Sustainable Ultrasound-Assisted Extraction and Encapsulation of Phenolic Compounds from Sacha Inchi Shell for Future Application

Abstract

Sacha inchi shell (SIS), an underutilized by-product of sacha inchi oil processing, is a rich source of phenolic compounds. In this study, ultrasound-assisted extraction (UAE) was optimized by response surface methodology (RSM) with a Box–Behnken design to investigate the effects of time (15–25 min), temperature (25–45 °C), and ethanol concentration (40–80%) on the total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity by DPPH assay of the obtained extracts. The maximum TPC was at 15 min, 45 °C and 60%, TFC at 25 min, 45 °C and 60% and DPPH at 15 min, 35 °C and 80%. The optimized condition selected for encapsulation purposes was at 25 min, 35 °C and 80% ethanol concentration. RSM analysis showed that all models analyzed for all three assays were significant at 95% confidence. The SIS extract had a greater inhibitory zone against Escherichia coli measuring 15.34 mm at a concentration of 30 µg/mL than Staphylococcus aureus among the samples. The spray-dried microcapsules using different combinations of gum arabic and maltodextrin (GMM 1 and GMM2) resulted in a proper encapsulation layer and a smoother surface and shape obtained at 1000× magnification. Also, GMM 1 and GMM2 had particle sizes ranging from 2.95 ± 0.02 to 27.73 ± 0.38 and from 5.20 ± 0.01 to 29.30 ± 0.42 µm, respectively. The microcapsules were in the acceptable ranges for moisture content (<5%) and water activity (<0.6). It has been concluded that SIS extract showed high antioxidant and antimicrobial properties and its encapsulation could be further used in food and nutraceutical formulations.

1. Introduction

Agricultural waste can be re-utilized to promote the concept of a circular economy (grow-make-use-restore) and to support the SDGs set by the United Nations. Non-thermal processing of fruit juices or plant extracts promote sustainable food production by minimizing the waste valorization and maximizing the use of resources. These technologies also support the implementation of a circular economy and the accomplishment of the Sustainable Development Goals (SDGs) established by the United Nations [1].

Sacha inchi (Plukenetia volubilis L.) is a Euphorbiaceous plant that is grown in the Amazonian forest at altitudes ranging from 200 to 1500 m and is commonly cultivated in Southern Columbia, Peru, and Southeast Asian countries such as Thailand [2]. Sacha inchi shell (SIS) is the outer covering of sacha inchi seeds and consists of 30–35% of the seed weight. SIS is produced as a by-product; when sacha inchi oil is extracted, the remaining seeds are discarded without any further use [3]. However, this by-product could be an important source of bioactive compounds that can be extracted and incorporated into human nutrition for various health benefits. As per the recent studies of Kittibunchakul [4], SIS is high in protein (43.12/100 g), energy (552.89 kcal/100 g) fat (37.87/100 g) and ash (4.46/100 g) with a total phenolic content (TPC) of about 503.96 mg GAE/100 g.

The extraction of bioactive compounds (e.g., phenolic compounds) from plant materials has piqued attention in recent years because these biomolecules have been shown to lower the risk of a variety of chronic diseases [5]. There are number of adopted techniques for the extraction of bioactive components from plant sources such as Soxhlet extraction, maceration and hydrodistillation, microwave-assisted extraction, and ultrasound-assisted extraction (UAE) [6]. Among these extraction techniques, UAE is an alternative to the conventional extraction technique which works on the principle of acoustic cavitation and has been successfully reported to extract phenolic compounds from different nutshell powder [7]. Ultrasound-based extraction of bioactive compounds is preferred over the traditional method because it is a widely used method, yielding more extracts within less extraction time than the conventional method [8]. Additionally, this method offers multiple ways of contributing to the SDGs, including lowering the energy and solvent requirements for extraction processes, raising productivity, and producing higher-quality extracts as previously mentioned, lessening the impact on the environment, and encouraging innovation in sustainable extraction methods. A number of SDGs, including those pertaining to sustainable development, innovation, responsible consumption, and resource efficiency, could be addressed by incorporating ultrasound-assisted extraction into different industries [9]. The previous study showed that UAE is an appropriate extraction technique in fruits and vegetables preventing damage to volatile compounds [9]. Beside extraction, an optimization of extraction is also important to observe the effect of experimental factors and their interaction on the phenolic content of an extract [10]. According to Hayat [11], extract recovered using optimization was significantly different in total phenolic content, total flavonoid content, and radical scavenging activity than extract recovered by using a conventional extraction procedure.

Response surface methodology (RSM) has already been reported for the optimization of extraction of phenolic compounds from SIS by microwave-assisted extraction (MAE) [3]. The influence of the extraction time (X₁), temperature (X₂) and ethanol concentration (X₃) on the total phenolic content (TPC) and antioxidant activity of extracts from SIS obtained by UAE by means of the response surface methodology (RSM) has not yet been explored, although RSM has been used as an optimization tool for many years.

Encapsulation plays a great role in providing protection for bioactive compounds by entrapment in a food-grade carrier such as gum arabic (GA) and maltodextrin (MD) and can prevent degradation when exposed to adverse environmental conditions and control its release at targeted systems [12]. This process also contributes to the SDGs by reducing waste, enhancing product efficacy, fostering innovation, and promoting sustainable practices across various industries [13]. There are number of processes involved in the encapsulation of bioactive compounds in suitable carriers and these include spray drying, extrusion and emulsification, fluidized-bed coating, spray cooling/chilling, liposomes, complex coacervation, and oil-in-water emulsion [14]. However, spray drying is the most preferred encapsulation technique to trap polyphenols in a suitable matrix like GA and MD because this technique is cost effective, simple and can produce good quality particles in large quantities [15]. Meena, Prasad, Khamrui, Mandal, & Bhat [16] reported that GA can produce a stable emulsion at a very low pH and is compatible with most of the wall material such as carbohydrates, starch, gums and different proteins; whereas, MD has a good taste, low cost, high solubility in water, and low solution viscosity [16]. GA and MD have been certified as generally recognized as safe (GRAS) and permitted by regulatory agencies such as the FDA and EFSA [17]. Estupiñan-Amaya, Fuenmayor, & López Córdoba [18] highlighted that the MD:GA ratio of 75:25 and vice-versa used as an encapsulating agent of Andean blueberry (Vaccinium meridionale) juice had high total phenolic content and antioxidant activity.

We as well as others have conducted some experiments on the extraction and encapsulation of bioactive compounds from agricultural waste in suitable carrier materials. Among these studies conducted by our team, one was on ultrasound-assisted extraction and encapsulation of a functional compound from the mulberry leaf as a functional food [19]. Another study was published recently by our colleagues Jafari et al. [17] on the optimization of ultrasound-assisted extraction by RSM to enhance the TPC and encapsulation of bioactive compounds from cocoa shell in GA and MD by spray drying to preserve and protect the extract for a long time and which could be further used in the food matrix. Though there are tons of SIS produced by sacha inchi oil production companies each year, there is no updated information available on its utilization, recovery of important components (polyphenols), or the encapsulation of extract from sacha inchi shell (SIS) until today.

Therefore, we highlighted the optimizing of the UAE of bioactive compounds from SIS by RSM and encapsulated the optimized extract in GA and MD by spray drying for future application as functional food ingredients in the food industry.

2. Materials and Methods

2.1. Sample Preparation

The sample of SIS was procured from the Sacha Inchi Oil Production Company, Saraburi province, Thailand to the Department of Food Technology in Chulalongkorn University. To reach a moisture content of less than 5%, the SIS was dried in hot air at 60 °C (DS_Memmert Universal oven UF110, Schwabach, Germany) for a whole night before being ground to a 45-mesh size (W.S. Tyler, Mentor, OH, USA). Following that, the samples were vacuum-packed in a laminated aluminum bag and kept at −20 °C, for further analysis.

2.2. Maximizing UAE Extraction Using RSM

Response surface methodology (RSM) with a Box–Behnken design (BBD) was used at three levels (−1, 0, 1). There were three variables: time (X₁: 15–35 min), temperature (X₂: 25–45 °C), ethanol concentration (X₃: 40–80%) and three responses: total phenolic compound (TPC), total flavonoid content (TFC), and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) antioxidant activity [19]. BBD was used to optimize the extraction parameters for UAE using an ultrasound bath-type extractor of a frequency of 80 kHz and power consumption of 970 W, with tank internal dimensions of W/D/H (mm) 505/137/100 and a control system including temperature and time. RSM produced 17 experimental runs using Design Expert 11 to manage the process (

Table 1. The values for Box–Behnken design (BBD).

Independent Variables	Code Symbols	Levels
		−1	0	1
Time (minutes)	X1	15	20	25
Temperature (°C)	X2	25	35	45
Ethanol Concentration (%)	X3	40	60	80

and

Table 2. The functional properties of sacha inchi shell (SIS) extract under different extraction conditions.

	Independent Variable			Responses
Run	X1: Time min	X2: Temperature °C	X3: EthanolConc %	TPC (mg GAE/mL)	TFC (mg QE/mL)	DPPH (mM Trolox/mL)
1	25.00	35.00	80.00	4.99	9.55	39,001.1
2	20.00	35.00	60.00	3.51	5.75	54,979.9
3	25.00	45.00	60.00	3.76	9.56	40,435.5
4	15.00	35.00	80.00	3.34	0.69	74,402
5	20.00	35.00	60.00	4.14	3.11	65,221.3
6	15.00	45.00	60.00	5.25	1.15	66,487.8
7	20.00	25.00	80.00	5.11	8.36	45,611.5
8	15.00	25.00	60.00	3.51	1.54	71,327.6
9	20.00	35.00	60.00	2.91	8.57	44,783.4
10	20.00	45.00	40.00	3.97	7.28	49,680.4
11	25.00	35.00	40.00	2.67	1.4	71,821.8
12	25.00	25.00	60.00	3.97	3.05	65,750.4
13	20.00	35.00	60.00	4.71	7.08	49,963.4
14	20.00	35.00	60.00	3.89	1.3	72,411.2
15	15.00	35.00	40.00	5.21	3.25	64,736.1
16	20.00	25.00	40.00	3.91	2.76	67,082.1
17	20.00	45.00	80.00	5.09	2.01	69,591.1

).

For UAE, 4 g of sample was dissolved in 100 mL ethanol (EMSURE^®, Darmstadt, Germany) and extracted in an ultrasonic bath-type extractor (Elmasonic bath P70, Singen, Germany) at various experimental conditions. Then extract was filtered through Whatman filter paper No.1 to obtain the clarified extract and concentrated using a rotary evaporator (Oilbath B-485, BÜCHI, Uster, Switzerland) at 45 °C. Following vacuum evaporation, the residual extract was reconstituted using distilled water and adjusted to a final volume of 10 mL. Further extract was stored at 4 °C in a 50 mL centrifuge vial for further analysis.

2.3. Microencapsulation Experiment

The microencapsulation experiment was conducted following the methodology previously outlined by Insang et al. [19]. To summarize, the optimized SIS extract was combined with a mixture of maltodextrin DE10-12 (Zhucheng Dongxiao Biotechnology, Weifang, China) and gum arabic (Agrigum, UK); GMM 1: GA 75% + MD 25% and GMM2: GA 25% + MD 75% at a ratio of 1:3 (optimized SIS extract:coating material). Then, the mixture was homogenized for 10 min in a high-speed blender (Ystral, model X10, Ballrechten-Dottingen, Germany). Following that, the solutions were placed into a spray dryer (Mobile Minor Niro-Atomizer, Søborg, Denmark) that had an intake temperature of 155 °C and an output temperature of 90 °C with a hot air flow rate of 1.54 m³/min. After that, the microcapsule powders were kept at −20 °C for later examination.

2.4. Functional Characteristics in SIS Extracts and Microcapsules

2.4.1. Total Phenolic Compound (TPC) and Total Flavonoid Content (TFC)

For TPC analysis, 0.5 mL of sample was pipetted into a test tube and 10 mL of distilled water was added. After this, 0.5 mL of 10% Folin–Ciocalteau’s reagent was added and mixed well using a vortex mixer. This solution was incubated for 5 min in the dark. A further 2 mL of 20% (w/v) sodium carbonate solution was added and mixed. Then, this solution was incubated at room temperature for 10 min. Finally, absorbance was measured at 765 nm using a spectrophotometer (Thermo Scientific GENESYS 20, model: 4001/4, Waltham, MA, USA) and TPC was expressed in extracts as mg gallic acid equivalent (GAE)/mL.

The aluminum tri-chloride method with slight modification was considered for TFC determination. 100 µL of extract was added with 400 µL of methanol into the test tube and spun on the vortex mixer to mix well. This analysis was followed by adding 100 µL of 10% aluminum chloride and 100 µL of 1 M sodium acetate solution and mixing with a vortex mixer. This solution was incubated in a dark condition for 40 min at room temperature. The absorbance was recorded at 430 nm and TFC was expressed in extracts as mg quercetin equivalents (QE)/mL. TPC and TFC were analyzed as per the procedure developed by Bhave & Dasgupta [20] with some modifications.

2.4.2. Antioxidant Activity by DPPH Assay

For determination of antioxidant activity by DPPH assay, 1.2 × 100 M fresh solution of DPPH was prepared, and absorbance (A_initial) should be 1.1. Then, 250 µL of DPPH solution was mixed with 450 µL of SIS extract and stored in a dark place for 15 min. After this, absorbance (A_final) was taken at 515 nm using a spectrophotometer. DPPH was determined following the protocol of Insang, Kijpatanasilp, Jafari, & Assatarakul [19]. DPPH was expressed as mM Trolox/mL.

2.5. Physicochemical Properties of Microcapsules

The moisture content (%) of spray-dried microcapsules was analyzed using a halogen moisture analyzer (Model HB43-S Halogen, Mettler Toledo, Port Melbourne, Australia). The encapsulation efficiency (%) was as described by Saénz et al. [21]. A water activity analyzer (model MS1, Novasina, Lachen, Switzerland) was used to measure the water activity (aw). The water solubility was measured as described by Jafari et al. [21] by calculating the weight of dried supernatant as a percentage of the initial powder. The yield was calculated using the percentage of total microcapsules obtained on the initial extract mixed with the coating agent taken for encapsulation.

For color determination, the CIE LAB system (L*, a*, and b*) by chroma meter Minolta CR-400 color meter, which uses illuminant D65 to display the color, was used at room temperature. A scanning electron microscope (SEM) and energy dispersive X-ray spectrometer—SEM-EDS (Model JEOL JSM-IT300) with a magnification of 1000× was used to analyze the morphology of microcapsules. The particle size of the microcapsule was determined using a laser diffraction particle size analyser (Mastersizer 3000, Malvern Instruments Ltd., Malvern, UK).

2.6. Antimicrobial Activity Experiment

2.6.1. Growth Condition for Initial Culture

Standard strains of Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were obtained from the Department of Food Technology, Chulalongkorn University. The inoculum of each microorganism was prepared by inoculating with 10 mL sterile growth medium (Muller Hinton Broth (MHB), Muller Hinton Agar (MHA)). The cultures were grown on MHB and incubated at 37 °C overnight.

2.6.2. Antimicrobial Screening of SIS Extract

For antimicrobial activity of SIS extract, the pour plate method was adopted for which a bacterial sample (20 µL of 24 h fresh culture of S. aureus and E. coli) was added to the Petri plate and the molten nutrient agar medium was poured over and then plates were swirled quickly to properly mix the sample with the medium, which was then allowed to solidify. The sterile discs were placed into the Petri dish and fitted with forceps. Finally, about 20 µL of SIS extract was added onto each disc. The plates were left for about 25–30 min for SIS extract diffusion into the inoculated agar plates. Then, these plates were incubated at 37 °C for 24 h in an upside-down position [22]. After incubation, the plates were observed for the number of colony forming units (CFU) and the zone of inhibition was measured for the determination of antimicrobial activity. Ampicillin antibiotic (50 mg/mL) was used as the positive control and ethanol at 80% was used as the negative control.

2.6.3. Inhibition Assay-Minimum Inhibitory Concentration (MIC)

After the antimicrobial screening, the extracts with sensitivity to bacterial strains of S. aureus and E. coli were further selected for inhibition assay as previously described by Mostafa et al. [22]. A broth dilution method was adopted in which all the test tubes containing equal amounts of nutrient broth were autoclaved and cooled. Further SIS extract was added into each test tube as per increasing concentration. Then, each dilution was inoculated with fresh 24 h bacterial suspension (1.5 × 10⁸ CFU/mL) and incubated at 37 °C for 24 h. A microplate reader (Biochrom Asys Expert 96 microplate reader, Biochrom, UK) was used to detect the optical density at 600 nm. When there was no obvious development of the test microorganisms, the minimum inhibitory concentration (MIC) of the sample was determined.

2.7. Statistical Analysis

The optimization of extraction was conducted using the Design Expert 11 program (Stat-Ease, Inc., Minneapolis, MN, USA). All physicochemical properties of the microcapsules were performed in triplicates, and the data were analyzed using SPSS version 22.0 statistical software and one-way analysis of variance (one-way ANOVA). Tukey’s honestly significant difference (HSD) test was used to distinguish the significant differences (p ≤ 0.05) among the microcapsules.

3. Results and Discussion

3.1. Optimization of UAE Using RSM

The effect of different extraction conditions (time, temperature, and ethanol concentration) designed by RSM on the antioxidant activity assays by DPPH, as well as the TPC and TFC is shown in Table 2. The TPC ranged from 2.67 to 5.25 mg GAE/mL and TFC was between 0.69 and 9.56 mg QE/mL. There is much variation in TFC at different extraction conditions compared to TPC and this is supported by the research of Mokrani & Madani [23] which showed that TPC and TFC were affected by all the extraction parameters. The antioxidant activity (DPPH) was from 39,001.1 to 74,402 mM trolox/mL. All models had p-values of below 0.05, indicating that the models were significant at about 95% confidence (

Table 3. Analysis of variance (ANOVA) for determination of optimization model fit.

	TPC (mg GAE/mL)					TFC (mg QE/mL)					DPPH (mM Trolox/ mL)
Source	Sum of Squares	df	Mean Square	F- Value	p-Value Prob > F	Sum of Squares	df	Mean Square	F- Value	p-Value Prob > F	Sum of Squares	df	Mean Square	F- Value	p-Value Prob > F
Model	7.07	6	1.18	3.26	0.0481	6.56	6	1.09	3.24	0.0492	1.58 × 109	6	2.63 × 108	3.25	0.0485
X1	0.46	1	0.46	1.28	0.2851	2.17	1	2.17	6.42	0.0297	4.49 × 108	1	4.49 × 108	5.55	0.0402
X2	0.31	1	0.31	0.85	0.3775	0.068	1	0.068	0.2	0.6621	6.95 × 107	1	6.95 × 107	0.86	0.376
X3	0.96	1	0.96	2.66	0.1343	0.098	1	0.098	0.29	0.6022	7.64 × 107	1	7.64 × 107	0.94	0.3543
X1 X2	0.95	1	0.95	2.63	0.1358	0.57	1	0.57	1.7	0.2219	1.05 × 108	1	1.05 × 108	1.29	0.2817
X1 X3	4.39	1	4.39	1.22 × 10	0.0059	2.07	1	2.07	6.14	0.0327	4.51 × 108	1	4.51 × 108	5.58	0.0399
X2 X3	1.60 × 10−3	1	1.60 × 10−3	4.43 × 10−3	0.9482	1.58	1	1.58	4.67	0.0561	4.28 × 108	1	4.28 × 108	5.29	0.0443
Residual	3.61	10	0.36			3.38	10	0.34			8.09 × 108	10	8.09 × 107
Lack of Fit	1.79	6	0.3	0.65	0.6944	1.28	6	0.21	0.41	0.8425	3.03 × 108	6	5.04 × 107	0.4	0.8496
Pure Error	1.82	4	0.46			2.09	4	0.52			5.07 × 108	4	1.27 × 108
Cor Total	10.68	16				9.93	16				2.39 × 109	16
C.V.%					14.61					29.39					15.09
R2					0.6618					0.66					0.6611
Adj-R2					0.4589					0.456					0.4578

). The best fitted model was the 2FI (two-factor integration) model, and the lack of fit was non-significant for this model. Additionally, the R² value was 0.66; this indicates that the model was nicely suited to the response.

The three-dimensional response surface plots utilized in this study are shown in Figure 1, Figure 2 and Figure 3. It can be noticed that as the temperature increases, TPC production increased, which was parallel with a previous study [15]. As per the surface plot, the percentage of water in ethanol (from 60% to 80% v/v) was noticed to be a major determinant in phenolic component extraction, with extracts being recovered less when the solvent level was larger than 60% (v/v). The TPC results matched with the previous study of Insang et al. [19] which found that bioactive compounds (such as TPC) extracted from mulberry leaf using ultrasonic extraction were at their greatest concentration at 60% v/v ethanol [19]. Tungmunnithum et al. [24] also confirmed that the optimal condition for ultrasound-assisted extraction of total phenolic compound from almond oil residues was at 53.0% (v/v) ethanol, as green solvent solutions with water. These two studies suggest that UAE extraction with 50–60% ethanol could be capable of penetrating cells to dissolve phenolic compounds, as compared to a higher ethanol concentration, which can cause protein denaturation and decrease the rate of dissolution of phenolic compounds.

As the highest extraction was at the minimum time (i.e., 15 min) and highest temperature (45 °C), this concludes that a lower extraction time is required when using higher temperatures for extraction from SIS. According to Dzah et al. [25], an increase in temperature with fixed time or ethanol concentration caused the total TPC of the extracts to reach a maximum. Whereas, applying the higher temperature and a longer extraction time resulted in decreased TPC content present in the extract.

The regression coefficient of the predicted second order polynomial models (BBD) for bioactive properties is shown in

Table 4. Regression coefficient of the predicted second order polynomial models (BBD) for bioactive properties.

Factor	TPC	TFC	DPPH
Intercept	4.11	1.98	59,605.11
Linear
X1	−0.24	0.52	−7493.09
X2	0.2	0.093	−2947.09
X3	0.35	0.11	−3089.34
Cross product
X1 X2	−0.49	0.38	−5118.78
X1 X3	1.05	0.72	−10,621.7
X2 X3	−0.02	−0.63	10,345.32

Level of significance p ≤ 0.05. Time (X₁), temperature (X₂), ethanol concentration (X₃).

.

TPC could be expressed using the following binomial equation:

TPC = +4.11 − 0.24 × X₁ + 0.2 × X₂ + 0.35 × X₃ − 0.49 × X₁ × X₂ + 1.05 × X₁ × X₃ − 0.020 × X₂ × X₃

(1)

The TFC was revealed to be statistically significant (p ≤ 0.05) (Table 3) at the maximum chosen time and temperature, whereas at a medium level of ethanol concentration, similar findings were reported by Mokrani and Madani [23] that an ethanol concentration of 60% was significantly the best solvent for extracting TFC. It has been noticed that increasing temperature and time had a positive impact on TFC output and this finding was comparable with a previous study by Hajiaghaalipour et al. [25] which showed that TFC increased with an increase in both extraction time and temperature. The extraction yield of TFC could be expressed using the following polynomial equation:

TFC = +1.98 + 0.52 × X₁ + 0.093 × X₂ + 0.11 × X₃ + 0.38 × X₁× X₂ + 0.72 × X₁ × X₃ − 0.63 × X₂ × X₃

(2)

The antioxidant activity analyzed by DPPH assay was affected by time in 2FI models (p ≤ 0.05). The highest and lowest DPPH was reported at 15 min, 35 °C and 80% ethanol concentration and at 25 min, 35 °C and 80% ethanol concentration, respectively (Table 2). As per the findings of Safdar et al. [26], which were parallel with our analysis, the high antioxidant activity exhibited in three antioxidant assays with 80% ethanolic extracts was obtained by ultrasound-assisted extraction of bioactive components from mango peel at a concentration of 80% (v/v) and ultrasound-assisted extraction was found to be an efficient technique with higher polyphenols recovery content than maceration.

The binomial equation for antioxidant activity by DPPH assay was described as follows:

DPPH = +59605.11 − 7493.09 × X₁ − 2947.09 × X₂ − 3089.34 × X₃ − 5118.78 × X₁ × X₂ − 10621.65 × X₁× X₃ + 10345.32 × X₂ × X₃

(3)

3.2. Antimicrobial Activity of Optimized Extract

Many plant extracts have demonstrated antimicrobial activity. Plants produce a variety of secondary metabolites like alkaloids, flavonoids, phenolics, terpenoids, and others, which often possess antimicrobial properties. These compounds help plants defend against pathogens and pests [27]. On the other hand, the public health concern regarding foodborne illness caused by the ingestion of food contaminated by pathogenic bacteria has been critically significant. In addition, S. aureus and E. coli stand out as the primary pathogens responsible for causing foodborne outbreaks globally [27]. SIS extract had a larger inhibitory zones against E. coli measuring 15.34 mm at concentration of 30 mg/mL than S. aureus among the other samples (

Table 5. Antimicrobial activity of the optimized SIS extract.

Parameter	Escherichia coli	Staphylococcus aureus
Zone of inhibition (mm)
SIS extract	15.3 ± 0.27	3.35 ± 0.30
Ampicillin (50 µg/mL)	58.50 ± 0.13	4.43 ± 0.26
Control (sterile water)	8.80 ± 0.18	8.80 ± 0.18
MIC (mg/mL)
SIS extract	0.77 ± 0.02	1.45 ± 0.07

Three replications were used for SIS extract per microorganism.

). Furthermore, no significant difference (p > 0.05) was observed between SIS extract and Ampicillin (positive control). Similarly, Fajrih et al. [28] demonstrated that the extract of banana corm had a strong inhibitory zone against S. aureus, E. coli and other pathogens extracted using ethanol as a solvent. It has been shown that antimicrobial activity of plant extracts can be influenced by various factors, one of which is the extraction method (e.g., UAE, maceration, distillation etc.). Based on the findings of previous studies [29,30], it can be deduced that UAE (ultrasonic-assisted extraction) stands out as a dependable technique for achieving increased antimicrobial activity.

After conducting an initial assessment of the antimicrobial activity of the SIS extract, the agar disk diffusion method was employed to evaluate its impact on two microorganisms under consideration, namely, E. coli and S. aureus. The examination involved the identification of inhibition zones, and subsequent investigation through cultivation in nutrient broth (NB) was carried out to ascertain the minimum inhibitory concentration (MIC). The results revealed that E. coli displayed a high level of susceptibility to the SIS extract, as indicated in Table 5. Gram-negative bacteria have an outer membrane made of lipopolysaccharides, which allows molecules to transfer across the cell membrane and serves as a barrier to foreign materials [28]. The fact that SIS extract had the lowest MIC value (0.77) suggests that it worked well against E. coli. In a similar study, Jarriyawattanachaikul et al. [31] demonstrated that Thai herbs had potent antimicrobial activity against E. coli and S. aureus. In another study and consistent with ours, the antibacterial efficacy of Croton macrostachyus extract against E. coli and S. aureus was demonstrated through both in vitro and in vivo studies, highlighting its potent antibacterial properties [32]. The potential utilization of plant extracts as a treatment for illnesses caused by S. aureus and E. coli pathogens was suggested.

3.3. Characterization of Microcapsules

Microencapsulation is a valuable technique used in the food and beverage industries as it protects delicate components like vitamins, minerals, flavors, and active compounds against degradation induced by factors such as exposure to light, oxygen, moisture, and other environmental elements [13]. The results indicated that the moisture content was 3.79% and 4.36% for GMM 1 and GMM 2, respectively (

Table 6. Effects of encapsulation by spray drying on quality properties of microcapsules.

Parameters	GMM 1	GMM 2
Encapsulation efficiency (%)	51.50 ± 2.18	55.9 ± 2.28
Moisture content (%)	3.79 ± 0.29	4.36 ± 0.28
Water activity	0.23 ± 0.01	0.24 ± 0.01
Solubility (%)	90.4 ± 4.02	98.4 ± 4.02
Color values
L*	85.66 ± 1.19	83.28 ± 1.17
a*	−1.31 ± 0.03	−1.26 ± 0.03
b*	14.11 ± 0.27	14.64 ± 0.26

Data are presented as mean ± SD. Three replicates were used for each microcapsule per each analysis. GMM: gum arabic + maltodextrin microcapsule (SIS extract coated with a mixture of GA + MD).

), and the water activity was between 0.23 and 0.24. Water activity for dried food products with enhanced stability should be lower than 0.6 and the moisture content should be less than 8%; our results fell within this range. The percentage of encapsulation efficiency was 51.50% and 55.90% for GMM 1 and GMM 2, respectively. The color values of microcapsules after spray drying were cream with a light-yellowish appearance whereas the color values measured in terms of L* (lightness), a* (green, red), and b* (blue, yellow) were 85.66 and 83.28, −1.31 and −1.26, and 14.11 and 14.64 for GMM 1 and GMM 2, respectively. Similar outcomes in microcapsule characteristics were observed when comparing the current study to our earlier research on encapsulating bioactive products from cocoa shells [17] and functional compounds from mulberry leaves [19] for use in functional foods.

The optimum microcapsules are spherical in shape, have a smooth and uniform surface, and lack wrinkles or dents. It has been identified that the microcapsules (GMM 1 and GMM 2) have a spherical shape (Figure 4). A similar observation was mentioned in a study by Akram et al. [33] where GA, MD, or a mixture of them was used to prepare spray-dried microcapsules of polyunsaturated oils by oil-in-water emulsions, and both microcapsules were found to have crystalline structures and a spherical form with surface shrinkage. Generally, microcapsules become dented by the pressure of steam created on the interior structure during spray drying which causes shrinking of the microcapsules due to moisture loss. The extract/GA + MD ratio utilized in this investigation produced an appropriate encapsulation layer, which in turn produced a smoother surface and shape for the microcapsules. The two different microcapsules (GMM 1 and GMM 2) were compared, and it was seen that the microcapsules of GMM 2 at 1000× were slightly bigger in size and compactly packed as compared to the microcapsules of GMM 1 at the same magnification. GMM 2 microcapsules were attached to one another due to the high amount of GA used in the encapsulation. The most important aspect of microcapsules that affect their appearance is particle size. GMM 1 had particle sizes ranging from 2.95 ± 0.02 to 27.73 ± 0.38 µm obtained at 1000× magnification (Figure 5). Similarly, GMM 2 had particle sizes ranging from 5.20 ± 0.014 to 29.30 ± 0.42 µm obtained at same magnification. The variation in particle size may be due to an increase in inlet temperature and change in formulation of MD and high feed viscosity of emulsion formulation [34]. There were not any significant changes (p > 0.05) noticed in the particle size distribution of microcapsules in the recent investigation. The combination of highly viscous MD with low viscosity GA resulted in the formation of particles of different sizes which was the case for GMM in this study. This result was also supported by San, Jaturanpinyo, & Limwikrant [35]: when compared to formulations that combined low viscosity GA with highly viscous MD, the combination of these two polymers resulted in increased emulsion viscosity.

4. Conclusions

Optimizing extraction processes is crucial in harnessing the full potential of plants for their bioactive compounds. This optimization ensures higher yields of active constituents, enhancing the efficiency and economic viability of extracting valuable compounds. In this study, experimental factors (time, temperature, and solvent concentration) conditions approach was used to determine the optimization of the extraction of phenolic compounds from sacha inchi shell (SIS). There were no research data previously reported about the effect of extraction parameters on the recovery of phenolic compounds from SIS using UAE and its encapsulation for future food application.

The conditions for UAE extraction of bioactive compounds from an underutilized SIS were optimized by RSM. The optimized extract was obtained at 25 °C, 15 min and 80% ethanol concentration. The SIS extract had antimicrobial properties against S. aureus and E. coli with an MIC of 1.45 and 0.77 mg/mL, respectively. The antimicrobial activity of plant extracts holds significant importance in various fields, including medicine, agriculture, and food preservation. As we combat antibiotic resistance, exploring and utilizing plant extracts’ antimicrobial properties may offer sustainable solutions to address this global challenge while paving the way for safer, eco-friendly alternatives in diverse applications. The optimized extract was also successfully encapsulated by spray drying in a combination of GA and MD with a smooth shape. Furthermore, the moisture content and water activity of the microcapsules were within the acceptable limit for dried food (moisture < 5% and water activity < 0.60). As encapsulation plays a pivotal role in harnessing the full potential of plant extracts, this study has concluded that microcapsules of SIS extract have the potential to be used as a functional food additive. Furthermore, these encapsulated microcapsules could be further studied for their compatibility with food products to be used as preservatives.