Vitamin C (99.66% sodium ascorbate) and vitamin D3 (98.50% cholecalciferol) were purchased from Sigma-Aldrich (New York, NY, USA). In addition, sunflower lecithin (≥40% phosphatidylcholine) was obtained from Jedwards International, Inc. (Braintree, Chicago, IL, USA). Potassium sorbate (PS) (99.47 ± 0.5%) and glycerin (99.80%) were purchased from Quadra Chemicals (Delta, BC, Canada) and EZ Chemicals Inc. (Mississauga, ON, Canada), respectively. Olive oil (100.00%) (Kirkland, WA, USA) and distilled water (ELGA LabWater (Woodridge, IL, USA)) were used for the preparation of all solutions. Methanol (99.80% HPLC grade) and acetonitrile (99.99% HPLC grade) were purchased from VWR international, LLC. Phosphoric acid (85.00% HPLC grade) was obtained from EMD Millipore Corporation (Kankakee, IL, USA). All solvents and samples were filtered through 0.2 µm wwPTFE filter (Pall Corporation (New York, NY, USA)) before injection into the HPLC. purchased from Sigma-Aldrich (New York, NY, USA). In addition, sunflower lecithin (≥40% phosphatidylcholine) was obtained from Jedwards International, Inc. (Braintree, Chicago, IL, USA). Potassium sorbate (PS) (99.47 ± 0.5%) and glycerin (99.80%) were purchased from Quadra Chemicals (Delta, BC, Canada) and EZ Chemicals Inc. (Mississauga, ON, Canada), respectively. Olive oil (100.00%) (Kirkland, WA, USA) and distilled water (ELGA LabWater (Woodridge, IL, USA)) were used for the preparation of all solutions. Methanol (99.80% HPLC grade) and acetonitrile (99.99% HPLC grade) were purchased from VWR international, LLC. Phosphoric acid (85.00% HPLC grade) was obtained from EMD Millipore Corporation (Kankakee, IL, USA). All solvents and samples were filtered through 0.2 μm wwPTFE filter (Pall Corporation (New York, NY, USA)) before injection into the HPLC.

vitamin C and vitamin D, which is homogenous and stable without the requirement of

#### *2.1. Preparation of Liposomal Vitamin C 2.1. Preparation of Liposomal Vitamin C*

A solution was prepared at room temperature (25 ◦C), by adding 0.134 g of potassium sorbate (PS) (99.47 ± 0.5%), and dissolving in purified water (45 mL). Then, 1124 (+5%) mg of vitamin C (99.66% sodium ascorbate) was added to the aqueous solution, and stirred until completely dissolved. There was 5% overage of material input for vitamin C, according to the sample product formula. Next, 55 mL glycerin (99.80%) was added to an aqueous solution, by stirring; the solution was then placed in a blender (Hamilton Beach (Avalon, NJ, USA)), and was mixed thoroughly at a high speed (3000 rpm). Then, sunflower lecithin (≥40% PC) (1.66 g) was added to the aqueous solution, and was blended at 3000 rpm to obtain liposome encapsulated vitamin C (cf. Scheme 1a). A solution was prepared at room temperature (25 °C), by adding 0.134 g of potassium sorbate (PS) (99.47 ± 0.5%), and dissolving in purified water (45 mL). Then, 1124 (+5%) mg of vitamin C (99.66% sodium ascorbate) was added to the aqueous solution, and stirred until completely dissolved. There was 5% overage of material input for vitamin C, according to the sample product formula. Next, 55 mL glycerin (99.80%) was added to an aqueous solution, by stirring; the solution was then placed in a blender (Hamilton Beach (Avalon, NJ, USA)), and was mixed thoroughly at a high speed (3000 rpm). Then, sunflower lecithin (≥40% PC) (1.66 g) was added to the aqueous solution, and was blended at 3000 rpm to obtain liposome encapsulated vitamin C (cf. Scheme 1a).

**Scheme 1.** Illustration of the liposome preparation method: (**a**) liposomal vitamin C, and (**b**) liposomal vitamin D3. **Scheme 1.** Illustration of the liposome preparation method: (**a**) liposomal vitamin C, and (**b**) liposomal vitamin D3.

#### *2.2. Preparation of Liposomal Vitamin D3 2.2. Preparation of Liposomal Vitamin D3*

A hydrophilic solution was prepared at room temperature (25 °C), by weighing 0.134 g of PS (99.47 ± 0.5%), and dissolving this amount in 45 mL of purified water. Next, A hydrophilic solution was prepared at room temperature (25 ◦C), by weighing 0.134 g of PS (99.47 ± 0.5%), and dissolving this amount in 45 mL of purified water. Next, 55 mL of glycerin (99.80%) was added to the aqueous solution, by stirring; the solution was then placed in a blender container (Hamilton Beach (Avalon, NJ, USA)), and was thoroughly mixed at high speed (3000 rpm). Then, 1.25 (+10%) mg of vitamin D3 (98.50% cholecalciferol) was dissolved in 0.3 mL of olive oil (100.00%), which was added to the mixture, and blended at 3000 rpm for 30 min. There was 10% overage of material input for

vitamin D3, according to the sample product formula. Next, 1.66 g of sunflower lecithin (≥40% phosphatidylcholine; PC) was added to the above solution, followed by blending, to obtain liposome-encapsulated vitamin D3 (cf. Scheme 1b).

#### *2.3. Characterization of Vitamin C and Vitamin D3 in Liposome Forms* 2.3.1. Transmission Electron Microscopy (TEM) Analysis

TEM analysis was performed by negative staining. Briefly, 5 µL of liposomal sample was placed on a copper–formvar-coated TEM grid, and was allowed to settle on the grid surface for 2 min. The excess liquid was removed using an absorbent tissue. Staining of the grid was done using 0.5% phosphotungstic acid for 20 s, and excess staining was removed. Imaging was achieved using an HT 7700 TEM (Tokyo, Japan) at 80 kV.

#### 2.3.2. Particle Size Distribution and Zeta Potential

The particle size and zeta potential of the liposomes were measured using a Malvern Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, UK). The sample size distribution was obtained by measurement of scattered light (θ = 173◦ ) by particles (dynamic light scattering, DLS) illuminated with a laser beam. Zeta potential was established by an electrophoretic light scattering method based on the Doppler effect [62]. Zeta potential charge values were derived from triplicate measurements, where each consisted of a minimum of ten individual runs.

#### 2.3.3. Encapsulation Efficiency (*EE*%) and Vitamins Loading (*VL*%)

The encapsulation efficiency (*EE*%) of the vitamins in the liposomes was calculated by Equation (1) [63–65]:

$$EE\left(\%\right) = \frac{\text{Total vitamin content (mg)} - \text{Solution vitamin content (mg)}}{\text{Total utilization content (mg)}} \times 100\qquad(1)$$

Nonencapsulated vitamins in the solution were quantified by high-performance liquid chromatography, after ultrafiltration (10 kDa) and centrifugation (Nuaire, NU-C200R, Plymouth, MA, USA) at 2500× *g* for 20 min at 4 ◦C [63]. The absorbance demonstrated the nonencapsulated vitamin content; thus, the amount of trapped vitamin was calculated indirectly by Equation (1).

Vitamin loading (*VL*%) means the amount of vitamin that has been encapsulated in hydrated liposomes, and it was obtained by employing Equation (2) [64–66]:

$$VL\text{ (\%)} = \frac{\text{Vitamin content in lipsomes (mg)}}{\text{Weight of lipsomes (mg)}} \times 100\tag{2}$$

#### 2.3.4. Instrumentation and Chromatographic Conditions

The UHPLC system consisted of flexible pumps, multicolumn thermostats, vial samplers, and diode array detector (Agilent 1290 Infinity II (Santa Clara, CA, USA)). The separation of vitamin C was carried out with a gradient elution program at a flow rate of 0.5 mL min−1 at 30.0 ◦C, by using a Luna C18 (100 mm × 3.0 mm, 2.5 µm) column supplied by Phenomenex (Torrance, CA, USA). The injection volume in the UHPLC system was 1 µL, where the mobile phase consisted of 0.05% (*v*/*v*) phosphoric acid (A) and methanol (B). The following linear gradient was applied: 0–3 min: 100% A; 3–5 min: 100–0% A, followed by re-equilibration of the column for 5 min before the next run. A wavelength of 245 nm was used for analyte detection of vitamin C. The separation of vitamin D3 was carried out with an isocratic elution program at a flow rate of 0.5 mL min−1 at 30.0 ◦C, by using a Luna C18 (100 mm × 3.0 mm, 2.5 µm) column supplied by Phenomenex, USA. The injection volume in the UHPLC system was 1 µL, and the mobile phase consisted of 75% acetonitrile (A) and 25% methanol (B). The total running time of the isocratic elution was 10 min. A wavelength of 280 nm was used for the detection of vitamin D3.

#### 2.3.5. Sample Preparation for UHPLC Analysis

Samples of liposomal vitamin C from the top and bottom regions of the centrifuge vial were mixed with 50% methanol, at volume ratio 1:1000 (*v*/*v*), and were sonicated for 30 min before filtration and injection. A volume of 0.8 mL of liposomal vitamin D3 from the top and bottom portions of the sample tube was mixed with 100% methanol, and then the volume was brought to 10 mL in a volumetric flask. The sample was sonicated for 30 min before filtration and injection.

#### 2.3.6. Liposomal Stability

The stability of the liposomes was assessed by determining the remainder of the vitamin in the liposomes after 3 months of storage at a refrigerated temperature of 4 ◦C, using Equation (3) below [63]:

$$\text{Stability } (\%) = \frac{\text{Remaining amount of vitamin in liquids (mg)}}{\text{Initial amount vitamin in liposome (mg)}} \times 100 \tag{3}$$

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

As outlined above, a key goal of this study was to develop a robust lipid-based formulation for vitamin C and vitamin D, where the resulting system is homogenous and stable without requiring any harmful chemicals or solvents. This section describes the characterization of the lipid-based formulations that contain vitamin C and D, according to a range of methods (TEM imaging, light scattering, and HPLC). Various parameters including particle size, zeta potential, encapsulation efficiency, and vitamin loading—which provide a measure of the stability of liposomal formulations, are discussed in the following sections.

#### *3.1. Optimization of Formulation*

Based on previous research [67], this study developed an optimized lipid-based formulation, to achieve high stability and homogeneity, where the liquid composition was critical— especially the ratio of water to glycerin, which played a key role in the sample preparation. By comparison of samples with different water–glycerin ratios, it was determined that a 45:55 (*v*/*v*) ratio should be used as the optimized water–glycerin volume ratio. For the conditions in which glycerin had a higher volume than water, the liposomes could be formed after homogenization, where the order of mixing of the respective ingredients also had a dramatic impact on the uniformity of the liposome product.

#### *3.2. Morphology by TEM*

The TEM imaging results in Figure 1 show that the liposomes were spherical in size, and that small fragments of liposomes were highly dispersed throughout them, which may indicate vesicle fracture [68,69]. Image J software (version 1.52, National Institutes of Health, Bethesda, MD, USA) was used to estimate the mean particle size of the liposomes, which was anticipated to be near 200 nm for liposomal vitamin C, and near 100 nm for liposomal vitamin D3. It is generally recognized that the size of liposomes has a significant role on tissue cell targeting: larger liposomes are usually taken by phagocytes, whereas smaller liposomes (100 to 200 nm) are more permeable to tumor cells [66,70]. The TEM images of liposomal vitamin C and D3 indicate that the liposomes were bilayer (unilamellar) vesicles, where the average sizes ranged between 100 nm to 200 nm.

**Figure 1.** Transmission electron microscopy (TEM) images: (**a**) liposomal vitamin C with an average diameter near 200 nm; (**b**) liposomal vitamin D3 with an average diameter near 100 nm. Samples of bilayer (unilamellar) vesicles are indicated by a red square. The scale bars in panels (**a**,**b**) represent a 500 nm scale range (bottom right). **Figure 1.** Transmission electron microscopy (TEM) images: (**a**) liposomal vitamin C with an average diameter near 200 nm; (**b**) liposomal vitamin D3 with an average diameter near 100 nm. Samples of bilayer (unilamellar) vesicles are indicated by a red square. The scale bars in panels (**a**,**b**) represent a 500 nm scale range (bottom right).

#### *3.3. Size and Zeta Potential 3.3. Size and Zeta Potential*

The surface charge, electrostatic repulsion between neighboring particles, colloidal stability, and entrapment efficiency were correlated with the zeta potential (ζ) [71]. According to the ζ-value results, the liposomes that contained vitamin C had a lower ζ-value (0.8 mV) than the pristine vitamin C solution [72], which related to the enhancement effect of the carrier between the phospholipids and the active compounds. The stability of the vitamin D3 liposomes could also be characterized by the ζ-value. Based on the result, a negative ζ-value (−4.0 mV) for liposomal vitamin D3 indicated stronger particle repulsion; therefore, there was no driving force for the particles to associate with, resulting in a more stable and dispersed liposome without coagulation [63,73,74]. The PDI values were close in magnitude, and exceeded 0.2 for liposomal vitamin C (0.2) and liposomal vitamin D3 (0.53), which indicated monodisperse and polydisperse particle size distributions, respectively [75]. Liposomes may vary in size, between 200–3000 nm for liposomal vitamin C, and 100–1000 nm for liposomal vitamin D3, which is related to multiple lipid layers, fusion or aggregation phenomena [75,76]. The surface charge, electrostatic repulsion between neighboring particles, colloidal stability, and entrapment efficiency were correlated with the zeta potential (ζ) [71]. According to the ζ-value results, the liposomes that contained vitamin C had a lower ζ-value (0.8 mV) than the pristine vitamin C solution [72], which related to the enhancement effect of the carrier between the phospholipids and the active compounds. The stability of the vitamin D3 liposomes could also be characterized by the ζ-value. Based on the result, a negative ζ-value (−4.0 mV) for liposomal vitamin D3 indicated stronger particle repulsion; therefore, there was no driving force for the particles to associate with, resulting in a more stable and dispersed liposome without coagulation [63,73,74]. The PDI values were close in magnitude, and exceeded 0.2 for liposomal vitamin C (0.2) and liposomal vitamin D3 (0.53), which indicated monodisperse and polydisperse particle size distributions, respectively [75]. Liposomes may vary in size, between 200–3000 nm for liposomal vitamin C, and 100–1000 nm for liposomal vitamin D3, which is related to multiple lipid layers, fusion or aggregation phenomena [75,76].

#### *3.4. Evaluation of Encapsulation Efficiency (EE%) and Vitamin Loading (VL%)*

*3.4. Evaluation of Encapsulation Efficiency (EE%) and Vitamin Loading (VL%)*  Table 1 summarizes the encapsulation efficiency and vitamin loading of liposomal vitamin C and liposomal vitamin D3. These parameters depended mostly on the composition of the liposomes, and may have been affected by other factors, such as liposome size and type, surface charge, bilayer rigidity, preparation method, and the properties of the vitamins. In turn, such factors may have helped to enhance the stability of the liposomes, and to have enhanced bioavailability on a physiological level without sacrificing Table 1 summarizes the encapsulation efficiency and vitamin loading of liposomal vitamin C and liposomal vitamin D3. These parameters depended mostly on the composition of the liposomes, and may have been affected by other factors, such as liposome size and type, surface charge, bilayer rigidity, preparation method, and the properties of the vitamins. In turn, such factors may have helped to enhance the stability of the liposomes, and to have enhanced bioavailability on a physiological level without sacrificing its potency on the cellular level in the digestive system [77,78].

**Table 1.** Encapsulation Efficiency (EE%) and Vitamin Loading (VL%) of lipid-based vitamin C and lipid-based vitamin D3. **Table 1.** Encapsulation Efficiency (EE%) and Vitamin Loading (VL%) of lipid-based vitamin C and lipid-based vitamin D3.

its potency on the cellular level in the digestive system [77,78].

#### *3.5. Determination of Vitamin C Potency in Liposomes 3.5. Determination of Vitamin C Potency in Liposomes*

The potency of ascorbic acid in the lipid-based formulation of vitamin C was measured by UHPLC equipped with a DAD detector, at a wavelength of 245 nm. Table 2 indicates the concentration of vitamin C from the top and bottom regions of the sample tube (cf. inset in Figure 2a,b); the vitamin recovery values were 97.38% and 91.86%, respectively. Based on these results, the sample was homogenous, and did not show any significant phase separation; however, the concentration of the top portion was greater than the bottom region. According to Health Canada's regulation of Natural Health Products, a variation of 20% in composition is allowed for industrial production of such vitamin formulations. The potency of ascorbic acid in the lipid-based formulation of vitamin C was measured by UHPLC equipped with a DAD detector, at a wavelength of 245 nm. Table 2 indicates the concentration of vitamin C from the top and bottom regions of the sample tube (cf. inset in Figure 2a,b); the vitamin recovery values were 97.38% and 91.86%, respectively. Based on these results, the sample was homogenous, and did not show any significant phase separation; however, the concentration of the top portion was greater than the bottom region. According to Health Canada's regulation of Natural Health Products, a variation of 20% in composition is allowed for industrial production of such vitamin formulations.

**Figure 2.** Representative liquid chromatograms obtained using UHPLC–DAD for liposomal vitamin C, sampled from the two separate sample regions shown in the inset: Top (**a**) and Bottom (**b**). **Figure 2.** Representative liquid chromatograms obtained using UHPLC–DAD for liposomal vitamin C, sampled from the two separate sample regions shown in the inset: Top (**a**) and Bottom (**b**).

#### *3.6. Determination of Vitamin D3 Potency in Liposomes*

*3.6. Determination of Vitamin D3 Potency in Liposomes*  The potency of cholecalciferol in liposomal vitamin D3 was assessed by UHPLC equipped with a DAD detector, at a wavelength of 280 nm. Table 3 shows the concentration of vitamin D3 determined from the top and bottom regions of the sample tube (cf*.* insets in Figure 3a,b); the vitamin recovery values were 100.95% and 96.73%, respectively. Based on these results, the sample was homogenous, and did not display any notable phase separation. According to Health Canada's regulation of Natural Health Products, a The potency of cholecalciferol in liposomal vitamin D3 was assessed by UHPLC equipped with a DAD detector, at a wavelength of 280 nm. Table 3 shows the concentration of vitamin D3 determined from the top and bottom regions of the sample tube (cf. insets in Figure 3a,b); the vitamin recovery values were 100.95% and 96.73%, respectively. Based on these results, the sample was homogenous, and did not display any notable phase separation. According to Health Canada's regulation of Natural Health Products, a variation of 20% in composition is allowed for industrial production of such vitamin formulations.

formulations. **Table 3.** Concentrations from top and bottom layer of liposomal vitamin D3.

Recovery value (%) 100.95% 96.73%

variation of 20% in composition is allowed for industrial production of such vitamin

**Figure 3.** Representative liquid chromatograms obtained using UHPLC–DAD for liposomal vitamin D3 sampled from the two sample regions shown in the insets: Top (**a**) and Bottom (**b**). **Figure 3.** Representative liquid chromatograms obtained using UHPLC–DAD for liposomal vitamin D3 sampled from the two sample regions shown in the insets: Top (**a**) and Bottom (**b**).

#### *3.7. Comparative Analysis of Vitamin Formulations*

*3.7. Comparative Analysis of Vitamin Formulations*  Size is a critical factor in determining the stability, biological origin, and effectiveness of formulated bioactives [79,80]. In this comparison study of liposomal vitamin C and liposomal vitamin D3, the particle size of liposomal vitamin C (~200 nm) was greater than that estimated for liposomal vitamin D3 (~100 nm), due to the higher bioactive–lipid weight ratio (B/L ratio), suggesting that the concentration of vitamin C was much greater than that of vitamin D3 [81]. While a 1:1 ratio is advantageous from a commercial standpoint, less phosphatidyl choline is required for its formulation. A 1:2 ratio, by comparison, achieves a smaller particle size (below 200 nm), which is optimal for liposomal stability: the latter is an ideal vesicle size (less than 200 nm) that is consistent with various food-based liposomes [82–84]. As a result, the upper region has a concentration of liposomal vitamin C that is higher than the bottom region of the sample. This trend indicates less homogeneity, unlike liposomal vitamin D3, where the concentration near the top region was very similar to the bottom region of the sample, indicating that a more uniform and stable solution was achieved with vitamin D3. This comparison of results from different sampling regions is consistent with the established relationship of the particle size and B/L ratio, which consequently leads to its stability [81]. We have further confirmed that at higher B/L ratios, colloids with larger size will be generated. Liposomes Size is a critical factor in determining the stability, biological origin, and effectiveness of formulated bioactives [79,80]. In this comparison study of liposomal vitamin C and liposomal vitamin D3, the particle size of liposomal vitamin C (~200 nm) was greater than that estimated for liposomal vitamin D3 (~100 nm), due to the higher bioactive–lipid weight ratio (B/L ratio), suggesting that the concentration of vitamin C was much greater than that of vitamin D3 [81]. While a 1:1 ratio is advantageous from a commercial standpoint, less phosphatidyl choline is required for its formulation. A 1:2 ratio, by comparison, achieves a smaller particle size (below 200 nm), which is optimal for liposomal stability: the latter is an ideal vesicle size (less than 200 nm) that is consistent with various food-based liposomes [82–84]. As a result, the upper region has a concentration of liposomal vitamin C that is higher than the bottom region of the sample. This trend indicates less homogeneity, unlike liposomal vitamin D3, where the concentration near the top region was very similar to the bottom region of the sample, indicating that a more uniform and stable solution was achieved with vitamin D3. This comparison of results from different sampling regions is consistent with the established relationship of the particle size and B/L ratio, which consequently leads to its stability [81]. We have further confirmed that at higher B/L ratios, colloids with larger size will be generated. Liposomes with reduced homogeneity concur with liposome systems that display reduced stability of the lipid-based formulation.

#### with reduced homogeneity concur with liposome systems that display reduced stability of the lipid-based formulation. *3.8. Stability*

*3.8. Stability*  The results of the present study, shown in Tables 4 and 5, reveal that the liposomal The results of the present study, shown in Tables 4 and 5, reveal that the liposomal entrapped vitamins leaked out only slightly over a 3-month period, for both 4 and 25 ◦C, as compared with the samples that were assessed immediately after the liposomal preparation.

**Table 5.** Stability of lipid-based vitamin D3 upon storage at 4 ◦C and 25 ◦C.

No significant differences in vitamin concentration were found between the top and bottom, for intervals ranging from 0 days to 90 days. This trend indicates that the formulations of both lipid-based vitamin C and vitamin D3 are homogenous, and stable for a minimum time of 3 months.

#### **4. Conclusions**

HPLC has been widely used for the identification and quantification of vitamins in diverse matrices, due to its relatively rapid separation capability, high sensitivity, precise quantification, and effective analytical performance; therefore, HPLC was chosen as the ideal method for assessing the potency of vitamin C and D3 in liposomal forms, and as a suitable analytical method for regular laboratory testing in the pharmaceutical industry. Based on a previous study [66], the encapsulation efficiencies of vitamin C were generally around 50%, which compares favorably to our results. A comparison of hydrophilic vitamin C to lipophilic vitamin D3 revealed a higher encapsulation efficiency (>90%) for vitamin D3, due to its high lipophilicity versus the more water-soluble (hydrophilic) vitamin C [85]. This trend was consistent with similar studies, and is related to the decreased particle size and polydispersity index, due to the role of the relative proportion of the constituents, the viscosity of the lipid phase, and the production conditions [86,87].

Our results provide support for both types of liposomal vitamin C and D3, which are homogenous colloids that remain very stable without phase separation, for a minimum of 3 months. Lipid-based formulations of this type are considered to be an effective and versatile approach to addressing the fortification of nutraceuticals and other bioactive components with variable molecular properties that cover a range of values on the hydrophile–lipophile scale. Our results concur with those of a recent study of liposomalbased vitamin C that had been readily prepared with food grade materials and simple equipment, and which provided a well-tolerated natural health product. The uptake of ingested liposomes from the gut to the mesenterial vessels and liver vessels was supported by a microbubble-enhanced ultrasound (MEU) method with real-time imaging [76]. Similarly, liposomes with a well-defined size distribution were used to achieve efficient absorption of hydrophobic vitamin D3. Clinical trials indicate that orally delivered liposomal vitamin D3 enhances the concentration of calcidiol in the serum rapidly, whereas no such function is found in oil-based vitamin D3 liquid [88].

This work focused mainly on the optimization of a lipid-based formula of watersoluble and fat-soluble vitamins, using a simple method and food-grade materials. Further studies could be extended to other applications of liposomal assemblies: for example, skin permeation studies could be carried out for both lipid-based vitamin C and lipidbased vitamin D3, in order to evaluate liposomes cellular internalization, and also to provide insight regarding the behavior of nanoparticles upon interaction with biological membranes [66,89,90]. In the case of nanocarriers, these systems have advantages for the topical and transdermal application of vitamin D3 (active 7-dehydrocholesterol or cholecalciferol), which is transformed primarily in dermal cells [51,66].

**Author Contributions:** Conceptualization, Y.-C.M. and L.D.W.; methodology, J.C. and L.D.; software, J.C.; validation, Y.-C.M. and L.D.W.; formal analysis, L.D. and J.C.; investigation, J.C. and L.D.; resources, Y.-C.M. and L.D.W.; data curation, J.C. and L.D.; writing—original draft preparation, J.C. and L.D.; writing—review and editing, L.D.W. and Y.-C.M.; visualization, J.C. and L.D.; supervision, Y.-C.M. and L.D.W.; and project administration, Y.-C.M. and L.D.W. All authors have read and agreed to the published version of the manuscript.

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

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Dr. Ma's Laboratories Inc. (DML) is acknowledged for supporting this research. All liposomal samples and HPLC analyses were carried out at Dr. Ma's Laboratories Inc. L.D acknowledges Eiko Kawamura for technical support with TEM imaging at WCV Image Centre (University of Saskatchewan). L.D.W. acknowledges Deysi J. Venegas-García for technical support with zeta potential and light scattering analysis.

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

#### **References**

## **Pomegranate Pomace Extract with Antioxidant, Anticancer, Antimicrobial, and Antiviral Activity Enhances the Quality of Strawberry-Yogurt Smoothie**

**Nouf H. Alsubhi 1,\* , Diana A. Al-Quwaie 1 , Ghadeer I. Alrefaei 2 , Mona Alharbi 3 , Najat Binothman 4 , Majidah Aljadani 4 , Safa H. Qahl 2 , Fatima A. Jaber 2 , Mashael Huwaikem 5 , Huda M. Sheikh 2 , Jehan Alrahimi 6,7, Ahmed N. Abd Elhafez 8 and Ahmed Saad 9,\***

**Abstract:** Valorizing the wastes of the food industry sector as additives in foods and beverages enhances human health and preserves the environment. In this study, pomegranate pomace (PP) was obtained from the company Schweppes and exposed to the production of polyphenols and fiber-enriched fractions, which were subsequently included in a strawberry-yogurt smoothie (SYS). The PP is rich in carbohydrates and fibers and has high water-absorption capacity (WAC) and oilabsorption capacity (OAC) values. The LC/MS phenolic profile of the PP extract indicated that punicalagin (199 g/L) was the main compound, followed by granatin B (60 g/L) and pedunculagin A (52 g/L). Because of the high phenolic content of PP extract, it (*p* ≤ 0.05) has high antioxidant activity with SC50 of 200 µg/mL, besides scavenging 95% of DPPH radicals compared to ascorbic acid (92%); consequently, it reduced lung cancer cell lines' viability to 86%, and increased caspase-3 activity. Additionally, it inhibited the growth of pathogenic bacteria and fungi i.e., *L. monocytogenes*, *P. aeruginosa*, *K. pneumonia*, *A. niger*, and *C. glabrata*, in the 45–160 µg/mL concentration range while killing the tested isolates with 80–290 µg/mL concentrations. These isolates were selected based on the microbial count of spoiled smoothie samples and were identified at the gene level by 16S rRNA gene sequence analysis. The interaction between Spike and ACE2 was inhibited by 75.6%. The PP extract at four levels (0.4, 0.8, 1.2, and 1.4 mg/mL) was added to strawberry-yogurt smoothie formulations. During 2 months storage at 4 ◦C, the pH values, vitamin C, and total sugars of all SYS decreased. However, the decreases were gradually mitigated in PP-SYS because of the high phenolic content in the PP extract compared to the control. The PP-SYS3 and PP-SYS4 scored higher in flavor, color, and texture than in other samples. In contrast, acidity, fat, and total soluble solids (TSS) increased at the end of the storage period. High fat and TSS content are observed in PP-SYS because of the high fiber content in PP. The PP extract (1.2 and 1.6 mg/mL) decreases the color differences and reduces harmful microbes in PP-SYS compared to the control. Using pomegranate pomace as a source of polyphenols and fiber in functional foods enhances SYS's physiochemical and sensory qualities.

**Keywords:** pomegranate; pomace; extraction; biological activity; functional beverages; sensory quality

**Citation:** Alsubhi, N.H.; Al-Quwaie, D.A.; Alrefaei, G.I.; Alharbi, M.; Binothman, N.; Aljadani, M.; Qahl, S.H.; Jaber, F.A.; Huwaikem, M.; Sheikh, H.M.; et al. Pomegranate Pomace Extract with Antioxidant, Anticancer, Antimicrobial, and Antiviral Activity Enhances the Quality of Strawberry-Yogurt Smoothie. *Bioengineering* **2022**, *9*, 735. https://doi.org/10.3390/ bioengineering9120735

Academic Editors: Minaxi Sharma, Kandi Sridhar, Zeba Usmani and Xiaohu Xia

Received: 16 October 2022 Accepted: 19 November 2022 Published: 28 November 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/).

#### **1. Introduction**

Agriculture and the food industry produce a huge amount of waste that poses a threat to health and the environment, although it is rich in bioactive components. Because of the global food shortage, we must reuse the waste in various applications, such as foods and feedstuffs [1–3]. Unfortunately, separating bioactive compounds from the consumable parts of the agricultural product could have an excessive cost for the starting material. Accordingly, using agro-industrial waste (such as fruit peels) for separating such compounds to control the spread of foodborne and human pathogens is more economical [4,5]. Pomegranate produces about 1,500,000 tons of waste worldwide per annum [6] and is expected to reach 12,500,000 tons by 2022; about 60% of the fruit weight corresponds to peels, which are often discarded as agro-waste.

Pomegranate (*Punica granatum* L.) has a well-known medicinal history. It is rich in bioactive molecules, phenolic compounds, and flavonoids and has many medicinal properties [7]. The level of phenolic compounds changes according to cultivars and fruit parts [8]. Gözlekçi et al. [9] demonstrated that peel extract contains higher total phenolics, flavonoids, and anthocyanins content than pulp and juice extracts and possesses stronger biological activities. The phytochemical constituents of the ethanolic extract of pomegranate juice were determined by Sorrenti et al. [10], who identified many phytochemical compounds; phenolic compounds were the main constituents. On the other hand, Khaleel et al. [11] demonstrated the presence of 292 different phytocompounds with different retention times and chemical structures. Eleven elements were considered most significant as antimicrobials.

In this regard, Priyanka et al. [12] studied the antimicrobial activity of methanol, chloroform, and methanol chloroform (1:1) extracts of peel and juice of pomegranates in different bacterial and fungal strains. They found that peel methanolic extract showed the highest antimicrobial activity, while chloroform extract was less effective against the tested microorganisms. Other studies have also reported that pomegranate methanol peel extract showed maximum growth inhibition (85.71%) compared to all tested organic solvents against some pathogenic and drug-resistant bacterial strains [13]. Li et al. [14] determined the MICs of punicalagin, as the main active component in pomegranate peel, for ten *Salmonella* strains and found the minimum inhibitory concentrations (MICs) in the range of 0.20–1.0 mg/mL. Moreover, Zam and Khaddour [15] detected a reduction in the adhesion index of up to 80% and a reduction in *E. coli* motility in the urinary tract using aqueous pomegranate peel extract at MIC. Abou El-Nour [16] recorded a large decrease in biofilm formation of *Pseudomonas aeruginosa* and inhibition in swarming motility when treated with aqueous and alcoholic pomegranate peel extracts.

Pomegranate extracts also exhibit antioxidant activity. It has been reported by many investigators that there is a relationship between chemopreventive agents and antioxidant activity; during the oxidation of biological materials, superoxide, H2O2, and hydroxyl radicals (OH˙) are produced. Reducing the destructive effect on human cells and tissues stops unorganized radical reactions. Mekni et al. [17] illustrated that methanol peel extracts of five pomegranate varieties have high antioxidant power (above 80%) against DPPH radicals. Ohshima et al. [18] demonstrated that oxidative stress generates toxic metabolites which can initiate and stimulate cancers. Breast cancer is the most important life disease a woman may face during her lifetime. Rocha et al. [19] found that the growth of two breast cancer cell lines was completely inhibited by a 1% concentration of pomegranate juice, which also can decrease cell migration to bones. Li et al. [14] recorded a reduction in cell viability in different human cancers by methanol PPE, and MCF-7 breast cells were the most responsive to the antitumor effect (growth inhibition of 81.0% at 5 µg/mL) compared to A549 lung cell cancer (80.0% at dose 250 µg/mL). A recent study by Mahmoudi et al. [20] suggested a decrease in the viability of MCF-7 and MDA-MB-468 breast cancer by slowing down their progression when treated with pomegranate seed oil.

Because of all the above-mentioned significant activities, pomegranate peels can be added to commercial tomato and orange juices that contain strawberries. The extract of dried pomegranate peel substantially boosted the antioxidant content of both flavored juices.

Due to the astringent flavor of pomegranate peel, the maximum extract concentration in the juices had the lowest sensory acceptability. According to the results, adding 0.5% dried pomegranate peel extract to strawberries was approved in tomato and orange juice [21]. This study investigated the antioxidant, total phenolic, tannin, and phytic acid levels of four fruit peels (pomegranates, grapes, apples, and mocha palms). The maximum antioxidant activity and phenolic content were found in pomegranate peel. It was somewhat poor in flavonoids and vitamin C. The sensory assessment of guava juice supplemented with pomegranate peel did not vary from the control. Incorporating pomegranate peel into guava juice is therefore highly well received, increases the antioxidant components, protects health, and enhances quality of life [22]. Altunkaya et al. [23] formulated apple juice supplemented with pomegranate peel extract (PPE) at concentrations ranging from 0.5% to 2%. The most pomegranate peel was found in the juice, as well as the most antioxidant activity.

In comparison, only 0.5 g PPE per 100 mL was determined to be acceptable for color, smell, and taste. The conclusion was that PPE-enriched apple juice is superior to regular apple juice. Therefore, adding PPE at concentrations between 0.5% and 1.0% proved acceptable and generated a healthier product without altering apple juice's sensory qualities or toxicological safety.

No available studies cast light on the pomegranate pomace and its application in the food industry, in which it may be more valuable than peels. Therefore, this study aimed to complete the evaluation of pomegranate pomace concerning the phenolic content by LC/MS, and antioxidant, anticancer, antibacterial, antifungal, and antiviral properties. Additionally, it encourages using natural and safe alternatives to control pathogenic microorganisms and component oxidation. Therefore, highly functional pomegranate pomace extract was added to the strawberry-yogurt smoothie to enhance its color, flavor, and texture, besides extending its shelf life and quality.

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

#### *2.1. Preparation of Pomegranate Pomace Aqueous Extract (PP)*

Pomegranate pomace was obtained from the juice company. The PP was homogenized and washed with tap water, followed by distilled water. The pomace was dried in an oven at 50 ◦C for 2 days, then powdered and kept dry until extraction. Ten grams of pomegranate pomace powder was stirred in 150 mL of sterilized distilled water for 12 h, then filtered by Whatman No. 1 paper. The filtered extracts were centrifuged at 4000 rpm/15 min and kept at 4 ◦C.

#### *2.2. Proximate Analysis of Pomegranate Pomace Powder*

Moisture, protein, fat, and ash levels were determined according to AOAC guidelines [24]. The carbohydrates were determined by differential analysis. As per Saad et al. [25], the water-absorption capacity (WAC) and oil-absorption capacity (OAC) were evaluated as follows: PP powder (1 g) was stirred in 10 mL of water or oil for 30 min, then centrifuged at 7500 rpm/30 min in weighted test tubes. The residue was weighed, and WAC and OAC were estimated as mL of water or oil retained per gram of sediment.

#### *2.3. Phenolic Profile in PP Extract*

#### 2.3.1. Total Phenols and Total Flavonoids Content

Total phenolics were determined in PP extracts by the Folin–Ciocalteu method described by Saad et al. [26], and the data were calculated as mg of gallic acid equivalents (GAE)/g. The total flavonoid content was estimated using the AlCl3 colorimetric method following Namir et al. [27]. The data were estimated as mg quercetin equivalents (QE)/g.

#### 2.3.2. High-Resolution LC/MS Profile of Phenolic Content of PP Extract

Accela U-HPLC system (Thermo Fisher Scientific, USA) was used to identify the phenolic compounds in PP. LC/MS data were obtained by an Executive Orbitrap mass spectrometer linked with the HPLC system. The compounds were separated following

Colantuono et al. [28]. Separation column (Gemini C18-110, Phenomenex, Torrance, CA, USA), 150 × 2.0 mm, 5 µm and mobile phases were (A) aqueous Formic acid 0.1% and (B) Formic acid in acetonitrile 0.1%. The column was heated to 30 ◦C, and the flow rate was adjusted at 0.2 mL/min. The PP extract was dissolved in a mixture of methanol and water (50:50). Then, 10 µL of the prepared sample was injected into the column. Negative ionization methods collected MS data in the mass range of 100–1400 m/z. The capillary temperature was 275 ◦C, the auxiliary gas had a pressure of 15 arbitrary units, and the sheath gas had a pressure of 30 arbitrary units. Linear calibration curves of PC (1–50 µg/mL) and EA and GA (0.1–5 µg/mL) were established. With a mass tolerance of ±5 ppm, the metabolites were identified using accurate mass values to the fifth decimal place.

#### *2.4. Pomegranate Pomace Extract Activity*

#### 2.4.1. DPPH-Radical-Scavenging Activity

The antioxidant activity was determined using the DPPH technique, as described by Abdel-Moniem et al. [29]. A total of 0.5 mL of ethanolic DPPH was added to 1 mL PP extract and incubated for 30 min in the dark; the produced color was read spectrophotometrically at a wavelength of 517 nm. The absorbance was applied as in Equation (1). The lowest concentration that scavenges 50% of DPPH radical was calculated as IC50 [30].

$$\% \text{DPPH saving} \cdot \text{acvenging activity} = \frac{\text{Abs control} - \text{Abs sample}}{\text{Abs control}} \times 100\tag{1}$$

#### 2.4.2. Anticancer

The lung carcinoma cell line (A-549) was assayed for cytotoxicity; the doxorubicin reference standard was used as a positive control. According to Mosmann [31] and Gomha et al. [32], the cytotoxicity evaluation was carried out using a viability assay. The apoptosisinducing potential of PP extract was assessed by measuring its caspase-3 activity. After treating the 25 cm2 flasks with DMSO or PP at the right doses and times, the A-549 cells were collected. For each experiment, 2 × 106 cell lysates were used and analyzed as specified. After 45 min of incubation with substrate Z-DEVD-R110 in a microplate reader, fluorescence was seen, and the excitation and emission were measured at 485/530 nm [33].

#### 2.4.3. Antimicrobial

Twelve pathogenic microorganisms were employed for the antimicrobial screening test. *S. aureus*, *L. monocytogenes*, *B. cereus*, *P. aeruginosa*, *K. pneumonia*, *E. coli*, *A. niger*, *A. flavus*, *C. glabrata*, *C. albicans*, *C. davenportii*, and *P. expansum*. These strains were selected based on the microbial count of spoiled yogurt samples. It was found during microbial examination with a light microscope and biochemical and morphological definitions that these isolates are the most isolates that cause smoothie spoilage. These isolates were confirmed by identification at the gene level through isolating DNA and using PCR to detect genes. The bacterial isolates were identified based on 16S rRNA and the fungal isolates on 18S rRNA gene sequence analysis. Sequencing was performed via the automated DNA sequencer (ABI Prism 3130 Genetic Analyzer by Applied Biosystems Hitachi, Japan). Genomic DNA was obtained by the hexadecyltrimethylammonium bromide (CTAB) technique, and the integrity and level of purified DNA were established by agarose gel electrophoresis. The DNA level was customized to 20 ng/µL for PCR amplification. The forward primer used with the isolates is (5 AGA GTT TGA TCC TGG CTC AG 3), and the reverse is (5 GGT TAC CTT GTT ACG ACT T 3). PCR products were isolated by electrophoresis on 1.5% agarose gels stained with ethidium bromide and documented in the Alphaimager TM1200 documentation and analysis system. The obtained polymorphic differences were analyzed via the program NTSYS-PC2 by assessing the distance of isolates by Jaccard's Similarity Coefficient. All the bacterial strains were maintained by sub-culturing on nutrient agar or potato dextrose slant and stored at 4 ◦C. The PP extract was evaluated for its antibacterial and antifungal activity by the well-disc-diffusion method, according to Dahham et al. [34] and El-Saadony et al. [35]. Next, 50 mL of molten Mueller–Hinton agar (MHA) or potato

dextrose agar (PDA) was poured into plates. A loopful of bacterial or fungal inoculum was spread over the surface of the plates. Then, 8 mm wells were punched into each plate and filled with 50 µL of PP extract concentrations (200, 400, 600, 800, and 100 µg/mL), and negative control wells were filled with water. MHA and PDA plates were incubated at 37 ◦C for 24–48 h (bacteria) or 28 ◦C for 5 days (fungi) [36]. The resultant inhibition zone diameters (mm) indicated the antimicrobial activity [37]. The MIC, MBC, and MFC values were determined by Saad et al. [25].

#### 2.4.4. Antiviral

ACE2 was created as a recombinant protein in human cells. SARS-CoV-2 Inhibitor Screening Assay kit (Adipogen, San Diego, CA, USA) was used to inhibit the binding between the Spike and ACE2. The SARS-CoV-2 antiviral activity of the PP extract was evaluated as follows: 100 µL of Spike RBD was added to each well of a 96-well plate and kept for 16 h at 4 ◦C. Then, 100 µL of PP concentrations (200, 400, 600, 800, and 1000 µg/mL) was added to wells containing Spike and incubated for one hour at 37 ◦C in the presence of Inhibitor Mix Solution ((0.2% BSA, 0.05% Tween R 20 in PBS, and biotin-conjugated ACE2 (0.5 g/mL)). After incubation, HRP-labeled streptavidin (1:200 dilution) was added to each well and incubated for one hour at room temperature. To end the reaction, 100 µL of tetramethyl benzidine was added and incubated for 5 min. The absorbance was read at 450 nm by the microplate reader (BioTek Elx808, Winooski, VT, USA).

#### *2.5. PP-Strawberry-Yogurt Smoothie Processing and Preservation*

#### 2.5.1. Preparation of Strawberry Smoothie

Fresh strawberry fruits were washed, cleaned, and processed for juice production using a Moulinex mixer following Saad et al. [26] with some modifications. In a blender, 50 mL of PP extract (25 g/100 mL) at different concentrations and yogurt (40 g) were mixed well with filtered strawberry juice (100 mL). The water and sugar were added as shown in Table 1. The smoothies, SYSC, PP-SYS1, PP-SYS2, PP-SYS3, and PP-SYS4, were transferred into 250 mL bottles and pasteurized in a TOMY Sx-700 autoclave (Tokyo, Japan) at 95 ◦C for 2 min under 50 MPa. The bottles were cooled and stored at 4 ◦C for two months.

**Table 1.** The ingredients of pomegranate pomace-enriched strawberry-yogurt smoothie.

Strawberry-yogurt smoothie (SYS), control (C), smoothie supplemented with pomegranate pomace extract 0.4 mg/mL (PP-SYS1), smoothie supplemented with pomegranate pomace extract 0.8 mg/mL (PP-SYS2), smoothie supplemented with pomegranate pomace extract 1.2 mg/mL (PP-SYS3), smoothie supplemented with pomegranate pomace extract 1.6 mg/mL (PP-SYS4).

#### 2.5.2. Physiochemical Properties

The pH values of the smoothies, SYSC, PP-SYS1, PP-SYS2, PP-SYS3, and PP-SYS4, were calculated using a pH meter (pH 211, HANNA, Nusfalau, Romania). Standard method 942.15 was used to estimate the titratable acidity, which was recorded as a percentage of citric acid. A refractometer was used to calculate the TSS. AOAC [24] was also used to assess vitamin C concentration.  The total sugars were estimated by El-Saadony et al. [38].

#### 2.5.3. Color Differences and Sensory Properties

Hunter spectrophotometer (AA Color Flex EZ) was used to determine the color parameters (*L\**, *a\**, and *b\**) of PP-SYS (HunterLab, Murnau, Germany). The following parameters were considered: Equation (2) calculated the color difference (∆E) between smoothie samples during the storage period [39].

$$
\Delta \mathbf{E} = \sqrt{\left(\Delta L\right)^2 + \left(\Delta \mathbf{a}\right)^2 + \left(\Delta \mathbf{b}\right)^2} \tag{2}
$$

The sensory characteristics (color, flavor, texture, and overall acceptability) of PP-SYS were estimated using a 9-point hedonic scale. PP-SYS samples were randomly coded and served in one-use cups to 30 semi-trained panelists, 20 males and 10 females (aged 21–30), in a fluorescent-lit laboratory with an air-conditioning temperature of 23 ◦C. Water was used to change the mouth contact areas after each judgment to avoid interfering with the results.

#### 2.5.4. Microbial Load

Ten PP-SYS samples were stirred in 90 mL of peptone water for 30 min to obtain a 10−1 suspension. Serial dilutions were generated up to 10−8 . The dilutions were put into disposable Petri plates with a specific medium [40]. The total viable count (TBC) was determined after 24 h incubation at 30 ◦C using plate count agar. The results for microorganisms were converted to logarithms (CFU/mL).

#### *2.6. Statistical Analysis*

A one-way ANOVA test was used to distinguish the significant differences between sample means at a probability level (*p* ≤ 0.05). The LSD test determined significant differences between means. All statistical calculations were performed with SPSS 20 for Windows.

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

#### *3.1. Chemical Composition*

The findings revealed that pomegranate pomace is a rich source of carbohydrates and fiber with very low-fat content. Table 2 shows the proximate analysis of pomegranate pomace, which comprised 0.61% fat, 4.12% ash, 6.98% moisture, 9.11% protein, and 20.66% fiber, with total carbohydrates accounting for 58.52% of the sample. There are no studies on pomegranate pomace, but the findings are comparable with those of Ranjitha et al. [41], except for pomegranate peels that contain 0.85% fat, 4.32% ash, 7.27% moisture, 3.74% protein, 17.31% fiber, and 66.51% carbohydrates. Moreover, El-Hadary and Taha [42] found that the chemical composition of pomegranate peel was crude carbohydrates (78%), fiber (12%), protein (3.5%), ash (3.4%), and lipids (2.25%).

**Table 2.** Physiochemical parameters of pomegranate pomace powder.

Data are presented as mean ± SD, lightness (*L\**), redness (*a\**), yellowness (*b\**), water-absorption capacity (WAC), and oil-absorption capacity (OAC).

The high content of carbohydrates and fibers in PP influences the WAC and OAC values; in this study, the WAC of pp was 8.21 mL/g, compared to lower WACs in cantaloupe waste, dates, pear, and tomato pomace with values of 6.17, 5.70, 4.90, and 4.12 mL/g, respectively [43]. The OH in polysaccharide chains can build hydrogen bonds with water, enhancing fiber-rich materials' capacity to retain moisture [44]. Regarding the OAC of PP, it was 7.77 mL/g, which is higher than lemon byproducts (6.60 mL/g) and tiger nut pomace (6.90 mL/g) [45]. The OAC is crucial since lipids serve as taste preservers and enhance the texture of food. The OAC is also a technological property related to the chemical structure of the plant polysaccharides. It depends on surface properties, overall charge density, thickness, and hydrophobic nature of the fiber particle [46]. Regarding the color parameters of PP, *L\** and *b\** values are high, which is a good indicator for enhancing the color when applied to food formulation.

#### *3.2. Phenolic Compounds in Pomegranate Pomace Aqueous Extract (PP)*

Phenolics are chemicals generated from the secondary metabolism of plants that are frequently used for their biological effects, notably their antioxidant properties. Phenolic compounds are antioxidants with biological and chemical actions, the most significant of which is free radical scavenging [47]. Figure 1 shows the significant (*p* ≤ 0.05) phenolic content (total phenolic content and total flavonoids) in PP. The concentration of TPC and TF significantly (*p* ≤ 0.05) increased in a concentration-dependent manner when PP (1 mg/mL) contained 205 mg of gallic acid equivalent/mL and 110 mg of quercetin equivalent/mL. There are no studies on the antioxidant content of PP extract; previous studies have focused on pomegranate peels and waste extracts with a high concentration of phenolics and flavonoids; hence, pomegranate peels can be considered a major source of antioxidants. The results may agree with Yassin et al. [48], who found that acetone extract of pomegranate peels contained 246 and 227.7 mg GAE/g. In comparison, the methanolic extract of pomegranate peels has a maximum total phenolic content of 18.89 g/100 g and a total flavonoid content of 13.95 mg QE/kg [42]. There is a preference for our PP in phenolic content, with a relative increase of 11%. *Bioengineering* **2022**, *9*, x FOR PEER REVIEW 8 of 23

**Figure 1.** Total phenolic and flavonoid content in pomegranate pomace aqueous extract (PPE). TPC, total phenolic content; TF, total flavonoids; PP, pomegranate pomace. Data are presented as mean ± SE. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05. **Figure 1.** Total phenolic and flavonoid content in pomegranate pomace aqueous extract (PPE). TPC, total phenolic content; TF, total flavonoids; PP, pomegranate pomace. Data are presented as mean ± SE. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05.

To determine the exact and active phenolic compounds in pomegranate pomace, high-resolution LC/MS was processed (Table 3). The detected compounds belong to three groups: ellagitannins (ETs), ellagic acid derivatives (EA), and gallic acid (GA); their values

LC/MS profile, causarinin, punicalin, galloyl-HHDP-hexoside, granatin B, pedunculagin A, and pedunculagin B were expressed as punicalagin (PC) equivalents. EA equivalents were EA hexoside, pentoside, and deoxyhexoside. Gallic acid was calculated with the corresponding standard. Total polyphenols accounted for 500 g/L; PC derivatives represent 95% of total phenolics, while EA derivatives and GA represent only 5%. Similar results were observed by Zivkovic et al. [49]; they found that pomegranate wastewater extract contains punicalin (197.13 mg/g), punicalagin (54.23 mg/g), and ellagic acid (25.42 mg/g) found to be dominant in peel extract. On the other hand, Coronado-Reyes et al. [50] found that pomegranate peel is a more important source of punicalagin and ellagic acid than aryls, where the punicalagin content of the crust ranged from 114.6 to 282 mg/g dry weight. The ellagic acid content varied from 1.07 to 2.49 mg/g dry weight. Additionally, a previous study on pomegranate aril showed that ellagic acid was detected in different extracts, and it was the highest detected phenolic compound with 34.5 (μg/mg) when a combination of ethanol: ether: water (8:1:1) was used [51]. These compounds can potentially be used in

**Table 3.** Phenolic compounds profile in pomegranate pomace extract (PP) achieved by LC/MS.

foods and medical applications [52].

**RT Compound Equivalent MW PP (g/L)** 6.3 Gallic acid - 169.014 1.20 ± 0.2 j 6.5 Punicalin - 781.053 65.35 ± 0.9 b 7.8 Punicalagin - 1083.059 199.85 ± 1.2 a 8.2 Pedunculagin A bis-Hexahydroxy diphenic acid (HHDP) Punicalagin 783.069 52.98 ± 0.8 c 8.2 Causarinin Punicalagin 935.080 22.96 ± 0.8 f 8.5 Galloyl-HHDP-hexoside Punicalagin 633.073 48.15 ± 0.7 d

To determine the exact and active phenolic compounds in pomegranate pomace, high-resolution LC/MS was processed (Table 3). The detected compounds belong to three groups: ellagitannins (ETs), ellagic acid derivatives (EA), and gallic acid (GA); their values are 479.35, 24.29, and 1.20 g/L, respectively. The main compound in LC/MS profile is punicalagin (199 g/L), followed by granatin B (60 g/L) and pedunculagin A (52 g/L). In the LC/MS profile, causarinin, punicalin, galloyl-HHDP-hexoside, granatin B, pedunculagin A, and pedunculagin B were expressed as punicalagin (PC) equivalents. EA equivalents were EA hexoside, pentoside, and deoxyhexoside. Gallic acid was calculated with the corresponding standard. Total polyphenols accounted for 500 g/L; PC derivatives represent 95% of total phenolics, while EA derivatives and GA represent only 5%. Similar results were observed by Zivkovic et al. [49]; they found that pomegranate wastewater extract contains punicalin (197.13 mg/g), punicalagin (54.23 mg/g), and ellagic acid (25.42 mg/g) found to be dominant in peel extract. On the other hand, Coronado-Reyes et al. [50] found that pomegranate peel is a more important source of punicalagin and ellagic acid than aryls, where the punicalagin content of the crust ranged from 114.6 to 282 mg/g dry weight. The ellagic acid content varied from 1.07 to 2.49 mg/g dry weight. Additionally, a previous study on pomegranate aril showed that ellagic acid was detected in different extracts, and it was the highest detected phenolic compound with 34.5 (µg/mg) when a combination of ethanol: ether: water (8:1:1) was used [51]. These compounds can potentially be used in foods and medical applications [52].

**Table 3.** Phenolic compounds profile in pomegranate pomace extract (PP) achieved by LC/MS.

RT, Retention time (min); molecular weight (MW), pomegranate pomace extract (PP). Lowercase letters in the same columns indicate significant differences between detected polyphenols *p* ≤ 0.05. Uppercase letters indicate the differences (*p* ≤ 0.05) between punicalagin derivatives, ellagic acid derivatives, and gallic acid.

#### *3.3. Activity of Pomegranate Pomace Aqueous Extract*

#### 3.3.1. Antioxidant

The antioxidant potential of the pomegranate pomace aqueous extract was evaluated according to a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay; the SC50 (antioxidants needed to reduce 50% of the initial DPPH concentration) was identified. Effective free radicalscavenging activity was recorded in a concentration-dependent manner, with considerable SC50 = 200 µg/mL (Figure 2). Increase in the extract concentration resulted in increased inhibition percentage (decrease in the concentration of DPPH) and reached almost complete inhibition (95%) at 1 mg/mL of PP compared to ascorbic acid. Lower DPPH radicalscavenging activity values were found with 93% in PP ethanolic extract [42], 83.6% in PP ethyl acetate extract [53], and 78.23% in pomegranate waste methanolic extracts [54]. Bopitiya and Madhujith [55] concluded that significant amounts of phenolic and flavonoid compounds (such as ellagic and gallic acids) were attributed to the antioxidant properties of pomegranate peel extracts. scavenging activity values were found with 93% in PP ethanolic extract [42], 83.6% in PP ethyl acetate extract [53], and 78.23% in pomegranate waste methanolic extracts [54]. Bopitiya and Madhujith [55] concluded that significant amounts of phenolic and flavonoid compounds (such as ellagic and gallic acids) were attributed to the antioxidant properties of pomegranate peel extracts.

*3.3. Activity of Pomegranate Pomace Aqueous Extract*

RT, Retention time (min); molecular weight (MW), pomegranate pomace extract (PP). Lowercase letters in the same columns indicate significant differences between detected polyphenols *p* ≤ 0.05. Uppercase letters indicate the differences (*p* ≤ 0.05) between punicalagin derivatives, ellagic acid

The antioxidant potential of the pomegranate pomace aqueous extract was evaluated according to a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay; the SC50 (antioxidants needed to reduce 50% of the initial DPPH concentration) was identified. Effective free radicalscavenging activity was recorded in a concentration-dependent manner, with considerable SC50 = 200 µg/mL (Figure 2). Increase in the extract concentration resulted in increased inhibition percentage (decrease in the concentration of DPPH) and reached almost complete inhibition (95%) at 1 mg/mL of PP compared to ascorbic acid. Lower DPPH radical-

*Bioengineering* **2022**, *9*, x FOR PEER REVIEW 9 of 23

8.7 Ellagic acid hexoside Ellagic acid 463.052 4.50 ± 0.6 h 8.8 Pedunculagin B (digalloyl- HHDP) Punicalagin 785.084 31.66 ± 0.9 e 8.8 Ellagic acid dihexoside Ellagic acid 625.105 1.21 ± 0.1 ij 9.0 Lagerstannin B Punicalagin 949.059 1.93 ± 0.2 i 9.3 Granatin B Punicalagin 951.075 62.42 ± 0.8 b 9.5 Ellagic acid pentoside Ellagic acid 433.041 3.12 ± 0.1 h 9.6 Ellagic acid deoxyhexoside Ellagic acid 447.057 3.89 ± 0.1 h 10.5 Ellagic acid - 300.999 12.55 ± 0.3 g Total punicalagin derivatives 479.35 A Total ellagic acid derivatives 24.29 B Gallic acid 1.20 C Total phenolic compounds 504.4

derivatives, and gallic acid.

3.3.1. Antioxidant

**Figure 2.** DPPH-radical-scavenging activity of pomegranate pomace aqueous extract (PP). Data are presented as mean ± SE. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05.

Considerable antioxidant activity is essential to inhibit lipid oxidation in the food system. Free radicals are unstable and can react with biomolecules of living cells, causing various diseases such as cardiovascular diseases and cancer [56]; the antioxidants help the body to protect itself against this oxidative damage. The considerable amounts of hydrolyzable tannins in pomegranate peel are responsible for their antioxidant power [57]. Mekni et al. [17] found a significant correlation between the best antioxidant activity of various pomegranate extracts and the extract's highest phenolic and flavonoid contents; furthermore, this antioxidant activity can be related to the presence of different functional groups, such as hydroxyl and carbonyl groups.

#### 3.3.2. Anticancer

In vivo anti-proliferative activity was assessed against lung carcinoma (A-549) using different concentrations of PP extract. The results indicated that the extract displayed potent anticancer activity and cytotoxic effects against A-549 lung carcinoma. The doxorubicin drug was also tested as a positive control for comparison. As illustrated in Figure 3A,B, increasing inhibition of cell viability with increasing extract concentrations was observed (dose-dependent manner). Maximum growth inhibition of 86% was detected for lung carcinoma cells at a dose of 1000 µg/mL. The 50% inhibition of cell viability (IC50) for A-549 was 250 µg/mL, representing a valuable result compared to the IC50 value of doxorubicin of 300 µg/mL. Compared to carcinoma cells, a less cytotoxic effect was recorded against normal lung cells. The anticancer activity of the pomegranate peel ethanolic extract was carried out against the oral cancer cell line (KB 3-1), and this peel extract exhibited promising anticancer properties. The MTT assay showed 94.53% inhibition on the oral cancer cell line, and the clonogenic assay showed a decrease in the colonies after treatment with the peel extract [58]. Moreover, against various kinds of cancer, for example, in prostate cancer and renal cell carcinoma tissues grown in vitro, pomegranate extracts inhibit the activity

of NF-kB [59]. Punicalagin, generated from pomegranate extracts, has anti-proliferative activity by inducing apoptosis of cancer cells such as lung carcinoma and breast and cervical cancer cell lines [60]. Induction of the apoptosis process by punicalagin through caspase activation and poly ADP ribose polymerase (PARP) inhibition. Caspase-3, -8, and -9 protein expressions exhibited a dose-dependent increase after 50 and 75 µM punicalagin treatment. Furthermore, A549 cells treated with 50 and 75 µM concentrations of punicalagin showed cleavage of PARP protein [61]. *Bioengineering* **2022**, *9*, x FOR PEER REVIEW 11 of 23

**Figure 3.** (**A**) Microscopic image of the effect of PP at different levels (0.2–1 mg/mL) on the lung cancer line viability compared to the negative and positive control, (**B**) histogram of PPE effect on cancer cell lines viability, (**C**) % caspase 3 activity in response to cancer cell death. Data are presented as mean ± SE. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05. **Figure 3.** (**A**) Microscopic image of the effect of PP at different levels (0.2–1 mg/mL) on the lung cancer line viability compared to the negative and positive control, (**B**) histogram of PPE effect on cancer cell lines viability, (**C**) % caspase 3 activity in response to cancer cell death. Data are presented as mean ± SE. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05.

Figure 3C shows that the activity of caspase-3 increased with cancer cell death, showing high activity when lung cancer cell lines were treated with PP extract (1 mg/mL). Caspase-3 is essential for normal brain development and other apoptotic conditions in a manner that is tissue-, cell-, or death-stimulus-specific. It is also necessary for apoptotic chromatin condensation and DNA fragmentation in every examined cell type. Caspase-3 is therefore required for cellular disintegration and the formation of apoptotic bodies. However, it may also function before or during cell death [62]. Pomegranate fruit and its extracts have been found to combat cancer in many in vitro and in vivo studies; modulating various signaling pathways is the mechanism of action against cancer growth. The introduction

of targeted medicines has revolutionized the treatment of patients with A-549 carcinoma. When combined with natural extracts with stratagems, repurposing non-cancer drugs into new therapeutic niches presents a cost-effective and efficient technique with enhanced outcomes for discovering novel pharmacological activity to potentially increase cure rates for lung and liver cancers [63,64].

#### 3.3.3. Antimicrobial

Table 4 shows the antibacterial and antifungal activities of PP extract against some foodborne and multidrug-resistant pathogens. The extract has a wide spectrum of antimicrobial activity; the inhibition zone diameters (IZDs) significantly increased *p* ≤ 0.05 in a concentration-dependent manner and were 9–35 mm against the tested bacteria compared to penicillin. The most sensitive bacteria to PP extract (1 mg/mL) were *S. aureus* with IZD, 35 mm. However, *K. pneumonia* were the most resistant bacteria to PP extract (1 mg/mL), where IZD was 27 mm, with a relative decrease of about 30% of the *S. aureus* inhibition zone.

**Table 4.** Antimicrobial activity of PP extract expressed as IZDs (mm) against pathogenic microorganisms.

Data are presented as mean ± SD, lowercase letters (a, b, c, d, . . . ) in the same columns indicate significant differences (effect of each pp concentration on pathogenic bacteria and fungi) *p* ≤ 0.05. \* St, penicillin (1000 µg/mL) for bacteria and clotrimazole (1000 µg/mL) for fungi.

The resistant Gram-negative bacteria have lower IDZs than the Gram-positive ones because of the unique structure of the Gram-negative bacteria's membrane.

On the other hand, as shown in Table 4, PP extract exhibited considerable antifungal activity against *Candida, Aspergillus,* and *Penicillium* species in this study, with IZDs ranging from 13 to 35 mm compared to clotrimazole (1000 µg/mL). *A. niger* and *C. glabrata* were the most resistant fungi to PP extract, while *A. flavus* and *P. expansum* were the most vulnerable. Figure S1 shows the IZDs image of PP extract 0.6, 0.8, and 1.0 mg/mL against tested bacteria (*S. aureus*, *L. monocytogenes*, *B. cereus*, *P. aeruginosa*, *K. pneumonia*, *E. coli*), Candida (*C. glabrata*, *C. albicans*, *C. davenportii*), and fungi (*A. niger*, *A. flavus*, *P. expansum*). Kesur et al. [12] demonstrated that the high efficiency of the methanolic extract of pomegranate peel against the *Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Escherichia coli*, *and Aspergillus niger* showed inhibition zones between 6 and 17 mm. At the same time, no activity was detected by chloroform extract against the same microorganisms. On the other hand, pomegranate waste extract has powerful antifungal activity against *Candida gelberta* and *Candida apis* [65].

The PP extract successfully inhibited the bacterial and fungal isolates in the 45–160 µg/mL concentration range while killing the tested isolates with 80–290 µg/mL concentrations

(Table 5). Kesur et al. [12] found that the minimum inhibitory concentration of the methanolic extracts of peels of pomegranate on *B. cereus*, *B. megaterium*, *P. vulgaris*, and *P. aeruginosa* was observed in the range of 50,0000 to 12,500 (µg/mL). In contrast, the MIC of peel extracts on *B. subtilis*, *S. typhi*, *S. typhi A*, and *S. typhi B* was found to be in the range of 50,000 to 25,000 (µg/mL), while MIC against fungi is higher than that. An efficient antimicrobial effect expressed as MIC of the crude extract was observed against the studied strain, for which we recorded 312.5 µg/mL. It was reported by Oliveira et al. [66] that strong inhibitors have MIC values below 500 µg/mL, moderate inhibitors have values between 600 and 1500 µg/mL, and weak inhibitors are above 1600 µg/mL. Hasan et al. [67] confirmed that pomegranate flavonoids and phenolic compounds are extracted more efficiently with methanol due to their polarity. Naziri et al. [68] showed that active antimicrobial substances have different solubility in various solvents and that using different solvents for extraction changes the extent of the antibacterial effect of the extract.

**Table 5.** The minimum concentration of PP extract that inhibits or kills bacteria or fungi.

Lowercase letters (a, b, c, d, . . . ) in the same columns indicate significant differences (effect of each pp concentration on pathogenic bacteria and fungi) *p* ≤ 0.05. \* MIC, minimum inhibitory concentration; \* MBC, minimum bactericidal concentration; \* MFC, minimum fungicidal concentration of PP extract against tested pathogens.

Pomegranates are a good source of potentially bioactive phytochemicals such as phenolics, flavonoids, anthocyanins, alkaloids, and tannins [8]. The results of this study clearly showed a good relationship between the IDZs of the studied bacteria and pomegranate varieties. Rosas-Burgos et al. [69] consistently observed different inhibition levels for the studied pomegranate peel extracts, where the *Salmonella* strain was the most sensitive. They concluded that the highest punicalagin and ellagic acid concentrations were detected in sour and sweet pomegranate cultivars with the highest inhibitory activity. Vasconcelos et al. [70] numerous microbial enzymes in culture filtrates or pure forms are inhibited by tannic, and the toxicity of tannins may be connected to their effect on microbes' membranes. High antibacterial activity for the studied methanolic extracts was observed, while chloroform extracts were less effective, with highly significant differences. Suresh et al. [71] observed a gradual decline in the protein content of bacterial cells incubated with different concentrations of pomegranate peel extract (PPE) compared to the control.

Based on the polynomial model in (Figure 4), employing a higher concentration of PP, i.e., 2 mg/mL, will increase the IZDs by 58.6, 96.4, 20.3, and 42.5% against *Listeria monocytogenes*, *E. coli*, *C. albicans*, and *A. flavus*, respectively, compared to PP (1 mg/mL). We noticed that *C. albicans* were more sensitive to PP extract (2 mg/mL), followed by *E. coli.*

*Bioengineering* **2022**, *9*, x FOR PEER REVIEW 14 of 23

**Figure 4.** Polynomial model for forecasting the antimicrobial activity with increasing PP concentrations. **Figure 4.** Polynomial model for forecasting the antimicrobial activity with increasing PP concentrations. **Figure 4.** Polynomial model for forecasting the antimicrobial activity with increasing PP concentrations.

#### 3.3.4. Antiviral 3.3.4. Antiviral 3.3.4. Antiviral

Figure 5 shows the inhibition activity of PP extract on the binding between Spike and ACE2 compared to a SARS-CoV-2 inhibitor test kit. At doses of 200, 400, 600, 800, and 1000 µg/mL, PP extract inhibited the interaction between Spike and ACE2 by as much as 75.6%. This impact was dose-dependent compared to the positive control, AC384, a monoclonal antibody that prevented the binding between Spike and ACE2 by identifying ACE2 itself and inhibiting 70% of the interaction. Surucic et al. [72] found that punicalagin in pomegranate peel extract inhibited 50% of S-glycoprotein and ACE2 contact only in the highest concentration sample (1 mg/mL). Figure 5 shows the inhibition activity of PP extract on the binding between Spike and ACE2 compared to a SARS-CoV-2 inhibitor test kit. At doses of 200, 400, 600, 800, and 1000 µg/mL, PP extract inhibited the interaction between Spike and ACE2 by as much as 75.6%. This impact was dose-dependent compared to the positive control, AC384, a monoclonal antibody that prevented the binding between Spike and ACE2 by identifying ACE2 itself and inhibiting 70% of the interaction. Surucic et al. [72] found that punicalagin in pomegranate peel extract inhibited 50% of S-glycoprotein and ACE2 contact only in the highest concentration sample (1 mg/mL). Figure 5 shows the inhibition activity of PP extract on the binding between Spike and ACE2 compared to a SARS-CoV-2 inhibitor test kit. At doses of 200, 400, 600, 800, and 1000 µg/mL, PP extract inhibited the interaction between Spike and ACE2 by as much as 75.6%. This impact was dose-dependent compared to the positive control, AC384, a monoclonal antibody that prevented the binding between Spike and ACE2 by identifying ACE2 itself and inhibiting 70% of the interaction. Surucic et al. [72] found that punicalagin in pomegranate peel extract inhibited 50% of S-glycoprotein and ACE2 contact only in the highest concentration sample (1 mg/mL).

pomace extract. Data are presented as mean ± SE. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05. **Figure 5.** PP concentrations (200–1000 µg/mL) affect Spike/ACE2 binding compared with control and antibody inhibitor AC384 (5 µg/mL). ACE2, angiotensin-converting enzyme 2; PP, pomegranate pomace extract. Data are presented as mean ± SE. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05. **Figure 5.** PP concentrations (200–1000 µg/mL) affect Spike/ACE2 binding compared with control and antibody inhibitor AC384 (5 µg/mL). ACE2, angiotensin-converting enzyme 2; PP, pomegranate pomace extract. Data are presented as mean ± SE. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05.

There are no studies on the antiviral activity of PP extract; however, most earlier investigations focused on pomegranate peel extracts, which showed antibacterial and antiviral activity and suppressed influenza and herpes viruses, which can also be employed effectively as an antiviral agent against SARS-CoV-2. In vitro, the aqueous–alcoholic pomegranate peel extract stopped SARS-CoV-2 S-glycoprotein from binding to ACE2, showing that the extract can stop SARS-CoV-2 from entering the host cells [73].

#### *3.4. Changes in Physiochemical Properties of PP-Strawberry-Yogurt Smoothie* 3.4.1. Physiochemical Properties

## The functional milk beverage fortified with pomegranate pomace extract during

storage influences the pH and beverage acidity. During two months of storage at 4 ◦C, the pH values of PP-smoothie samples decreased by 8–13% compared to the control smoothie; therefore, the pp-smoothie acidity increased by 16–21% compared to the control. Thus, in agreement with Al-Hindi and Abd El Ghani [74], pH levels of the functional milk beverages with and without POPE decreased significantly from 4.5 to 4.1 with increasing storage time. The higher counts of viable bacteria at the end of the storage period resulted in lower pH levels in the functional milk beverages fortified with or without POPE. In addition, the beverage's acidity increased markedly from 0.2 to 0.35 with increasing storage time.

Massive decreases in vitamin C content in the control sample (133%) after 2 months of storage, but these reductions were considerably inhibited (*p* ≤ 0.05) in PP-smoothies by 11–55% over control. The high phenolic content in PP extract inhibited the oxidation of vitamin C, and total sugars were decreased with storage period by up to 53% in the control sample, but these reductions decreased in PP-SYS1, PP-SYS2, PP-SYS3, and PP-SYS4 by 4–28% compared to the control. The high phenolic content in PP extract inhibited the fermentative action of microbes against sugars. The reductions in sugars were followed by an increase in TSS (28–38%) in PP smoothies compared to the control. In addition, the fat content was elevated based on pomegranate pomace concentrations (Table 6). During three weeks of cold storage, a substantial increment in fat content was detected between untreated and treated yogurt (*p* < 0.05) [3]. Saad et al. [75] observed the same trend in cucumber beverages supplemented with polyphenolic extracts, i.e., decrements in vitamin C and total sugars while increasing TSS percentage. They noticed that the decrements in vitamin C and total sugars were mitigated by polyphenolic extract addition.

**Table 6.** Changes in physiochemical parameters of smoothie supplemented with pomegranate pomace extract at different addition levels (0.4, 0.8, 1.2, 1.6 mg/mL) during two-month storage at 4 ◦C.

Data are presented as mean ± SD, lowercase letters (a, b, c, d, . . . ) in the same columns indicate significant differences (between the changes in physiochemical parameters of SYS samples during 2-month storage period) at *p* ≤ 0.05.

Strawberry-yogurt smoothie (SYSC) supplemented with pomegranate pomace extracts 0.4 mg/mL (PP-SYS1), smoothie supplemented with pomegranate pomace extract 0.8 mg/mL (PP-SYS2), smoothie supplemented with pomegranate pomace extract 1.2 mg/mL (PP-SYS3), smoothie supplemented with pomegranate pomace extract 1.6 mg/mL (PP-SYS4). 3.4.2. Total Phenolic Content Polyphenols have a crucial role in the color and flavor of foodstuffs while being very volatile and easily oxidized [76]. Adding PP extract with different levels (0.4, 0.8, 1.2, and 1.6 mg/mL) to the tested smoothie enhanced the antioxidant activity from 50% in the con-

*Bioengineering* **2022**, *9*, x FOR PEER REVIEW 16 of 23

Strawberry-yogurt smoothie (SYSC) supplemented with pomegranate pomace extracts 0.4 mg/mL (PP-SYS1), smoothie supplemented with pomegranate pomace extract 0.8 mg/mL (PP-SYS2), smoothie supplemented with pomegranate pomace extract 1.2 mg/mL (PP-SYS3), smoothie supplemented with pomegranate pomace extract 1.6 mg/mL

#### 3.4.2. Total Phenolic Content trol smoothie to 88% in PP-SYS4 (data not shown). The antioxidant activity of smoothies

(PP-SYS4).

Polyphenols have a crucial role in the color and flavor of foodstuffs while being very volatile and easily oxidized [76]. Adding PP extract with different levels (0.4, 0.8, 1.2, and 1.6 mg/mL) to the tested smoothie enhanced the antioxidant activity from 50% in the control smoothie to 88% in PP-SYS4 (data not shown). The antioxidant activity of smoothies was enhanced due to the presence of phenolics and flavonoids that were boosted by the PP extract. The total phenolic and flavonoid content in PP-SYS4 increased by 170% compared to the control at zero months of storage. However, after 2 months of storage, the activity further increased, estimated at 98%, despite decreasing the phenolic and flavonoid content by 14% (Figure 6). No studies have focused on using PP extract as an additive in foods. However, our findings may be correlated with Ahmed et al. [77], who added pomegranate peels to yogurt and observed their effects on total phenolic contents and antioxidant activity of the product; they found an average TPC in yogurt of (4.07 ± 0.37) mg GAE/g. However, the maximum content was detected in pomegranate yogurt (4.64 mg GAE/g compared to the control, which was 3.39 mg GAE/g). Antioxidant activity examination reveals that an average (70.58%) was calculated. Meanwhile, extreme activity was diagnosed in the pomegranate yogurt sample (83.87%), having 9% freeze-dried PPP. was enhanced due to the presence of phenolics and flavonoids that were boosted by the PP extract. The total phenolic and flavonoid content in PP-SYS4 increased by 170% compared to the control at zero months of storage. However, after 2 months of storage, the activity further increased, estimated at 98%, despite decreasing the phenolic and flavonoid content by 14% (Figure 6). No studies have focused on using PP extract as an additive in foods. However, our findings may be correlated with Ahmed et al. [77], who added pomegranate peels to yogurt and observed their effects on total phenolic contents and antioxidant activity of the product; they found an average TPC in yogurt of (4.07 ± 0.37) mg GAE/g. However, the maximum content was detected in pomegranate yogurt (4.64 mg GAE/g compared to the control, which was 3.39 mg GAE/g). Antioxidant activity examination reveals that an average (70.58%) was calculated. Meanwhile, extreme activity was diagnosed in the pomegranate yogurt sample (83.87%), having 9% freeze-dried PPP. Due to the abundance of phenolic components in several plant extracts, developing natural alternatives to synthetic preservatives in food is of special interest because these compounds possess antioxidant and antimicrobial properties that inhibit the process of lipid peroxidation in fatty foods and scavenging free radicals and prevent the growth of microorganisms [75,78,79].

**Figure 6.** Fluctuations in total phenolic content (TPC) of smoothie supplemented with pomegranate pomace extract at different addition levels (0.4, 0.8, 1.2, 1.6 mg/mL) during two-month storage at 4 °C. Lowercase letters above columns indicate significant differences between SYS samples. Lowercase letters with a star indicate a significant change during the storage period for each SYS. **Figure 6.** Fluctuations in total phenolic content (TPC) of smoothie supplemented with pomegranate pomace extract at different addition levels (0.4, 0.8, 1.2, 1.6 mg/mL) during two-month storage at 4 ◦C. Lowercase letters above columns indicate significant differences between SYS samples. Lowercase letters with a star indicate a significant change during the storage period for each SYS.

Due to the abundance of phenolic components in several plant extracts, developing natural alternatives to synthetic preservatives in food is of special interest because these compounds possess antioxidant and antimicrobial properties that inhibit the process of lipid peroxidation in fatty foods and scavenging free radicals and prevent the growth of microorganisms [75,78,79].

#### 3.4.3. Color Difference and Sensorial Properties

Analysis of the nutritional values and the quality of ingredients of the beverages are very important to ensure the safety and quality of the beverages. Sensory investigation using color, taste, odor, and textural analysis efficiently assesses the final beverage quality [80]. Based on the color analysis of PP in Table 2, the extract has a high redness value that enhances the whiteness and red color of the strawberry-PP yogurt smoothie. Nonetheless, at the end of storage, the color parameters faded (data not shown), but the addition of PP extract mitigates the color reduction, and that is clear in Figure 7, where color change (∆E) value was observed as limiting the color change in PP-supplemented smoothies; the ∆E value in the control was 6.14; however, in PP-SYS4, it decreased by 22%. Trigo et al. [81] found that with the addition of pomegranate peel extract to carrot beverage, the *whiteness* was not affected by PPE, but heat treatment led to higher *L*\* levels compared to the HPP treatment. The increase in *L*\* values was noticed in the first week of storage, but the *L\** remained constant within the next few days. Concerning ∆E, this difference was evident throughout the entire storage period. For example, on day 7, the ∆*E*\* changed between 1.2 and 2.0 for HPP-treated juice and between 22.1 and 32.1 for TP-treated juice. 3.4.3. Color Difference and Sensorial Properties Analysis of the nutritional values and the quality of ingredients of the beverages are very important to ensure the safety and quality of the beverages. Sensory investigation using color, taste, odor, and textural analysis efficiently assesses the final beverage quality [80]. Based on the color analysis of PP in Table 2, the extract has a high redness value that enhances the whiteness and red color of the strawberry-PP yogurt smoothie. Nonetheless, at the end of storage, the color parameters faded (data not shown), but the addition of PP extract mitigates the color reduction, and that is clear in Figure 7, where color change (ΔE) value was observed as limiting the color change in PP-supplemented smoothies; the ΔE value in the control was 6.14; however, in PP-SYS4, it decreased by 22%. Trigo et al. [81] found that with the addition of pomegranate peel extract to carrot beverage, the *whiteness* was not affected by PPE, but heat treatment led to higher *L*\* levels compared to the HPP treatment. The increase in *L*\* values was noticed in the first week of storage, but the *L\** remained constant within the next few days. Concerning ΔE, this difference was evident throughout the entire storage period. For example, on day 7, the Δ*E*\* changed between 1.2 and 2.0 for HPP-treated juice and between 22.1 and 32.1 for TP-treated juice.

*Bioengineering* **2022**, *9*, x FOR PEER REVIEW 17 of 23

**Figure 7.** Changes in the color of the smoothie supplemented with pomegranate pomace extract at different addition levels (0.4, 0.8, 1.2, 1.6 mg/mL) during two months of storage at 4 °C. Table 7 shows the sensory properties of tested smoothies. The results indicated that **Figure 7.** Changes in the color of the smoothie supplemented with pomegranate pomace extract at different addition levels (0.4, 0.8, 1.2, 1.6 mg/mL) during two months of storage at 4 ◦C. Different lowercase letters above columns indicate significant differences.

PP-SYS4 gained the highest scores by panelists, i.e., flavor and color (9), texture (8.8), and over acceptability (8.9) after a month of storage at 4 °C, but a slight decrease was noticed and reflected on for acceptability values that declined by 3% compared to the control, which decreased by 9%. The results for the other smoothies are in between PP-SYS4 and the control. Ahmed et al. [77] noticed low sensorial scores in relation to the yogurt with pomegranate peels, which were reduced by 9%, but decreasing the addition level increases the sensorial scores. Sensory evaluation of food products is important in determining what consumers might like. The main way to judge a juice's quality is by color [82]. Table 7 shows the sensory properties of tested smoothies. The results indicated that PP-SYS4 gained the highest scores by panelists, i.e., flavor and color (9), texture (8.8), and over acceptability (8.9) after a month of storage at 4 ◦C, but a slight decrease was noticed and reflected on for acceptability values that declined by 3% compared to the control, which decreased by 9%. The results for the other smoothies are in between PP-SYS4 and the control. Ahmed et al. [77] noticed low sensorial scores in relation to the yogurt with pomegranate peels, which were reduced by 9%, but decreasing the addition level increases the sensorial scores. Sensory evaluation of food products is important in determining what consumers might like. The main way to judge a juice's quality is by color [82].

**Table 7.** Fluctuations in sensory traits of smoothie supplemented with pomegranate pomace extract at different addition levels (0.4, 0.8, 1.2, 1.6 mg/mL) during two months of storage at 4 ◦C.

Data are presented as mean ± SD. Lowercase letters in the same columns indicate significant differences *p* ≤ 0.05. Smoothie supplemented with pomegranate pomace extract 0.4 mg/mL (PP-SYS1), smoothie supplemented with pomegranate pomace extract 0.8 mg/mL (PP-SYS2), smoothie supplemented with pomegranate pomace extract 1.2 mg/mL (PP-SYS3), smoothie supplemented with pomegranate pomace extract 1.6 mg/mL (PP-SYS4).

#### *3.5. Microbial Load Changes during Storage of Smoothie*

The microbial load in all smoothie samples was significantly increased with the storage period; however, it decreased with the gradual addition of levels of PP extract. The microbial load in control samples was 3.5 CFU/mL and increased by 56% after two months. However, in PP-SYS4, the microbial load significantly decreased (*p* ≤ 0.05) by 37% compared to the control after 2 months of cold storage (Figure 8). Ebrahimain et al. [83] studied microbiological stability and sensory properties of traditional Iranian butter with pomegranate peel extract incorporated into it, and they found that PPE could reduce the bacterial count in butter from 9.25 log CFU to 6.30 log CFU after 90 days of storage, while with increasing storage time, the total number of bacteria was increased in the treatments; this increase is most evident when dairy products are made with poor safety quality. *Bioengineering* **2022**, *9*, x FOR PEER REVIEW 19 of 23

**Figure 8.** Total bacterial count (TBC) in smoothie supplemented with pomegranate pomace extract at different addition levels (0.4, 0.8, 1.2, 1.6 mg/mL) during two months of storage at 4 °C. **Figure 8.** Total bacterial count (TBC) in smoothie supplemented with pomegranate pomace extract at different addition levels (0.4, 0.8, 1.2, 1.6 mg/mL) during two months of storage at 4 ◦C.

There is a current need to renew attention in looking for natural resources of bioactive active compounds to be used as safe food additives. Isolation of such compounds from agro-industrial wastes will provide a cost-effective solution for controlling the spread of

sesses great potential to scavenge DPPH free radicals with high antioxidant capacity, besides its potent antitumor activity in lung cell lines and potential cure of COVID-19. Adding PP extract (1.2 or 1.6 mg/mL) to SYS samples enhances color, texture, and quality and introduces a functional beverage that may benefit many patients. Considering all these details, we believe that pomace of *Punica granatum* may provide a safe and efficient alternative-perspective useful additive in functional food. We hope to continue further explo-

**Supplementary Materials:** The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Inhibition zones diameters of PP extracts (0.6, 0.8, and 1.0 mg/mL) against tested bacteria (*S. aureus, L. monocytogenes, B. cereus, P. aeruginosa, K. pneumonia, E. coli*); Can-

**Author Contributions:** N.H.A., D.A.A.-Q., and A.S. designed the experiments. G.I.A., M.A. (Mona Alharbi), N.B., M.A. (Majidah Aljadani), and A.S.: Writing—original draft and editing. S.H.Q., F.A.J., J.A., A.N.A.E., and A.S.: Methodology. N.H.A., D.A.A.-Q., and A.S.: Conceptualization. G.I.A., M.A. (Mona Alharbi), N.B., M.A. (Majidah Aljadani), and A.S.: resources, formal analysis. S.H.Q., H.M.S., M.H., F.A.J., J.A., A.N.A.E., and A.S.: Methodology and Formal Analysis. N.H.A., D.A.A.-Q., and A.S.: Conceptualization and Revision. All authors have read and agreed to the published version of

dida (*C. glabrata, C. albicans, C. davenportii*), and fungi (*A. niger, A. flavus, P. expansum*).

the manuscript. the article

rations of this attractive botanical species.

**Funding:** This research received no external funding. **Institutional Review Board Statement:** Not applicable.

**4. Conclusions**

Many studies have indicated that phenolic compounds in PP extract contribute to the direct inhibition of bacterial pathogens and higher antioxidant activity [84]. The ultracellular damage of tested bacteria treated with sour pomegranate peel extract was examined using TEM, illustrating severe damage in the bacterial cells and confirming the extract's potential antibacterial and lethal activity. Karaman et al. [85] also showed that lipophilic parts in phenolic acids permeate through the cell membrane by passive diffusion, enhancing membrane permeability by reducing intracellular pH. The triggering of protein denaturation, explained by Li et al. [14], confirmed that damage to the bacterial cell membrane is one of the possible modes of action for the phytochemical elements, followed by releasing intracellular material and cell death.

#### **4. Conclusions**

There is a current need to renew attention in looking for natural resources of bioactive active compounds to be used as safe food additives. Isolation of such compounds from agro-industrial wastes will provide a cost-effective solution for controlling the spread of human pathogens and improve waste reduction. Given that, pomegranate pomace was found to exert a marked reduction in multidrug resistance and food pathogens and possesses great potential to scavenge DPPH free radicals with high antioxidant capacity, besides its potent antitumor activity in lung cell lines and potential cure of COVID-19. Adding PP extract (1.2 or 1.6 mg/mL) to SYS samples enhances color, texture, and quality and introduces a functional beverage that may benefit many patients. Considering all these details, we believe that pomace of *Punica granatum* may provide a safe and efficient alternative-perspective useful additive in functional food. We hope to continue further explorations of this attractive botanical species.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bioengineering9120735/s1, Figure S1: Inhibition zones diameters of PP extracts (0.6, 0.8, and 1.0 mg/mL) against tested bacteria (*S. aureus, L. monocytogenes, B. cereus, P. aeruginosa, K. pneumonia, E. coli*); Candida (*C. glabrata, C. albicans, C. davenportii*), and fungi (*A. niger, A. flavus, P. expansum*).

**Author Contributions:** N.H.A., D.A.A.-Q. and A.S. designed the experiments. G.I.A., M.A. (Mona Alharbi), N.B., M.A. (Majidah Aljadani), and A.S.: Writing—original draft and editing. S.H.Q., F.A.J., J.A., A.N.A.E. and A.S.: Methodology. N.H.A., D.A.A.-Q. and A.S.: Conceptualization. G.I.A., M.A. (Mona Alharbi), N.B., M.A. (Majidah Aljadani), and A.S.: resources, formal analysis. S.H.Q., H.M.S., M.H., F.A.J., J.A., A.N.A.E. and A.S.: Methodology and Formal Analysis. N.H.A., D.A.A.-Q. and A.S.: Conceptualization and Revision. All authors have read and agreed to the published version of the manuscript the article.

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

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

**Sample Availability:** Samples of the compounds are not available from the authors.

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**Pabitra Chandra Das , Alec Roger Vista, Lope G. Tabil \* and Oon-Doo Baik \***

Department of Chemical and Biological Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon, SK S7N 5A9, Canada<EMAIL_ADDRESS>(P.C.D<EMAIL_ADDRESS>(A.R.V.) **\*** Correspondence<EMAIL_ADDRESS>(L.G.T<EMAIL_ADDRESS>(O.-D.B.)

**Abstract:** In recent years, cannabis (*Cannabis sativa* L.) has been legalized by many countries for production, processing, and use considering its tremendous medical and industrial applications. Cannabis contains more than a hundred biomolecules (cannabinoids) which have the potentiality to cure different chronic diseases. After harvesting, cannabis undergoes different postharvest operations including drying, curing, storage, etc. Presently, the cannabis industry relies on different traditional postharvest operations, which may result in an inconsistent quality of products. In this review, we aimed to describe the biosynthesis process of major cannabinoids, postharvest operations used by the cannabis industry, and the consequences of postharvest operations on the cannabinoid profile. As drying is the most important post-harvest operation of cannabis, the attributes associated with drying (water activity, equilibrium moisture content, sorption isotherms, etc.) and the significance of novel pre-treatments (microwave heating, cold plasma, ultrasound, pulse electric, irradiation, etc.) for improvement of the process are thoroughly discussed. Additionally, other operations, such as trimming, curing, packaging and storage, are discussed, and the effect of the different postharvest operations on the cannabinoid yield is summarized. A critical investigation of the factors involved in each postharvest operation is indeed key for obtaining quality products and for the sustainable development of the cannabis industry.

**Keywords:** cannabis; cannabinoids; postharvest; drying; curing; storage; biosynthesis; pre-treatments; sorption isotherm

#### **1. Introduction**

Cannabis, scientifically known as *Cannabis sativa* L. from the family Cannabaceae, has been cultivated for ages and utilized as a potential source of fiber, oil, protein, and popularly, psychoactive and medicinal purposes [1]. Historically, cannabis products have been illicitly grown for recreational and social activities to appreciate their euphoric effects [2]. Cannabis contains more than 120 active compounds, which are classified to belong in a secondary metabolite group known as phytocannabinoids or cannabinoids [3] of which cannabidiol (CBD), ∆9-tetrahydrocannabinol (THC), cannabigerol (CBG), and cannabinol (CBN) are the most studied [4]. Terpenes, which are secondary metabolites usually associated with the flavor and smell profile of biological materials have recently been found to be relevant in terms of the chemical make-up of cannabis [5]. *Cannabis sativa* L. is often classified into three subspecies depending on physical or chemical characteristics, namely *Cannabis sativa* ssp. *sativa*, *Cannabis sativa* ssp. *indica*, *Cannabis sativa* ssp. *ruderalis* [6]. The cannabis plant (Figure 1) is holistically utilized to undergo different treatments after harvest for specific uses.

As shown in Figure 1, the cola or inflorescence of cannabis contains the most abundant amount of trichomes. Trichomes are glandular membranes containing secondary metabolites, phytocannabinoids, and terpenes, and are found to be most abundant on the surface of the cannabis inflorescence.

**Citation:** Das, P.C.; Vista, A.R.; Tabil, L.G.; Baik, O.-D. Postharvest Operations of Cannabis and Their Effect on Cannabinoid Content: A Review. *Bioengineering* **2022**, *9*, 364. https://doi.org/10.3390/ bioengineering9080364

Academic Editors: Minaxi Sharma, Kandi Sridhar, Zeba Usmani and Xiaohu Xia

Received: 23 June 2022 Accepted: 28 July 2022 Published: 3 August 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/).

**Figure 1.** A cannabis plant: (**1**) mature bud/calyx/inflorescence, (**2**) stem, (**3**) fan leaves, (**4**) sugar leaves, and (**5**) trichomes (images were taken by M. Neufeldt and are used with permission).

Extracts or products of cannabis have been studied for the treatment of different illnesses including neurological and psychiatric disorders [2], prevention of vomiting and nausea of cancer patients, boosting of hunger in acquired immunodeficiency syndrome (AIDS) patients [7], and the treatment of muscle spasms, spasticity and neuropathic pain, and chronic pain [8]. On the other hand, hemp has been produced for textiles, biocomposites, papermaking, biofuel, functional foods, and cosmetics [6]. Hemp seeds have been used in the processing of different edible foods like cookies, chocolate, peanut-cannabis butter blends, coffee, etc. The benefits outweigh the detriments brought about by cannabis as it has been legalized by more than 50 countries and 12 states of the USA for production, processing, and use [9]. Today, the cannabis industry is developing around the world, resulting in economic gain. The total cannabis market was anticipated at \$3.5 billion in 2019, and a consistent increase up to \$20.2 billion is expected by 2025. In Canada, production and marketing of cannabis have been legalized since October 2018 for medicinal and recreational purposes, although special monitoring and licensing from Health Canada is required in many cases. Total cannabis sales in Canada were around \$210 million from mid-October to December 2018 and this value can reach multi-billions by 2021 [10]. This growing business sector should be given special focus to ensure safety and quality for the consumers.

Aside from positive effects, cannabis has been shown to cause detrimental health impacts in humans. Continuous usage of cannabis has been found to cause a variety of immediate and long-term detrimental consequences, including cardiovascular and respiratory problems, behavioral impairment, psychosis, schizophrenia, and mental illnesses [2]. People with cannabis use disorder have problems such as extreme mood shifts and memory-loss-related and concentration issues [11].

After harvest, different treatments are involved in the processing of cannabis-based food or non-food products. Drying is the most important postharvest operation involved in cannabis along with storage and curing. However, the present industrial drying involves a

hang-drying method, which is inefficient and can cause an inconsistent quality of products. Thus, it is urgent to identify suitable drying methods that can enhance consistency in terms of quality for large-scale operations. In this paper, the postharvest treatments involved in cannabis processing and preservation are critically discussed and interpreted. In a broader context, this paper includes the following sections: phytocannabinoids and their decarboxylation; the postharvest operations of cannabis, including trimming, drying of cannabis (and the technical factors associated with these operations, such as equilibrium moisture content and sorption isotherms), curing, and storage and packaging; and finally, the effects of different postharvest treatments on the cannabinoid profile of cannabis. The relevant published articles were collected using the search engines Google Scholar, Scopus, and the library website of the University of Saskatchewan by inputting the keywords "cannabis, postharvest, cannabinoids" and the published articles or books from 2011 to date were considered. However, for specific interests like biosynthesis, sorption, pre-treatments, etc., the keywords were changed, and a specific publication period was not considered for extracting basic information.

#### **2. Phytocannabinoids and Decarboxylation**

Because it has various natural ingredients, cannabis is a chemically complicated plant. It includes secondary metabolites or phytochemicals called cannabinoids, a special type of terpenophenolic molecule [12]. Apart from cannabis, few other plants, such as cinnamon, clove, oregano, cocoa, black truffles, and black pepper, among others, also have cannabinoids in minor amounts. There are currently more than 120 cannabinoids found in the genus Cannabis that have different properties, structures, and uses [13]. THC and CBD are the most popular.

Radwan et al. [12] grouped cannabinoid compounds into 11 subcategories: ∆9-THC type (23 compounds), ∆8-THC (5 compounds), CBG (16 compounds), cannabichromene (CBC, 9 compounds), CBD (7 compounds), cannabinodiol (CBND, 2 compounds), cannabielsoin (CBE, 5 compounds), cannabicyclol (CBL, 3 compounds), cannabinol (CBN, 11 compounds), and cannabitriol (CBT, 9 compounds), along with miscellaneous type cannabinoids (30 compounds). According to Addo et al. [4], ∆9-THC, CBD, CBG, and CBN are the major cannabinoid groups because of their distinct nature and application.

The biosynthesis process describes the formation of the neutral forms of major cannabinoid compounds from their acidic form(s). In the biosynthesis process (Figure 2), cannabigerolic acid (CBGA) is produced by combining the primary molecules olivetolic acid and geranyl-pyrophosphate (CBGA) with the action of geranyl-pyrophosphate-olivetolic acid geranyltransferase enzymes, which is then used to synthesize cannabinoids [14,15]. Oxidocyclase enzymes convert CBGA into acidic forms of THCA, CBDA, and CBCA [4].

All enzymatically generated cannabinoids (including CBG) start off at an acidic state, that is then decarboxylated through heating to yield the functional or active form. With the application of heat, THCA, CBDA, and CBGA change into their active forms of THC, CBD, and CBG, respectively. Active cannabinoids have several physiological or psychological functions that could be either beneficial or detrimental. THC is euphorigenic, hungertriggering, pain-relieving, and works against vomiting and nausea. CBD is not euphorigenic but works as an anti-inflammatory and anticonvulsant agent. On the other hand, CBG has antibacterial properties as well as working against neurologic disorders and inflammatory bowel disease [15].

A process called decarboxylation, which is the removal of a carboxyl group in the presence of heat, is required to convert acidic cannabinoids to their pure form. Thus, decarboxylation can be defined as the chemical reaction that removes a carboxyl (COOH) group from THCA and CBDA, resulting in THC and CBD, respectively. This reaction is facilitated by heating and vaporization. Wang et al. [13] reported that the decarboxylation of THCA to THC is more pronounced and very abrupt in a period of 60 min for temperatures greater than 100 ◦C. The lower temperature limit used for this study was 80 ◦C and it was reported that the concentration of THCA is still decreasing, following an almost linear

decreasing trend in a period of 1 h. CBDA also follows a similar pattern. Recalling the spontaneous deterioration of THC to CBN, which is a less psychoactive cannabinoid [16], a holistic approach is usually taken when drying cannabis; thus, the use of a low drying temperature is practiced.

**Figure 2.** Biosynthesis of cannabinoids (adapted from Nachnani et al. [15] with permission).

Entourage effect refers to the joint effects of taking these cannabinoids together with other minor cannabinoids and phytochemicals called terpenes (volatile aromatic compounds) being more beneficial than just taking pure THC or pure CBD [17,18]. Trichomes (as shown in Figure 1), which are the microscopic glandular membranes found on the surface of the cannabis inflorescence, contain cannabinoids and terpenes that are vital for psychoactive and medicinal effects. The glands enclosing these cannabinoids and terpenes allow them to be protected from external environmental factors that could hasten degradation [8]. Trichomes, not the buds, contain the cannabinoids that are vital for psychoactive and medicinal use; the temperature sensitivity of the cannabinoid- and terpene-containing trichomes is of prime importance when considering the postharvest unit operation conditions [19–21]. Terpenes have also been closely investigated as a possible solution to the taxonomic classification challenges that arise with the rampant genetic modification of cannabis cultivars to produce more secondary metabolites, focusing on high THC or CBD content in the illicit market in the years that cannabis was illegal. The chemical classification, or more specifically, the metabolomics of cannabis terpenes, is in the forefront of this emerging solution [18,22].

The stability data of these cannabinoids and phytochemicals during harvesting, drying, and storage are important assets for the emerging industry. According to Kader et al. [23], there are biological and environmental factors that influence the deterioration of biological commodities. Biological factors include: (a) respiration, which is the breaking down of

biological components, such as carbohydrates, proteins, and fats into simpler substances; (b) the production of ethylene, which encourages senescence or degradation; (c) changes in the composition (in the case of cannabis), which involves the spontaneous deterioration of THC to CBN; (d) further growth and development, which may do more harm than good to plant quality; (e) water loss or transpiration, although this could be beneficial for cannabis; (f) pathological breakdown due to the presence of moisture; (g) physiological breakdown; and h) physical damage. The physical factors that encourage deterioration are temperature, light, relative humidity (RH), the presence of ethylene, environmental conditions, and the addition of other chemicals, such as fertilizers and fungicides [16,23].

#### **3. Postharvest Operations Involved in Cannabis**

Maturity and harvesting of cannabis flowers depend on a number of factors similar to other plants, including day length, season, variety, soil fertility, temperature, etc. Generally, it takes six to sixteen weeks to harvest the necessary parts from cannabis plants [24]. The general operations involved in the processing of cannabis starting from harvesting and ending at marketing are depicted in Figure 3.

**Figure 3.** Postharvest operations involved in cannabis processing.

#### *3.1. Trimming*

During harvesting, different portions of cannabis are separated from the main plant and further processing is performed based on the target product types. The stems are used for the production of fiber, textiles, or animal bedding while the seeds are used for the extraction of oil, protein, and the processing of different food products. The cannabinoidcontaining parts, the inflorescence along with stems, are removed from the main plant for pharmacological or recreational use. Stems and long fan or sugar leaves are cut to detach the inflorescence. This also helps in increasing the surface area for drying. Dried cannabis sometimes undergoes the trimming process to discard the less desirable parts, such as the sugar leaves, which have less cannabinoid content compared to the inflorescence. Size reduction processes in the food and agriculture industry often involve the use of mechanical force and the large material to separate physical parts or to divide larger particles into smaller more manageable ones. Size reduction processes are often classified based on the nature and direction of force applied to the process, such as compression, shear, or impact [25].

Trimming in the cannabis industry is a niche size reduction operation of removing the sugar leaves that cover the inflorescence, especially during drying. There are two iterations: trimming before the extensive drying process (wet trimming) or trimming after drying and/or curing process (dry trimming). An effective trimming process is delicate enough to preserve the structure of trichomes that house major cannabinoids, but effective enough to remove most of the sugar leaves [26]. Size reduction operations can be done before or after drying, depending on the producer's preference and discretion and ease of flow of operations.

Current trimmer designs commonly use shearing techniques to reduce the amount of sugar leaves on the inflorescence. The mechanism for trimming uses a rotating cylinder with perforated walls or a set of pressurized wires that are designed to shear off the sugar leaves but not cut or slice through the inflorescence. The rotating cylinder is then inclined downwards to allow continuous flow with options for manual feeding and output collection or extra accessories for conveying the product into and from the trimming operation [27]. The perforated cylinders are then surrounded by a closed shell attached to a vacuum to collect the discarded sugar leaves, where ambient air drying may occur [27]. Improvements in the equipment design and final output quality in trimming operations are critical for better industry standards. These improvements should focus on the effectiveness of the process of sugar leaf removal with minimal damage and abrasion to the trichomes on the surface of the inflorescence.

#### *3.2. Drying of Cannabis*

The main postharvest operation of cannabis processing is drying. As heat is the main agent for the decarboxylation of cannabinoids, the appropriate drying temperature and conditions are very important to obtain good quality product with a maintained amount of THC, CBD, or other cannabinoids. The inflorescence of raw cannabis contains around 78 to 80% moisture, which needs to be reduced for safe storage and production of readyto-use dry products [28,29]. Researchers are trying to identify suitable drying technology, which can replace the existing hang-drying practice. In this section, insights on drying and the current industrial practices with their pros and cons are discussed. Additionally, equilibrium moisture content, sorption isotherm, sorption isotherm models, and possible pre-treatments for the improvement of drying are also discussed.

#### 3.2.1. Mechanism of Drying

Drying is a popular unit operation usually characterized by the presence of a solidliquid separation that is usually facilitated by the presence of heat, resulting in the evaporation of a liquid, which is water in the majority of cases. The definition of drying could also extend to solvent evaporation, dehydration of various biological materials such as in food and feed, the dehydration of salts, and the removal of hydroxyl groups from organic material [30–32].

Industrial drying involves the use of large amounts of energy usually in the form of heat, which can use different mechanisms, such as convection, conduction, radiation, or a combination of these. Certain amounts of sensible and latent heat need to be provided around the entirety of the material to achieve evaporation. Convection is characterized by a carrier gas or feed gas that is usually air, supplying the heat for evaporation of water or solvent. Conduction is the heat transfer mechanism during contact drying. The carrier gas in this context serves as the medium to which the solvent is evaporated. The radiation mechanism has different subtypes; penetrative radiation drying would consist of dielectric drying, such as radiofrequency or microwave drying, where heat is generated in the material rather than the heat diffusing into the material, which is non-penetrative [31,33].

Drying rate is the rate of moisture removal during a given drying process, obtained by the ratio of moisture removed to the time of drying. Total drying time is split into two major periods: (i) a constant rate period and (ii) one or more falling rate period(s). The constant rate period occurs when water is removed from the surface by evaporation and internal water movement is sufficient to keep the surface saturated. The surface temperature remains constant and is lesser than the circumambient drying air because the energy input rate is the same as the heat energy lost due to evaporation. In the falling rate period, the surface of a material is unsaturated, and the movement of interior moisture is lower than the rate of surface evaporation. During the first falling rate period, the drying rate is observed to fall as water content reduces due to increased resistance to evaporation, as well as a reduction in heat flowing into the material when the surface temperature rises to the heating medium temperature. However, the temperature of the material does not significantly increase or differ from the wet-bulb temperature. The second falling rate period commences when the partial pressure of moisture all throughout the material is lower than saturation. Heat flux from the hot air to the sample is very low as there is a small temperature gradient [32,33].

#### 3.2.2. Current Industrial Drying Practices for Cannabis

Cannabis drying operations through the years have not evolved too far from the slow-drying method or low-temperature drying (Figure 4) that usually takes 5–6 d at near ambient conditions. The commonly used drying temperature is 18–21 ◦C and relative humidity is 50–55%. Currently, no model has yet been established for the prediction of the drying endpoint and total drying time. Postharvest processes are currently considered more of an art than a science as there are no standards for which the endpoint of drying is defined, and practices are based on word of mouth and are usually still subject to deviation. In the slow drying technique, cannabis stalks are hung on wire liners with the top buds or the cola upside down in isothermal room conditions until desired water activity is achieved. Screen drying is a variation of the slow drying process wherein the trimmed buds are dried in an enclosed and properly ventilated room by placing them on trays or screens. In this screen drying method, drying conditions are almost the same as in slow drying processes, but this process takes around 4 to 5 d to dry the cannabis completely [10].

Present industrial drying practices have several drawbacks such as a long processing time, chances of contamination by molds or fungi, and inconsistent quality in the final products. Researchers are trying to introduce modern and novel technologies in this sector. According to Chen et al. [34], drying conditions and methods dominate the drying time and quality characteristics of cannabis. Air drying, microwave-assisted convective drying, freeze drying, vacuum drying, microwave assisted freeze drying, intermittent (non-isothermal) drying, conveyor drying, radio frequency, and electrohydrodynamic drying, among others are the suggested modern drying techniques for cannabis [4,10,35]. However, critical identification of psychrometric behavior for devolvement of new drying technologies is equally important.

Psychrometric terms predominantly govern postharvest operations in the cannabis industry. Psychrometrics is the study of atmospheric air and its thermodynamic properties, typically focusing on psychrometric terms or parameters [36–38]. The interaction and control of these parameters are vital for success in an optimum quality-controlled process, storage of products, and packaging. In the cannabis industry, four humidity terms

are used to describe the drying convective air: (a) vapor pressure, (b) relative humidity, (c) humidity ratio, and (d) water activity. These terms are usually common in psychrometric calculations together with dry and wet bulb temperature, enthalpy, dew point temperature, and specific volume [37]. However, relationships among the psychrometric parameters should be investigated as it would be helpful for cannabis drying and storage in different environmental conditions.

**Figure 4.** Hang-drying practice commonly used in large scale cannabis operations. Image taken by A. Vista (co-author).

#### 3.2.3. Equilibrium Moisture and Sorption Isotherms

Equilibrium moisture content (EMC) is the amount of moisture in a biological material when equilibrium occurs. The corresponding relative humidity is the equilibrium relative humidity (ERH). The plot of product moisture versus air relative humidity at a constant temperature is called the equilibrium moisture content curve [32].

The EMC sorption curve appears in a sigmoidal shape due to how moisture is bound in a material. At low relative humidity values, shown in Figure 5 (region A), moisture is assumed to be bound tightly to the biological material's surface. In this region, water exhibits a monolayer behavior where the amount of tightly held water is approximately like when a single water molecule is being held on each hydrophilic site across the surface of a biological material. The Langmuir isotherm, which is obtained by the monomolecular adsorption of vapor by porous particles in a finite volume of voids, can be used to approximate these areas [39,40].

The intermediate region (region B in Figure 5) is where water is more accurately described as "multimolecular" because water molecules are bound to different sites at different distances from the center of the biological material, and thus is easily pictured as multilayers of moisture across the biological material. The point of transition between the monolayer and multilayer occurs at relative humidity values of around 5 to 20%. The third region (region C in Figure 5) is the free water region, usually associated with water activity values greater than 0.9, where properties exhibited by dilute solutions are observed and water is loosely attached to the binding sites and is readily available for reaction to other substances.

**Figure 5.** Sorption isotherms in biological materials (reproduced from [32,39,41]).

Hysteresis is a phenomenon related to the means on which water is distributed across the biological material, and it explains the nature of biological materials to undergo structural rearrangements that affect the availability of hydrophilic sites to bind to water at re-adsorption as a result of the presence of an offset, as seen in Figure 5 [32,39].

Moisture activities in biological materials for drying processes are usually predicted from standard water activity thresholds to prevent the proliferation of unwanted biological contaminants. The United States Food and Drug Administration (FDA) defined water activity as the ratio between the vapor pressure of the substance itself, when in a completely undisturbed balance or equilibrium with the circumambient air media, and the vapor pressure of distilled water under similar conditions. This means that at different temperatures, the water activity of a substance may vary [42]. The FDA also defined water activity as equivalent to the equilibrium relative humidity:

$$\mathbf{a}\_{\rm W} = \frac{\text{ERH}}{100} \tag{1}$$

where ERH is the equilibrium relative humidity (%).

Sorption isotherms are equilibrium moisture content and equilibrium relative humidity values at a constant temperature and pressure. The knowledge of the stability of water in a biological material such as cannabis is important in designing and optimizing drying operations, designing packaging materials, predicting quality and stability, and estimating the loss of moisture during storage [39,43].

#### Sorption Isotherm Models

To predict EMC based on the measurements of ERH, isotherm equations are generated based on standardized equations. ASABE Standards ASAE D245.7 [44] summarized five isotherm equations.

• Modified Henderson equation:

$$\text{ERH} = 1 - \exp\left[-\text{A} \times (\text{T} + \text{C}) \times (\text{MC}\_{\text{D}})^{\text{B}}\right] \tag{2}$$

• Modified Chung–Pfost equation:

$$\text{ERH} = 1 - \exp\left[-\frac{\text{A}}{\text{T} + \text{C}} \exp(-\text{B} \times \text{MC}\_{\text{D}})\right] \tag{3}$$

• Modified Halsey equation:

$$\text{ERH} = \exp\left[-\frac{\exp(\mathbf{A} + \mathbf{B} \times \mathbf{T})}{\left(\mathbf{M}\mathbf{C}\_{\text{D}}\right)^{\text{C}}}\right] \tag{4}$$

• Modified Oswin equation:

$$\text{ERH} = \left[ \left( \frac{\text{A} + \text{B} \times \text{T}}{\text{MC}\_{\text{D}}} \right)^{\text{C}} + 1 \right]^{-1} \tag{5}$$

• Guggenheim–Anderson–de Boer (GAB) equation:

$$\text{ERH} = \frac{\text{A} \times \text{B} \times \text{C} \times \text{ERH}}{(1 - \text{B} \times \text{ERH}) \times (1 - \text{B} \times \text{ERH} + \text{B} \times \text{C} \times \text{ERH})} \tag{6}$$

where ERH = equilibrium relative humidity (decimal value); T = temperature (◦C); MCD = dry-basis moisture content (decimal value); and A, B, and C = equation constants, and the related data specifications based on which these constants were obtained are summarized in Table 2 of ASABE Standards ASAE D245.7 [44].

#### 3.2.4. Possible Pre-Treatments for Improvement of Drying of Cannabis

Pre-treatments are the physical or chemical treatments used prior to any process to enhance the quality of the product as well as to improve the process. Drying pretreatments can reduce the drying time as well as the energy consumption during the drying process [45]. A number of physical and chemical pre-treatments are frequently used for the drying of foods, medical plants, herbs, spices, etc. Physical pre-treatments include steaming, blanching, microwave heating, ohmic heating, ultrasonic treatment, pulsed electric field, high pressure processing, freezing, etc., while chemical pre-treatments are conducted by treating the samples with different chemicals, such as sulfur dioxide, carbon dioxide, ozone, alkali solution, acid solution, etc. [45]. Do these pre-treatments improve the cannabinoid profile of cannabis? Up to now, there is no clear answer and very little research is conducted so far on the analysis of the potential of pre-treatments for maintaining the level of cannabinoids and terpenes. However, these pre-treatments can reduce the drying time. As decarboxylation requires heat treatment for the conversion of cannabinoids from their acidic form into their neutral form, thermal or physical pre-treatments could accelerate the process. This section discusses some possible pre-treatments that can be applied in the cannabis industry.

#### Microwave Heating

Electromagnetic waves with wavelengths ranging from 0.001 to 1 m and frequencies ranging from 300 to 300,000 MHz are called microwaves [46]. The frequency of electromagnetic energy equaling 915 MHz is used in the industry due to better penetration depth. During MW heating, heat is generated throughout the volume simultaneously due to the absorption of electromagnetic waves of certain frequencies in the form of energy [47]. Microwave heating helps in the improvement of operational speed, product quality, and energy use efficiency, and it lowers the operational costs of a process [48]. In cannabis