduced using a calibration curve constructed by plotting the peak area against five different concentrations for each sugar. The peak corresponding to different sugars in the sample was confirmed by a comparison of retention time with that of the standards.

#### 4.3.10. HPLC Determination of Organic Acids

HPLC determination of organic acids was performed on an HPLC system (Agilent 1220, Agilent Technology, Santa Clara, CA, USA), with a binary pump and UV-Vis detector (Agilent Technology, USA). Separation was performed on an Agilent TC-C18 column (5 µm, 4.6 mm × 250 mm) at 25 ◦C. Twenty microliters of the extract, prepared as described in 4.3.9., were injected and eluted (1.0 mL/min) isocratically with a 25 mM solution of K2HPO<sup>4</sup> in water, whose pH was finely adjusted to 2.4 with H3PO4. The UV detector was set at 210 nm. The concentration of each organic acid in the sample was calculated using a calibration curve obtained by using five different concentrations for each acid. The peak corresponding to different acids was confirmed by a comparison of the retention time with that of the standards.

#### 4.3.11. HPLC Determination of Phenolic Components

Before analysis, the finely ground (<500 µm) sample (0.5 g) was mixed with 40 mL of a solvent containing 60% ethanol in 0.5% formic acid, and further constantly stirred at room temperature for 1 h. The mixture was centrifuged (6000× *g*, 20 min) and the supernatant was then used for HPLC analysis, total polyphenols, and in vitro antioxidant activity determination. Different phenolic components were determined using an HPLC system (Agilent 1220, Agilent Technology, USA), with a binary pump and UV-Vis detector (Agilent Technology, USA). Separation was performed on an Agilent TC-C18 column (5 µm, 4.6 mm × 250 mm) at 25 ◦C and a wavelength of 280 nm was used. The following mobile phases were used: 0.5% acetic acid (A) and 100% acetonitrile (B) at a flow rate of 0.8 mL/min. The gradient elution started with 14% B, between 6 min and 30 min, linearly increased to 25% B, and then to 50% B at 40 min. The identification of the compounds was confirmed by a comparison of retention times utilizing the standard solutions and standard calibration curves of different phenolics.

#### 4.3.12. Total Polyphenolic Content and In Vitro Antioxidant Activity

The total phenolic content was determined according to the method of Singleton and Rossi with Folin–Ciocalteu's reagent [47]. Gallic acid (10–200 µg/mL) was employed as a calibration standard. The oxygen radical absorbance capacity (ORAC) and hydroxyl radical averting capacity (HORAC) were measured according to the methodology used by Denev et al. (2010) [48]. Both analyses were carried out on a FLUOstar OPTIMA plate reader (BMG Labtech, Ortenberg, Germany).

#### 4.3.13. Scanning Electron Microscopy

The surface and internal topography and composition of the dried non- and 25 kGyirradiated RH fruit flesh (non-ground) were examined by a digitalized SEM Philips 515 (accelerating voltage 8 kV, magnification 500×, 0.1 mm) equipped with an Everhart–Thornley secondary and Robinson backscattered electron detector (Philips, Amsterdam, the Netherlands). An SC7620 Mini Sputter Coater (Quorum Technologies, Laughton, East Sussex, UK) was used for coating specimens prior to examination with the microscope. The preparation and examination of the specimens were carried out in the Laboratory of Electron Beam Microscopy, Institute of Optical Materials and Technologies "Acad. Jordan Malinowski" (Bulgarian Academy of Sciences, Sofia, Bulgaria).

#### *4.4. Extraction of the Polysaccharide Fraction*

The ground sample (dried non-, 10 kGy-, and 25 kGy-irradiated fruit flesh of RH) was extracted with distilled water at 80 ◦C for 1 h, mixing the plant material and solvent in a ratio of 1 to 25 (*w*/*v*). After cooling, the solid was separated from the liquid through centrifugation (4428× *g* for 30 min at 20 ◦C) and an additional Büchner funnel filtration (filter paper, KA-4, OP Papírna, s.r.o., Olšany, Czech Republic). To the filtrate, 3.0 volumes of 96 % (*v*/*v*) cold ethanol were added, and after standing overnight at 4 ◦C, the resulting precipitate was collected by centrifugation (20 min, 4 ◦C, 3490× *g*). Then, the precipitate was re-dissolved in distilled water, dialyzed (mwco 12,000–14,000 Da; VISKING®, SERVA Electrophoresis) for 72 h against distilled water (4 ◦C), and freeze-dried.

#### *4.5. Endo-Polygalacturonase (endo-PG) Hydrolysis of the Polysaccharide Fraction*

Initially, PSs (2 mg/mL), isolated as described in 4.4., were dissolved in 50 mM of sodium acetate buffer (pH 5.2) and incubated for 24 h at 40 ◦C with *endo*-PG-I-M2 (EC <IP_ADDRESS>; *Aspergillus aculeatus*; 5000 U/mL; 0.16 U/mL [S]) purchased from Megazyme International Ltd. (Bray, Co., Wicklow, Ireland). Finally, the enzyme was inactivated at 95 ◦C for 5 min, and after cooling the digest was centrifuged at 18.187× *g*, at 5 ◦C for 10 min. The supernatant obtained was further analyzed for the molecular weight distribution.

#### *4.6. Physico-Chemical Characterization of Polysaccharides*

#### 4.6.1. General Analytical Methods

For the estimation of the total uronic acid content of the PSs, appropriately diluted solutions of PSs were directly assayed by the colorimetric method as described above (4.3.3.). For the estimation of the degree of methyl esterification, PSs (1 mg/mL) were saponified (0.5 M NaOH) and after neutralization (1.0 M HCl) the quantity of methanol was evaluated using a combined alcohol oxidase/4-amino-5-hydrazino-1,2,4-triazole-3-thiol (Purpald®) method [49]. The analysis was carried out according to the methodology used by Anthon and Barrett [50]. The acetyl content was determined photometrically by the hydroxamic acid reaction method of McComb and McCready, using *β*-D-glucose pentaacetate (24–120 µg/mL) as a standard [51]. The degrees of methylation and acetylation were expressed as moles of methyl esters or acetyl groups per 100 moles of uronic acid, respectively. The protein content was estimated by the dye-binding method of Bradford using Coomassie® Brilliant blue G-250 dye (Amresco®) and bovine serum albumin as a standard [52].

#### 4.6.2. Monosaccharide Composition Analysis

The monosaccharide composition analysis of non-irradiated and 25 kGy-irradiated PSs was carried out according to the method of Honda et al. as modified by Yang et al. [53,54]. In brief, the PSs were hydrolyzed (4M TFA, 8 h at 110 ◦C) and then the released monosaccharides were derivatized employing 1-phenyl-3-methyl-5-pyrazolone (PMP) to UV-absorbing products. The resulting PMP derivatives were separated on an Agilent 1220 HPLC system with a UV detector (250 nm). The separation was conducted on an Agilent TC-C18 (4.6 × 250 mm, 5 µm) column with a mobile phase consisting of 50 mM phosphate buffer (Na2HPO4-NaH2PO4, pH 6.9) and acetonitrile using a gradient elution [54].

#### 4.6.3. Molecular Weight Distribution Analysis

PSs samples (initial and *endo*-PG-digested) were analyzed using an HPSEC Agilent 1220 Infinity LC system coupled with a 1260 RID detector using an Agilent Bio SEC-3 (300 Å, 4.6 × 300 mm, 3 µm) column. The elution was performed with a mobile phase of 150 mM NaH2PO<sup>4</sup> (pH 7.0) employing a flow rate of 0.5 mL/min. Pullulan standards (Shodex 159 standard P-82 kit, Showa Denko, Kawasaki, Japan) with molecular weights in the range of 0.59 <sup>×</sup> <sup>10</sup><sup>4</sup> to 78.8 <sup>×</sup> <sup>10</sup><sup>4</sup> g/mol were used for the construction of a standard curve by plotting the logarithm of the molecular weight and retention time.

#### *4.7. FT-IR Spectroscopy*

The sample (4 mg) was mixed with spectroscopic grade KBr and was then pressed into a pellet. FT-IR spectrum was collected on a Nicolet Avatar 330 (Thermo Electron Corp., Waltham, MA, USA) spectrometer. The spectrum was recorded over a wavenumber range of 4000–400 cm−<sup>1</sup> at 132 scans with a spectral resolution of 4 cm−<sup>1</sup> . The analysis and processing of the obtained spectrum were performed using the Spekwin32 software (version 1.71.5).

#### *4.8. Statistical Analysis*

Measurements were carried out in triplicate. The results are presented as a mean value of parallel determinations ± standard deviation and were compared by the Student's *t*-test (MS Excel 2010 software).

#### **5. Conclusions**

For the first time, an in-depth study entirely devoted to the examination of the effect of gamma irradiation on phytochemical constituents of dried RH fruits was carried out. What is more, the current study is the first provide valuable insight into the cell wall PS constituents of irradiated RH fruits. To our knowledge, the gathered data for carbohydrate, organic acids, and lipid composition are also reported for the first time in this study. The results of our experiment indicate that the gamma irradiation of dried RH does not significantly change the content of fructose, most organic acids, fatty acids, *β*-carotene, and total polyphenols. By contrast, cellulose and pectin, which constituted the majority of plant cell walls, were adversely affected proportionately to the irradiation dose (0 < 10 < 25 kGy), which was indicated by changes in the yield, sugar content, and molecular weight distribution pattern. Therefore, our findings could be interpreted in various ways. On the one hand, it seems reasonable to suggest that the exposition of dried RH fruits on doses less than or even equal to 25 kGy have a negligible effect on the lower molecular weight constituents (such as some lipids, sugars, pigments, organic acids, and polyphenols). This means that some nutritional and functional properties of food predetermined by these components would not significantly change. On the other hand, it seems that PSs constituents may be affected by doses even lower than 10 kGy. However, the fruits may change their textural properties when their PS components are changed/decomposed, thus exerting an influence on the way the fruit is further technologically processed. In addition, there is an expectation that pectin would change its jellifying properties. Nevertheless, it could be compromised that the exposition of dried RH up to 10 kGy is safe and can be used to decontaminate fruits, which are very often used as an ingredient in fruit-based teas and functional beverages.

**Author Contributions:** Conceptualization, S.M., P.D., and M.O.; methodology, M.O.; formal analysis, M.O.; investigation, M.O., D.T., Y.G., S.T., I.T., M.K.-N., and Y.N.; resources, S.M. and P.D.; data curation, M.O., Y.G., P.D., and S.M.; writing—original draft preparation, M.O.; writing—review and editing, P.D., S.M., and Y.G.; visualization, M.O.; supervision, S.M., P.D., and M.O.; project administration, S.M.; funding acquisition, S.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Bulgarian National Science Fund, grant number DN 19/14 from 12 December 2017.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The first author is very grateful to Galina Maleshkova and Daniela Karashanova from the Laboratory of Electron Beam Microscopy at the Institute of Optical Materials and Technologies "Acad. Jordan Malinowski" (Bulgarian Academy of Sciences, Sofia) for conducting the SEM of the samples and the useful discussions. He owes Irina Yanakieva a great debt of gratitude for her valuable technical assistance in this work.

**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**

10. Denev, P.; Lojek, A.; Ciz, M.; Kratchanova, M. Antioxidant activity and polyphenol content of Bulgarian fruits. *Bulg. J. Agric. Sci.* **2013**, *19*, 22–27.

## *Article* **L-Lysine-Based Gelators for the Formation of Oleogels in Four Vegetable Oils**

**Qiannan Li, Jieying Zhang, Guiju Zhang \* and Baocai Xu \***

School of Light Industry, Beijing Technology and Business University, No. 11 Fucheng Road, Haidian District, Beijing 100048, China<EMAIL_ADDRESS>(Q.L<EMAIL_ADDRESS>(J.Z.)

**\*** Correspondence<EMAIL_ADDRESS>(G.Z<EMAIL_ADDRESS>(B.X.)

**Abstract:** Supramolecular oleogel is a soft material with a three-dimensional structure, formed by the self-assembly of low-molecular-weight gelators in oils; it shows broad application prospects in the food industry, environmental protection, medicine, and other fields. Among all the gelators reported, amino-acid-based compounds have been widely used to form organogels and hydrogels because of their biocompatibility, biodegradation, and non-toxicity. In this study, four Nα, N<sup>ε</sup> -diacyl-L-lysine gelators (i.e., Nα, N<sup>ε</sup> -dioctanoyl-L-lysine; Nα, N<sup>ε</sup> -didecanoyl-L-lysine; Nα, N<sup>ε</sup> -dilauroyl-L-lysine; and Nα, N<sup>ε</sup> -dimyristoyl-L-lysine) were synthesized and applied to prepare oleogels in four kinds of vegetable oils. Gelation ability is affected not only by the structure of the gelators but also by the composition of the oils. The minimum gel concentration (MGC) increased with the increase in the acyl carbon-chain length of the gelators. The strongest gelation ability was displayed in olive oil for the same gelator. Rheological properties showed that the mechanical strength and thermal stability of the oleogels varied with the carbon-chain length of the gelators and the type of vegetable oil. The microstructure of oleogels is closely related to the carbon-chain length of gelators, regardless of oil type. The highest oil-binding capacity (OBC) was obtained in soybean oil for all four gelators, and Nα, N<sup>ε</sup> -dimyristoyl-L-lysine showed the best performance for entrapping oils.

**Keywords:** Nα; N<sup>ε</sup> -diacyl-L-lysine; vegetable oil; oleogel; gelator; oil-binding capacity

**1. Introduction**

Gel is a solid-like form of soft matter comprised of a liquid phase as the main component and a low concentration of a molecular gelator [1]. When the mass of the gelator is less than 2000 Da, the corresponding gel is defined as supramolecular gel or low-molecularweight gel (LMWG) [2–6], which is formed by small molecular gelators through supramolecular interactions, including hydrogen-bonding, van der Waals, π-stacking, coordination, donor-acceptor, and charge-transfer interactions.

Two types of gels are commonly described in the literature: organogels and hydrogels [7]. Recently, ionic liquid gels, i.e., gels obtained in ionic liquids [8–10] and eutectogels, i.e., gels obtained in deep eutectic solvents [11,12] have also been described. Organogels are gels in which the liquid phase is an organic solvent, unlike hydrogels, which are gels with a continuous liquid-aqueous phase. Supramolecular organogels can be used in various everyday applications, including processing petroleum, food, pharmaceuticals, or cosmetics [13–15]. If liquid oils are used as organic solvents, the resulting organogels are called oleogels. In general, the solid lipids possess specific characteristics over liquid oils for food products. Thus far, high-melting lipids containing *trans* fatty acids, and saturated fatty acid moieties have been commonly employed. However, it has recently been claimed that the intake of high levels of *trans* and saturated fats contribute to global epidemics related to metabolic syndrome and cardiovascular disease [16]. The mechanistic resemblance of oleogels to *trans* and saturated fats makes liquid oil gelation an ideal alternative in developing fat-based food products. In practical application, both the mechanical and aesthetic

**Citation:** Li, Q.; Zhang, J.; Zhang, G.; Xu, B. L-Lysine-Based Gelators for the Formation of Oleogels in Four Vegetable Oils. *Molecules* **2022**, *27*, 1369. https://doi.org/10.3390/ molecules27041369

Academic Editor: Mirella Nardini

Received: 14 January 2022 Accepted: 16 February 2022 Published: 17 February 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/).

properties of oleogels are vital for food and other products. Modulating various parameters for ideal mechanical and aesthetic properties has gained significant interest in recent years. Currently, a lot of research has been conducted on oleogels, to discover the relationship between gelator structure, the nature of the oil, and gelation ability [17,18]. However, it is still not well understood, due to the complicated systems. A better understanding of these oleogel systems will help to improve applications in cosmetic products, drug delivery and pharmaceuticals, and provide an alternative for making healthy foods that are free of trans fats and contain minimal saturated fats [19].

Among many small molecular gelators reported, amino-acid-based compounds have received tremendous attention because they are available in large quantities as inexpensive starting materials, and synthetic methods are relatively simple and well-established [20–22]. The demand for better and environmentally friendly gelators makes amino-acid-based amphiphiles very attractive, as these compounds are generally biodegradable and biocompatible. There is at least one amide linkage between the hydrophobic tail and the polar headgroup in the structure of amino-acid-based gelators, providing one of the interactions responsible for their supramolecular assembly, i.e., hydrogen bonding [23,24]. The gelation ability also depends on the hydrophobic interactions (van der Waals force) [25,26]. Seeking a balance between hydrogen bonding and van der Waals forces is very important for gelation.

Nα, N<sup>ε</sup> -diacyl-L-lysine and its derivatives are called all-powerful gelators [27–29]. Its ester derivatives can form organogels in a wide variety of organic fluids, such as alkanes, alcohols, ketones, esters, cyclic ethers, aromatic solvents, polar solvents, and mineral and vegetable oils. Its carboxylate derivatives can form hydrogels in an aqueous solution. Nα, N<sup>ε</sup> -diacyl-L-lysine itself has also displayed good organogelation ability. In this study, four Nα, N<sup>ε</sup> -diacyl-L-lysines (i.e., Nα, N<sup>ε</sup> -dioctanoyl-L-lysine; Nα, N<sup>ε</sup> -didecanoyl-L-lysine; Nα, N<sup>ε</sup> -dilauroyl-L-lysine; and Nα, N<sup>ε</sup> -dimyristoyl-L-lysine) were synthesized and used as gelators to prepare oleogels in four kinds of vegetable oils. As the hydrogen-bonding and hydrophobic interactions between gelators and vegetable oils are responsible for the gelation behaviors and the properties of formed oleogels, the chemical structures of both Nα, N<sup>ε</sup> -diacyl-L-lysines (i.e., the carbon-chain length) and oils (i.e., the fatty acid composition) are the key parameters of oleogel systems. The rheological properties, morphology, and oil-binding capacity of formed oleogels were studied, with hopes of providing better insight into the gelation abilities of L-lysine-based gelators with different carbon-chain lengths in different oils.

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

#### *2.1. Materials*

Octanoyl chloride (99%), decanoyl chloride (98%), lauroyl chloride (98%), myristoyl chloride (98%), and L-lysine (98%) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Petroleum ether (bp 60–90 ◦C), anhydrous ethanol (99.8%), sodium hydroxide (≥98%), and hydrochloric acid (37%) were obtained from Beijing InnoChem Science & Technology Co., Ltd. (Beijing, China). Corn germ oil was purchased from Shandong Xiwang Food Co., Ltd. (Tianjin, China). Soybean oil was purchased from COFCO Fulinmen Food Marketing Co., Ltd. (Zouping, China). Olive oil was purchased from Shanghai Jiage Food Co., Ltd. (Shanghai, China). Linseed oil was purchased from Hebei Jiafeng vegetable oil Co., Ltd. (Handan, China). All the reagents were used as received.

#### *2.2. Synthesis and Characterization of N*α*, N*<sup>ε</sup> *-diacyl-*L*-lysines*

The L-lysine and fatty acid chloride were weighed in a molar ratio of 1:2. L-lysine was added into a four-necked flask, followed by a mixture of anhydrous ethanol and deionized water (2:1, *v*/*v*). The reaction mixture was cooled in an ice-water bath under stirring. Then fatty acid chloride was added slowly via a dropping funnel. In the meantime, the pH of the reaction solution was adjusted by an aqueous sodium hydroxide solution (10 wt%) and maintained at 9–10. The reaction continued for 3 h after the dropwise addition. At last,

the reaction solution was taken out and allowed to stand at room temperature for 3–4 h. After acidification with dilute hydrochloric acid, the precipitate was filtered and washed with deionized water, then rinsed with petroleum ether three times, giving a white, solid appearance the final product.

Four Nα, N<sup>ε</sup> -diacyl-L-lysines were characterized by FT-IR, ESI-MS, and <sup>1</sup>H NMR measurements. IR spectra were obtained on a Nicolet iS10 FT-IR Spectrometer (Thermo Fisher Scientific, Madison, WI, USA) using KBr tablets at room temperature. ESI-MS spectra were recorded using an API3200 triple-quadrupole mass spectrometer (AB SCIEX, Redwood, CA, USA). The <sup>1</sup>H NMR spectra were acquired on a DRX-600 NMR spectrometer (Bruker, Ettlingen, German).

#### *2.3. Oleogel Preparation*

The oleogel formation of Nα, N<sup>ε</sup> -diacyl-L-lysines was investigated by the heating– cooling method [30]. The gelator was added to vegetable oil in a sealed vial. Then, the mixture was heated until a transparent solution was obtained, then cooled to room temperature by simply removing it from the heat. The gelation properties were evaluated by inversion of the vial, and the absence of gravitational flow was utilized to determine the successful achievement of gelation. The minimum gel concentration (MGC) for a gelator was determined by weighing up the minimum amount of gelator needed to form a stable gel through the heating–cooling cycle.

#### *2.4. Characterization of Vegetable Oils*

Analysis of the fatty acid composition of the oils was carried out using a 7890A/5975C gas chromatography–mass spectrometry system (GC-MS, Agilent, Santa Clara, CA, USA) equipped with a HP-5MS (30 m × 0.25 mm × 0.25 µm) capillary column. Helium (99.999%) was the carrier gas, with a flow rate of 1.2 mL/min. The initial temperature of 100 ◦C was held for 2 min and increased to 220 ◦C at 10 ◦C/min. The temperature was held at 220 ◦C for 1 min and increased to 230 ◦C at 2 ◦C/min, then held 2 min. Oils were trans-esterified to fatty acid methyl esters for GC-MS analyses. The injection (1 µL) was performed in the split mode at a split ratio of 1:20. The samples were run by electron ionization, and the source temperature and electron energy were 230 ◦C and 70 eV. The auxiliary heater temperature was 200 ◦C.

#### *2.5. Rheological Behavior Measurements*

The rheological analysis of the oleogels was measured by HAAKE MARS III rheometer (Thermo Electron GmbH, Dreieich, Germany). The time dependences of the storage modulus (G0 ) and loss modulus (G00) of oleogels were measured at 25 ◦C with a fixed rotational speed of 1 rad/s and a shear strain of 0.25 Pa. The temperature dependence of the storage modulus (G0 ) and loss modulus (G") for oleogels was determined at a heating temperature from 20 ◦C to 145 ◦C, with a constant rotational speed of 1 rad/s and a shear strain of 0.25 Pa.

#### *2.6. TEM Measurements*

The morphology of oleogels was observed using TEM measurements. The samples were prepared according to the literature method of [21]. Firstly, a small amount of oleogel was cast on carbon-coated copper grids (300 mesh), then the sample was vacuum dried at 50 ◦C, 0.08 MPa for 2 days to evaporate the oils before observation. The microscopic images of oleogels were monitored using an X-MAX JEM-2100 transmission electron microscope (JEOL, Tokyo, Japan), operating at 120 kV.

#### *2.7. Oil-Binding Capacity (OBC) Determination*

The oil-binding capacity was measured according to the literature method of [21,31]. This methodology allows us to understand the degree of oil retention in the oleogel structure. First, 2 mL of the melted oleogel (the concentration corresponded to the MGC of each

gelator) was put into a previously weighed (m1) centrifuge tube and kept at room temperature for 24 h. Then, the tube was weighed (m2) again. Finally, the tube was centrifuged at 10000 rpm for 15 min at room temperature and turned over to drain the released liquid oil. After drainage, the tubes were weighed (m3) again. The oil-binding capacity (OBC) values were calculated by the following equations.

$$\text{Released oil} \left( \% \right) = \frac{\left( \text{m}\_2 - \text{m}\_1 \right) - \left( \text{m}\_3 - \text{m}\_1 \right)}{\text{m}\_2 - \text{m}\_1} \times 100 \tag{1}$$

$$\text{OBC } (\%) = 100 - \text{Released oil } (\%) \tag{2}$$

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

#### *3.1. Preparation and Characterization of* L*-Lysine-Based Gelators*

Four Nα, N<sup>ε</sup> -diacyl lysine-based gelators were synthesized using the Schotten–Baumann reaction previously reported in the literature with minor modifications [32,33]. Briefly, fatty acyl chlorides were used as acyl donors, and reacted with L-lysine under alkaline conditions, followed by acidification with HCl aqueous solution. Four fatty acyl chlorides, i.e., octanoyl chloride, decanoyl chloride, lauroyl chloride, and myristoyl chloride, were applied in this paper. Both the α amino group and the ε amino group of L-lysine readily reacted with a fatty acyl chloride, forming chemically stable amide bonds. Four Nα, N<sup>ε</sup> diacyl <sup>L</sup>-lysine-based gelators were obtained with a yield of 42%−71% (Table 1). In the case of Nα, N<sup>ε</sup> -dimyristoyl-L-lysine, the reason for the low yield was perhaps a low solubility of the reactants in the solvent used, due to the longer carbon-chain length. *Molecules* **2022**, *27*, x FOR PEER REVIEW 9 of 17 *Molecules* **2022**, *27*, x FOR PEER REVIEW 9 of 17 **Table 1.** Product list of four N<sup>α</sup>, N<sup>ε</sup>-diacyl lysine-based gelators. *Molecules* **2022**, *27*, x FOR PEER REVIEW 9 of 17 **Table 1.** Product list of four N<sup>α</sup>, N<sup>ε</sup>-diacyl lysine-based gelators. **Gelator Structure Abbreviation Yield (%)**  *Molecules* **2022**, *27*, x FOR PEER REVIEW 9 of 17 **Table 1.** Product list of four N<sup>α</sup>, N<sup>ε</sup>-diacyl lysine-based gelators. **Gelator Structure Abbreviation Yield (%)**

Nα, Nε-dimyristoyl-L-lysine C14-lys-C14 42 The synthesized four Nα, Nε-diacyl lysines were characterized by FT-IR, ESI-MS, and The synthesized four Nα, N<sup>ε</sup> -diacyl lysines were characterized by FT-IR, ESI-MS, and <sup>1</sup>H NMR. The results are presented below.

Nα, Nε-dimyristoyl-L-lysine C14-lys-C14 42 The synthesized four Nα, Nε-diacyl lysines were characterized by FT-IR, ESI-MS, and 1H NMR. The results are presented below. *Nα, Nε-dioctanoyl-L-lysine (C8-lys-C8)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), The synthesized four Nα, Nε-diacyl lysines were characterized by FT-IR, ESI-MS, and 1H NMR. The results are presented below. *Nα, Nε-dioctanoyl-L-lysine (C8-lys-C8)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]−, while M is molar weight of Nα-octanoyl-Nε-octanoyl-L-lysine. The synthesized four Nα, Nε-diacyl lysines were characterized by FT-IR, ESI-MS, and 1H NMR. The results are presented below. *Nα, Nε-dioctanoyl-L-lysine (C8-lys-C8)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]−, while M is molar weight of Nα-octanoyl-Nε-octanoyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm : 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 18H, 9CH2 ), 1H NMR. The results are presented below. *Nα, Nε-dioctanoyl-L-lysine (C8-lys-C8)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]−, while M is molar weight of Nα-octanoyl-Nε-octanoyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm : 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 18H, 9CH2 ), 1.35 (m, 2H, CH2), 1.47 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.02 (t, *J* = 7.8 Hz, 2H, CH2), 2.10(t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.72 (t, *J* = Nα, N<sup>ε</sup> -dioctanoyl-L-lysine (C8-lys-C8). IR (*v*max, cm−<sup>1</sup> ): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M <sup>−</sup> H]−, while M is molar weight of Nα-octanoyl-N<sup>ε</sup> -octanoyl-Llysine. <sup>1</sup>H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 18H, 9CH2), 1.35 (m, 2H, CH2), 1.47 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.02 (t, *J* = 7.8 Hz, 2H, CH2), 2.10(t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.72 (t, *J* = 4.8 Hz, 1H, NH), 7.97 (d, *J* = 7.8 Hz, 1H, NH), 12.41 (s, 1H, COOH).

1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]−, while M is molar weight of Nα-octanoyl-Nε-octanoyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm : 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 18H, 9CH2 ), 1H NMR (DMSO-*d*6, 600 MHz) δppm : 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 18H, 9CH2 ), 1.35 (m, 2H, CH2), 1.47 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.02 (t, *J* = 7.8 Hz, 2H, CH2), 2.10(t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.72 (t, *J* = 1.35 (m, 2H, CH2), 1.47 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.02 (t, *J* = 7.8 Hz, 2H, CH2), 2.10(t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.72 (t, *J* = 4.8 Hz, 1H, NH), 7.97 (d, *J* = 7.8 Hz, 1H, NH), 12.41 (s, 1H, COOH). 4.8 Hz, 1H, NH), 7.97 (d, *J* = 7.8 Hz, 1H, NH), 12.41 (s, 1H, COOH). *Nα, Nε-didecanoyl-L-lysine (C10-lys-C10)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which Nα, N<sup>ε</sup> -didecanoyl-L-lysine (C10-lys-C10). IR (*v*max, cm−<sup>1</sup> ): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M <sup>−</sup> H]−, while M is molar weight of Nα-octanoyl-N<sup>ε</sup> -octanoyl-L-

1.35 (m, 2H, CH2), 1.47 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.02 (t, *J* = 7.8 Hz, 2H, CH2), 2.10(t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.72 (t, *J* =

was assigned as [M − H]-, while M is molar weight of Nα-octanoyl-Nε-octanoyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 26H, 13CH2),

Nα, Nε-dimyristoyl-L-lysine C14-lys-C14 42

*Nα, Nε-didecanoyl-L-lysine (C10-lys-C10)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O),

*Nα, Nε-didecanoyl-L-lysine (C10-lys-C10)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which

1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]-, while M is molar weight of Nα-octanoyl-Nε-octanoyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 26H, 13CH2), 1.35 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.01 (t, *J* = 7.2 Hz, 2H, CH2), 2.10 (t, *J* = 7.2 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.72 (t, *J* =

1.35 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.01 (t, *J* = 7.2 Hz, 2H, CH2), 2.10 (t, *J* = 7.2 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.72 (t, *J* =

1H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 26H, 13CH2), 1.35 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.01 (t, *J* = 7.2 Hz, 2H, CH2), 2.10 (t, *J* = 7.2 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.72 (t, *J* =

*Nα, Nε-dilauroyl-L-lysine (C12-lys-C12)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]-, while M is molar weight of Nα-lauroyl-Nε-lauroyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85(t, *J* = 7.2 Hz, 6H, 2CH3), 1.23 (m, 34H, 17CH2 ), 1.35 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.65 (m, 2H, CH2), 2.01 (t, *J* = 7.2 Hz, 2H, CH2), 2.10 (t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.71 (t, *J* = 5.4

*Nα, Nε-dilauroyl-L-lysine (C12-lys-C12)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]-, while M is molar weight of Nα-lauroyl-Nε-lauroyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85(t, *J* = 7.2 Hz, 6H, 2CH3), 1.23 (m, 34H, 17CH2 ), 1.35 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.65 (m, 2H, CH2), 2.01 (t, *J* = 7.2 Hz, 2H, CH2), 2.10 (t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.71 (t, *J* = 5.4

*Nα, Nε-dilauroyl-L-lysine (C12-lys-C12)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]-, while M is molar weight of Nα-lauroyl-Nε-lauroyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85(t, *J* = 7.2 Hz, 6H, 2CH3), 1.23 (m, 34H, 17CH2 ), 1.35 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.65 (m, 2H, CH2), 2.01 (t, *J* = 7.2 Hz, 2H, CH2), 2.10 (t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.71 (t, *J* = 5.4

*Nα, Nε-dilauroyl-L-lysine (C12-lys-C12)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]-, while M is molar weight of Nα-lauroyl-Nε-lauroyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85(t, *J* = 7.2 Hz, 6H, 2CH3), 1.23 (m, 34H, 17CH2 ), 1.35 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.65 (m, 2H, CH2), 2.01 (t, *J* = 7.2 Hz, 2H, CH2), 2.10 (t, *J* = 6.0 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 4.12 (m, 1H, CH), 7.71 (t, *J* = 5.4

*Nα, Nε-dimyristoyl-L-lysine (C14-lys-C14)*. IR (*v*max, cm−1): 3326.75 (N-H), 1724.29 (C=O), 1639.58 (C=O, amide band I), 1536.28 (δN-H, amide band II). ESI-MS: *m/z* = 396.8, which was assigned as [M − H]-, while M is molar weight of Nα-myristoyl-Nε-myristoyl-L-lysine. 1H NMR (DMSO-*d*6, 600 MHz) δppm: 0.85 (t, *J* = 6.6 Hz, 6H, 2CH3), 1.23 (m, 42H, 21CH2), 1.35 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.01 (t, *J* = 7.2