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Nutraceutical dark chocolate: A delivery system for double-encapsulated extracts of Crocus sativus L., Rosa damascena, Melissa officinalis L., and Echium amoenum

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Highlights

  • A low-calorie, nutraceutical dark chocolate fortified with herbal extracts was formulated.
  • Extracts of Crocus sativus L., Rosa damascena, Melissa officinalis L., and Echium amoenum were used.
  • Complex coacervation-spray drying approaches were utilized for the double encapsulation of the extracts.
  • The textural and sensory properties of the designed chocolate were enhanced.
  • The visual characteristics of the samples, in terms of brightness and Chroma, were improved.
Hamid Rajabi, Samineh Sedaghati
Incubator Center of Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

Received 30 October 2023, Revised 9 December 2023, Accepted 2 April 2024, Available online 6 April 2024, Version of Record 9 April 2024.

Abstract

This study aimed to develop a functional delivery system based on dark chocolate for double encapsulated extracts of four medicinal herbs, thereby creating a Nutraceutical Dark Chocolate (NDCh). Extracts of Crocus sativus L. (saffron), Rosa damascena (rosa), Melissa officinalis L. (balm), and Echium amoenum (borage) were encapsulated using a complexation-spray drying approach. The coacervates had a particle size ranging from 2.0 to 2.2 μm and a polydispersity index between 0.103 and 0.114. The first and second encapsulation efficiencies of spray-dried coacervates varied from 90.2 to 91.1% and 99.1–99.7%, respectively. NDCh samples, containing varying levels of microcapsules (500–885 mg per 33g of NDCh), exhibited an increase in Casson’s plastic viscosity and yield stress compared to the control sample. The fortified NDCh samples demonstrated improved brittleness and superior melting properties, although their whitening index was lower than that of the control sample. Sensory analysis indicated that NDCh samples fortified with minimal amounts of balm and borage microcapsules, and maximal levels of saffron and rosa, received higher scores than even the control sample. In conclusion, the fortification of dark chocolate with microcapsules containing extracts of well-known sedative medicinal herbs could potentially introduce a new category of nutraceutical products with suitable technological and sensory properties.

Keywords

Nutraceutical dark chocolate
Complex coacervation
Spray drying
Fortification
Sensory analysis

 Introduction

The consumption of delicious treats like chocolate, often seen as a great snack, has the potential to transition from a health risk to a highly functional food (Abedini et al., 2023). Dark chocolate, in particular, has gained popularity in recent decades among all age groups for its numerous health benefits. The European Union’s Directive 2000/36/EC defines dark chocolate as a product derived from cocoa products and sugars. It must contain a minimum of 35% total dry cocoa solids, including at least 18% cocoa butter and a minimum of 14% dry non-fat cocoa solids (Glicerina et al., 2016). It is packed with health-enhancing elements, including bioactive constituents such as polyphenols, flavonoids, procyanidins, and theobromines, as well as a variety of vitamins and minerals that positively impact the human immune system (Samanta et al., 2022). As such, it presents an intriguing option for delivering bioactive compounds to the body through fortification. It could be used to treat and reduce symptoms of various ailments, including obesity, hypertension, stress, cardiovascular disease, congestive heart failure, diabetes, certain cancers, and neurological disorders like Alzheimer’s and Parkinson’s disease (Abedini et al., 2023; Cimini et al., 2013; Martin & Ramos, 2021; Samanta et al., 2022).
Stress, anxiety, and depression are common psychiatric conditions that often coexist. They are characterized by negative emotional experiences and changes in biochemistry, cognition, behavior, and psychology. Due to the adverse effects of certain chemical medications, many patients prefer phytotherapy, which has been widely used for treating these disorders. Research on the effect of medicinal herbs on these disorders has increased in recent years. The results have shown their efficacy in improving the health state of patients. Among these herbs, Crocus sativus L. (saffron), Rosa damascena (rosa), Melissa officinalis L. (balm), and Echium amoenum (borage) have been proven to have significant therapeutic effects for declining mental-physical states caused by stress, anxiety, and depression (Ahmad et al., 2022; Farajdokht et al., 2020; Haybar et al., 2018; Jafari et al., 2020; Rasooli et al., 2021; Shafiee et al., 2018).
Recent research indicates a growing trend towards the use of chocolate fortified with high-value-added botanical substances as a therapeutic agent against non-communicable diseases. This is due to its enhanced health benefits, nutritional properties, and longevity. A variety of ingredients have been used for fortification purposes, including fish oil, cinnamon, phytosterols, probiotics, prebiotics, flavonoids, and flavan-3-ols (Hadnađev et al., 2023; Muhammad et al., 2018; Samanta et al., 2022; Sik et al., 2021).
Despite the undeniable functional benefits of fortified chocolate, there are challenges associated with its fortification. In other words, its functional characteristics, such as its rheological and sensory attributes, may be adversely affected. Encapsulation techniques such as spray drying may be able to overcome these challenges. As a result, numerous studies have focused on the design and development of nanoscale delivery systems for the purpose of fortifying chocolate. Accordingly, Gultekin-Ozguven et al. (2016), Dean et al. (2016), and Marsanasco et al. (2016) respectively utilized encapsulated black mulberry waste extract, peanut skin extracts, Omega (3 and 6) to fortify chocolate. More recently, fish oil was microencapsulated using a spray drying approach and the resultant powders were used to fortify dark chocolate (Hadnađev et al., 2023). On the other hand, the bioactive compounds that are responsible for therapeutic effects of medicinal herbs are very heat sensitive and spray drying microencapsulation may not provide full protection for them in products like chocolate. The production process of chocolate consists of several heating-mixing steps which can destroy/solve the wall material. In such a situation, the core material releases during the production process and not only is their efficacy decreased following exposure to the relatively severe production process (Jafari et al., 2017), but also the physical-sensorial properties of the final product may undergo adverse changes. So, it is crucial to design a more protective encapsulation system. Some researchers used a double encapsulation approach, in which the core material is initially encapsulated by complex coacervation and after that, a second process like spray drying or freeze drying is utilized. For instance, Bordón et al. (2021) used a double encapsulation system of complex coacervation-spray drying to produce microcapsules of chia seed oil.
The aim of this study was to develop a functional delivery system based on dark chocolate for the double encapsulation of extracts from four medicinal herbs. Consequently, a nutraceutical chocolate was produced to help prevent or decrease the symptoms of stress, anxiety, and depression. After a comprehensive literature review and consultation with traditional Iranian medicine, we selected four medicinal herbs: saffron, rosa, balm, and borage. These plants were chosen from a wide variety of options due to their minimal side effects on humans. Furthermore, their consumption limits for different age groups and physiological conditions are lower than those of other medicinal plants. These medicinal herbs were extracted and double encapsulated using Arabic gum (AG) and chitosan (CH) complexation, followed by maltodextrin (MD) and AG spray drying. Pertaining the selection of dark chocolate over milk chocolate, it will be a better option due to its higher polyphenol and flavonoid content. The total polyphenol and flavonoid content in dark chocolate is five times higher than in milk and white chocolate. The fat (30 g fat per 100 g) and sugar (52 g per 100 g) content is also significantly higher in milk and white chocolate (Samanta et al., 2022). The morphology, particle size, and particle size distribution of these microcapsules were characterized. Different levels of these microcapsules were introduced into dark chocolate formulations. The fortified samples were then subjected to analyses of their colorimetric properties, melting characteristics, rheological attributes, textural properties, and sensory profile.

2. Materials and methods

Saffron was donated by Mirmohannay (ArioRadMehr Torshiz Co., Iran). Rosa damascena, Melissa officinalis L., and Echium amoenum were purchased from well-known old local store of Mashhad (Iran). Chitosan (50–190 kDa), maltodextrin (DE = 16.5–19), and gallic acid were procured from Sigma-Aldrich (USA). Arabic gum, potassium chloride, sodium acetate, sodium carbonate, and Folin-Ciocalteu reagent were acquired from Merck Millipore (Germany). All the other reagents were of analytical grade.

2.1. Preparation of medicinal herb extracts

2.1.1. Preparation of saffron extract

The saffron extract was prepared using a method described by Rajabi et al. (2015), with slight modifications. The powdered saffron was mixed with a solvent and stirred continuously at room temperature for 2 h. After centrifuging and filtering the mixture, the solvent was removed using a rotary evaporator until 90% of it was gone. The concentrated extract was then dried using a freeze dryer (Operon-Korea) and stored in a dark, air-tight glass container at a temperature of 4 ± 1 °C.

2.1.2. Preparation of rosa extract

Dried petals (50 g) were milled and mixed with ethanol (400 mL, 80%, v/v). This mixture was magnetically stirred for 48 h at room temperature, then centrifuged and filtered. The solvent was removed using a rotary evaporator until the final volume of 80 mL was achieved. The concentrated extract was freeze-dried and stored in a dark, air-tight glass container at 4 ± 1 °C.

2.1.3. Preparation of borage extract

The dried flowers were milled and mixed with ethanol. This mixture was magnetically stirred for 24 h at room temperature, then centrifuged and filtered. The solvent was removed using a rotary evaporator until the final volume of 80 mL was achieved. The concentrated extract was freeze-dried and stored in a dark, air-tight glass container at a temperature of 4 ± 1 °C.

2.1.4. Preparation of balm extract

The leaves of balm were powdered and mixed with ethanol. This mixture was magnetically stirred for 24 h at room temperature, then centrifuged and filtered. The solvent was removed using a rotary evaporator until the final volume of 100 mL was achieved. The concentrated extract was freeze-dried and stored in a dark, air-tight glass container at a temperature of 4 ± 1 °C.

2.2. Determination of indicator compounds in herbal extracts

To determine the encapsulation efficiency of each herbal extract in subsequent steps, one or a group of bioactive compounds was quantified. Accordingly, the amount of crocin for saffron, total phenolic compounds for rosa and balm, and total anthocyanin for borage were determined as described below.
The amount of crocin in saffron extract/spray dried samples was determined by dissolving a specific amount of freeze-dried extract/microencapsulated powder in water. The amount of crocin was quantified using the following equation (Eq. 1), where  A1cm1%(λ440) signifies the absorbance of samples at 440 nm and the sample weight and moisture are symbolized by m and H, respectively (Rajabi et al., 2019).
(1) A1cm1%(λ440)=A×10000m(100−H)
The total amount of anthocyanin (TA) was quantified using the pH differentiation method (Lee, Durst, & Wrolstad, 2005), in which the absorbance of borage extract/spray-dried samples was determined in two different acidic media. Accordingly, two samples of extract/spray-dried powders (0.2 mL) were prepared and made up to 3 mL, one of them by adding a potassium chloride buffer (0.025 M, pH = 1) and the other by mixing with a sodium acetate buffer (0.4 M, pH = 4.5). The absorbance of these solutions was read at 520 and 700 nm, and the amount of TA was calculated using the following equation (Eq. 2), which includes absorbance (A), dilution factor (DF), cyaniding-3-glucosode molecular weight (MW, 449.2166 g mol−1), solvent volume (V, mL), cell path length (L, 1 cm), and molar absorptivity (a, 26,900 L. mol−1. cm−1).
(2) TA=A×Mw×DF×V×1000α×L
The amount of total phenolic compounds (TPC) in rosa and balm extract/spray-dried samples was quantified using the Folin-Ciocalteu colorimetric approach. Accordingly, the sample (100 μL), deionized water (6 mL), and Folin-Ciocalteu reagent (500 μL) were sequentially added to a 10 mL volumetric flask. After 1 min, the experiment was continued by adding sodium carbonate (1.5 mL, 0.2 mg L−1) and topping up to 10 mL with deionized water. The resulting mixture was allowed to sit undisturbed for 2 h before being spectrophotometrically analyzed at 760 nm. The amount of TPC, expressed as gallic acid equivalents (GAE; mg gallic acid/g extract), was calculated by interpolation from the gallic acid calibration curve (Singleton et al., 1999)

2.3. Encapsulation of herbal extracts

2.3.1. Encapsulation via complex coacervation

To achieve the highest coacervation efficiency of the complexation process, the proportion of biopolymers in the solution should be adjusted so that the zeta potential approaches zero (Rojas-Moreno et al., 2018). This is because when the functional groups of biopolymers interact at the highest rate, the surface charge approaches zero (or near zero). AG and CH were utilized to encapsulate the freeze-dried medicinal herbal extracts through complex coacervation (Rajabi et al., 2019). Accordingly, the zeta potential of different blends of AG and CH was measured over a pH range, showing that the lowest surface charge was obtained at a weight ratio of 1–4 (CH:AG) and pH ∼ 3.8 (data not given). Therefore, this ratio was used for the complexation encapsulation of herbal extracts. Subsequently, the specified amount of CH and AG was dissolved in an acetic acid solution (1%, v/v) and distilled water, respectively, and stirred for 2 h at room temperature. To achieve complete hydration, the solutions were refrigerated for 12 h. Each herbal extract was then separately mixed with CH at a constant weight ratio of 1:1 (herbal extract to biopolymers), and the mixture was homogenized using an Ultra-Turrax (IKA® T25) (10000 rpm, 2 min, ambient temperature). The AG solution was then added dropwise to the solution under continuous stirring (250 rpm, 60 min) to form coacervates around the herbal extract. The resultant mixtures were then ready to be dried through a spray drying process.

2.3.2. Encapsulation via spray drying

AG and MD were utilized for the second encapsulation process, as it has been shown that these wall materials effectively protect the encapsulated bioactive compounds through the spray drying approach (Rajabi et al., 2015; Ravichandran et al., 2014; Sukri et al., 2020). Immediately after the completion of complexation process and to uphold the structural soundness of coacervates, and also to encapsulate the unencapsulated bioactive compounds, a mixture of AG: MD (1:4, 5% w/w, relative to the mixture weight), as a drying aid was added into each mixture followed by magnetic stirring at room temperature for 15 min finally, each mixture transferred to a Buchi spray drier (mini, B 191, Switzerland) while the operating conditions were air flow 550 l/h, inlet air temperature 170 °C, outlet air temperature 85 °C, pump flow rate 6 mL/min, and atomization pressure 20 psi. Following the spray drying process, the encapsulated powders were collected from the cyclone and transferred into a dark air-tight glass and kept at −20 °C.

2.3.3. Characterization of microcapsules

2.3.3.1. Characterization of complex coacervates
The size properties of the coacervates, including particle size and particle size distribution, were examined before drying using a Malvern Zetasizer at ambient temperature (25 ± 1 °C). An optical microscope was used to visualize the morphological properties of the microparticles.
2.3.3.2. Characterization of spray dried coacervates
The moisture content (MC) of the spray-dried coacervates was determined under reduced pressure at 70 °C using a vacuum oven. The microcapsule yield (MY), one of the most important quantitative indices of the spray drying process, was calculated by weighing the produced powders and the total solid contents of the feed, as shown in the equation below (Eq. (3)). The ingredients used in this calculation include MC (wet basis), feed volume (V, L), total solid contents (Ts, g dry matter/L), and feed container weight before (g, W1) and after (g, W2) drying (Rajabi et al., 2015).
(3) MY(%)=(W2−W1)−MC(W2−W1)VTS×100
The first encapsulation efficiency (EEf) of each encapsulated herbal extract was calculated spectrophotometrically. For all samples, a specified amount of powder was first dispersed in absolute ethanol to release the core material. This was followed by ultrasonication in an Elmasonic P bath (30 min, 37 kHz, and 100% power). The mixture was then centrifuged (Digicen 21, Spain) for 10 min at 5000 rpm, and the supernatant was used to calculate the encapsulation efficiency.
For saffron, the amount of crocin, for rosa and balm, the amount of total phenolic compounds and for borage, the amount of anthocyanins were used as indicator for the determination of EE. Accordingly, the amount of these bioactive compounds was determined both in each herbal extract and in the corresponding spray dried samples. Then, the EE was calculated using the following equation (Eq. 4):
(4) EEf(%)=(BCsdBCex)×100 where, BCsd and BCex are the amount of index bioactive compounds in spray dried sample and herbal extract, respectively. After computing EE, the percentage of loss for each herbal extract was determined. An equivalent amount was then added to the feed to ensure that the specified concentration of each bioactive compounds keep in the microencapsulated powders and the second EE (EEs) was determined as below (Eq. (5)):
(5) EEs(%)=(BCesdBCex)×100 In this equation, BCesd represents the amount of index bioactive compounds in the enhanced spray-dried sample, and BCex is the quantity of index bioactive compounds in the herbal extract before the addition of the lost amount.
The morphology of the microcapsules was determined using a MIRA3 TESCAN microscope (scanning electron microscope, SEM).

2.4. Nutraceutical dark chocolate designing

In designing an NDCh, two fundamental aspects must be taken into account:
(1) The amount of bioactive compounds in each chocolate bar, and
(2) The weight of each chocolate bar. The proportion of each herbal extract to add to the chocolate formulation was determined based on their effective dosage for depression and anxiety reported in previous studies. Accordingly, the range of effective dosages for saffron, borage, balm, and rosa was 30–40, 375–500, 80–300, and 15–45 mg/day, respectively (Farajdokht et al., 2020; Haybar et al., 2018; Rasooli et al., 2021; Sayyah et al., 2006; Shafiee et al., 2018; Sik et al., 2021). Considering the importance of the sensory properties of this nutraceutical product, chocolate samples with different concentrations of herbal extract were produced and analyzed (Table 1).

Table 1. The amount of herbal extract microcapsules in different NDCh samples.

Chocolate sample code Microencapsulated herbal extract Total (mg)
Saffron (%) Borage (%) Balm (%) Rosa (%)
Control – – – – 0
F1 6 75 16 3 500
F2 4.5 56.5 33.9 5.1 885
F3 5.4 51.4 41 2.2 730
F4 4.6 76.3 12.3 6.8 655
F5 4 50 40 6 750
F6 7.4 69.4 14.8 8.4 540
F7 3.6 59.1 35.5 1.8 845
F8 6.3 78.7 12.6 2.4 635
From a commercial viewpoint, the weight of each chocolate bar is important and must be designed in such a way that all the bioactive compounds (in this study, microencapsulated herbal extract powders) are included in one serving. Through literature review, we found that the weight of dark chocolate recommended for therapeutic purposes ranges between 11.7 and 75 g (Engler et al., 2004; Jackson et al., 2019; Martin et al., 2009; Mursu et al., 2004; Shiina et al., 2009). Furthermore, tracking the weight of commercial chocolates on the market (dark, milky, etc.) showed that their average weight is about 33 g (1.16 oz). So, in this study, each NDCh is designed to weigh 33 g and contain all the microencapsulated herbal extract powders for daily consumption.
The dark chocolate formulation (without the addition of encapsulated powders) and processing conditions (duration and temperature) were optimized through preliminary experiments considering sensory attributes (data not given). To prepare the chocolate, cocoa butter (30%), cocoa powder (26.44%), MD and/or encapsulated powders (32%), inulin (11%), sucralose (0.06%), and soy lecithin (0.5%) were mixed and pre-refined by blending for 150 min at 100 rpm and 45 °C in a ball mill (Sepehr Machine Inc., Iran). The process continued with conching at 50 °C for 120 min. Based on the experimental design (Table 1), encapsulated herbal extract powders were added to the mixture in the last 15 min of the conching process. Afterward, the mixture was tempered at 30 °C for 30 min, molded (55 × 15 × 8 mm3), and cooled (5 °C, 30 min). Finally, the process was completed by demolding, packing in aluminum foil, and storing at 20 °C. To track the changes in dark chocolate following the addition of microencapsulated herbal extract powder, a control sample was made in a similar way, except for the addition of those powders.

2.5. Nutraceutical dark chocolate characterization

2.5.1. Color indices

The visual surface properties of the chocolate samples were examined by determining color indices (L*, a*, and b*), which were expressed as the whiteness index (WI) [Eq. (6)], Chroma (C) [Eq. (7)], and hue (h) [Eq. (8)], using a Chroma Meter colorimeter (CR-400, Konica Minolta) (Toker et al., 2018).
(6) WI=a*2+b*2+(100−L)2
(7) C=a*2+b*2
(8) h=tan−1(b*a*)

2.5.2. Melting properties

To predict the melting behavior of the chocolate sample once it’s placed in the mouth, the melting properties of the samples, i.e., Tonset, Tpeak, Tend, and enthalpy (ΔH), were quantified using a PerkinElmer differential scanning calorimeter (Model DSC 7). Accordingly, 6 mg of each chocolate sample was transferred into the DSC pan and then heated from 5 to 60 °C at a rate of 5 °C/min under nitrogen sparging. Tonset, Tpeak, and Tend represent the beginning of melting, highest melting rate, and liquefaction, respectively, while ΔH represents the amount of energy required to completely melt the fat.

2.5.3. Analysis of rheological and textural properties

A hysteresis loop test was used to track the rheological properties of melted chocolate at 40 °C over variations in shear rate. Accordingly, the shear rate was changed from 2 to 50 s−1 over 180 s, kept at the maximum shear rate for 60 s, and then reduced to the starting point over 180 s. The obtained data was fitted by the non-Newtonian fluid model of Casson, in which shear stress ( τ ), yield stress ( τCYS ) and plastic viscosity ( ηCPV ) of Casson, and shear rate ( γ. ) were employed (Eq. (9)).
(9) (τ)0.5=(τCYS)0.5+(ηCPVγ.)0.5
The textural analysis was carried out using a TA-XT plus texture analyzer to track the changes in hardness and brittleness of the samples as affected by the addition of microencapsulated powders. The experiment was performed using a three-point bend test, in which the chocolate samples were subjected to a force until they broke. The hardness (N) and brittleness (mm) were calculated by plotting the displacement over force under the following conditions: temperature 20 °C, trigger force 0.05 N, load cell 5 kg, and compression speed 2 mm s−1.

2.5.4. Assessment the stability of encapsulated bioactive compounds in chocolate sample

In the pursuit of understanding the impact of the chocolate-making process on bioactive compounds, an analysis was conducted on the quantity of these compounds present in chocolate. Dark chocolate, a known abundant source of phenolic compounds, has the potential to interfere with the quantification of phenolic compounds in borage and balm. Furthermore, crocin and anthocyanin, being more sensitive than phenolic compounds, are more likely to be destroyed in larger quantities during the chocolate-making process. Consequently, these compounds can serve as reliable stability indices to monitor the effect of the process on encapsulated bioactive compounds and evaluate the efficacy of chocolate as a delivery system. In light of this, the quantities of crocin and TA were ascertained in the chocolate sample that received the highest organoleptic scores. To determine the quantity of bioactive compounds, lipids were extracted from the chocolate using hexane. The TA quantity was then quantified using the pH differentiation method (Eq. (2)). With respect to crocin, the defatted and dried chocolate was heated to 60 °C and subjected to extraction with ethanol (50%, v/v), followed by ultrasonication (300 W, 6 min) to release the core material. Subsequently, the resultant mixture was centrifuged (10000 rpm, 5 min), filtered (Whatman filter paper No. 2), and the supernatant was analyzed spectrophotometrically (Eq. (1)). To mitigate the influence of other soluble compounds on the calculations, the control sample (without spray dried coacervates) was prepared using the same methodology, and the supernatant obtained was employed as the blank.

2.5.5. Sensory analysis

Having good sensory attributes is crucial for the development of nutraceutical products. In this context, we considered the different sensory properties of our designed nutraceutical chocolate. We trained a group of 20 individuals, consisting of 10 men and 10 women aged 20–40, to evaluate these properties. Over the course of three comprehensive training sessions, the panel members underwent a rigorous educational process. This process was designed to familiarize them with the specific definitions of sensory attributes and their associated references (Table 2). Informed consent was obtained from the participants through a statement that read, “I am cognizant of the confidentiality of my responses and I consent to participate in this survey.” An affirmative response to this statement was a prerequisite for participation in the survey. Participants were granted the liberty to withdraw their participation from the survey at any given time, without the obligation to provide a justification for their withdrawal. Furthermore, it is important to note that the products subjected to testing were deemed safe for consumption. They scored the sample on a scale from 1 to 7 (1 = not perceptible, 2 = barely noticeable, 3 = slightly prominent, 4 = moderately discernible, 5 = clear distinction present, 6 = moderately abundant, 7 = strong and plentiful, easily observable). The analyzed attributes were appearance (subdivided into blooming, surface brightness, and color), texture (subdivided into hardness, grittiness and aftertaste), and taste and aroma (subdivided into sweetness, astringency, and bitterness), as described in Table 2. After scoring each sample, the panelists rinsed their mouths with warm water. Each sample was assigned a unique three-digit code chosen at random (Sarfarazi & Mohebbi, 2020; Toker et al., 2018).

Table 2. Description of the sensory attributes.

Sensory attributes description
Appearance Brightness The perceived color of an object signifies the correlation between the light it reflects and the light it absorbs.
Color Intensity of brown color
Texture Hardness The force that is necessary to cleave the chocolate in two using one’s front teeth.
Grittiness The presence of fine or large particles in the molten chocolate
Melting The resistance of chocolate to being completely liquid
Taste and aroma Herbal Intensity of medicinal herbs extract taste
Astringency Intensity of causing tango to contract.

2.6. Statistical analysis

A 2-level factorial design was employed to explore varying ratios of microencapsulated herbal extract in the chocolate recipe. An ANOVA test was carried out to ascertain whether the use of different proportions of microencapsulated herbal extract had a significant impact on physicochemical characteristics of NDCh samples (p < 0.05). The data were analyzed using the MINITAB Release 16 software (Minitab Inc., PA, USA). Each experiment was conducted three times and the average values were reported.

3. Results and discussion

3.1. Complex coacervates characterization

3.1.1. Particle size and particle size distribution

Particle size and its distribution are key descriptive indices for micro/nano-sized food systems, contributing significantly to the texture of the systems they are contained in. As indicated in Table 3, the average particle size ranged from 2.0 to 2.2 μm, while the polydispersity index (PDI) varied between 0.103 and 0.114 (p > 0.05). The PDI values of the prepared complex coacervates, as depicted in Fig. 1, confirm the formation of a highly uniform micro-sized system. It can be inferred that the type of herbal extract did not significantly impact the particle size and PDI of the complex coacervates (p > 0.05). Previous studies have reported that particle size and PDI obtained through the complex coacervation method can vary from approximately 50 nm to 100 μm and 0.057 to 0.7, respectively. These variations depend on factors such as the type and physicochemical properties of polyelectrolytes, their ratio, and operational conditions like solution pH and stirring rate (Bordón et al., 2021; Ferreira & Nicoletti, 2021; Lv et al., 2014; Santos et al., 2014). For instance, encapsulated saffron extract using AG:CH (5:10 w/w, pH = 4.7, stirring rate = 200 rpm) resulted in a particle size of about 200 nm and a PDI of approximately 0.3 (Rajabi et al., 2019).

Table 3. Characterization of complex coacervates and spray dried complex coacervates of different herbal extracts.

Herbal extract Complex coacervates properties Spray dried complex coacervates properties
Mean particle size (μm) PDI MC (%) MY (%) First EE (%) Second EE (%)
Saffron 2.1 ± 0.1 a 0.111 ± 0.02 a 4.49 ± 0.33 a 92.1 ± 1.5 a 90.6 ± 1.7 a 99.2 ± 0.4 a
Borage 2.2 ± 0.1 a 0.105 ± 0.01 a 4.55 ± 0.18 a 91.9 ± 2.1 a 90.2 ± 1.9 a 99.7 ± 1.2 a
Balm 2.1 ± 0.1 a 0.103 ± 0.03 a 4.46 ± 0.47 a 92.5 ± 1.9 a 91.0 ± 2.2 a 99.1 ± 1.5 a
rosa 2.1 ± 0.1 a 0.114 ± 0.01 a 4.59 ± 0.63 a 91.6 ± 3.2 a 91.1 ± 1.8 a 99.5 ± 0.8 a
The values provided are the averages derived from three trials, with the standard deviation denoted as ±. In each column, values that are followed by distinct lowercase letters are statistically different from one another, with a significance level of less than 0.05.

Fig. 1. Particle size distribution of complex coacervates of herbal extracts. (A) Borage, (B) Saffron, (C) Balm, (D) rosa.

3.1.2. Morphology of coacervates

The morphological characteristics of the coacervates, as depicted in Fig. 2, reveal spherical particles with a narrow particle size, which is further confirmed by the PDI in Table 3. Similar observations have been reported by other researchers, who attribute this homogeneity to two key factors: the interactions between the functional groups of polyanionic and polycationic biopolymers, and changes in the micro-viscosity of the system due to the addition of core material. Both these factors contribute to a decrease in PDI and the formation of homogeneous microparticles (Lv et al., 2014; Rajabi et al., 2020; Rajabi et al., 2021). Furthermore, the formation of distinct and narrowly distributed spherical particles without aggregation, as well as flake-like structures, indicate that the ratio between biopolymers and the pH of the medium were appropriately selected (Ferreira & Nicoletti, 2021; Schröder et al., 2023).

Fig. 2. Optical microscopy images of complex coacervates of herbal extracts. (A) Borage, (B) Saffron, (C) Balm, (D) rosa.

Moreover, as depicted in Fig. 2, the color of the medium surrounding the coacervates is similar to that of the corresponding herbal extract. This suggests that the herbal extract was not completely encapsulated by the coacervates (Rajabi, Sedaghati, Rajabzadeh, & Sani, 2024). For instance, as shown in Fig. 2B, the background color is a pale red due to the presence of free saffron bioactive compounds, particularly crocin. Therefore, it is essential to encapsulate these compounds to ensure their full protection. To achieve this, MD and AG were used in a ratio of 1:4 (w/w) as a secondary wall material system.

3.2. Spray dried complex coacervates characterization

3.2.1. Moisture content

As indicated in Table 3, the moisture content (MC) of the spray-dried complex coacervates ranged between 4.46 and 4.59%, with no significant differences observed between the samples (p > 0.05). This trend was expected as all feed conditions were similar, except for the core materials. The MC of spray-dried powders is influenced by several factors, including the properties of wall materials, wall:core ratios, and spray drying operating conditions (Couto et al., 2013). However, in this study, the difference in core materials properties did not affect this feature. On another note, it has been reported that the optimal MC for food/pharmaceutical powders to meet stability requirements is less than 5% (Rajabi et al., 2015). Therefore, it can be concluded that all the produced microcapsules met these requirements and were stable.

3.2.2. Microcapsule yield and encapsulation efficiency

One of the key characteristics in the design of a spray drying microencapsulation system is achieving the highest possible microcapsule yield (MY) and encapsulation efficiency (EE), particularly when the core material is of high economic value. The MY of the process was found to be between 91.6 and 92.5%. MY in the spray drying process is influenced by several factors, including the physicochemical properties and ratio between wall materials, wall:core ratio, core properties, and operating conditions (Rajabi et al., 2015). As all these factors, except for the core material, were the same for all four microcapsules, it is predictable that no significant results could be obtained. The EE of spray-dried coacervates was calculated by tracking and quantifying the amount of index compounds in freeze-dried herbal extracts and their corresponding microcapsules. EE was in the range of 91.6–93.1% (Table 3), revealed the ability of the designed system to protect herbal extract bioactive compounds. Bordon et al. (2021) and Hernández-Nava, López-Malo, Palou, Ramírez-Corona, and Jiménez-Munguía (2020) reported lower EE efficiency for chia oil and oregano essential oil, respectively, prepared via complex coacervation and subsequent spray drying. Lower EE than that of this study may be due to differences between the first and especially second encapsulation systems. We utilized a combination of MD and AG to surround coacervates and unencapsulated herbal extract. It has been revealed that the combination of these two biopolymers provides superior protection over the core material, with EE exceeding 90% (Carlan et al., 2020; Rajabi et al., 2015; Ribeiro et al., 2020). Furthermore, to design an NDCh, we need to have a specific amount of each bioactive compound in the microencapsulated powders. Therefore, the amount of loss for each formulation was calculated, an equal amount was added to the feed, and the encapsulation efficiency was computed as second encapsulation efficiency (EEs). The results showed that EEs for all herbal extracts exceeded 99% (Table 3).

3.2.3. Morphological properties of spray dried coacervates

The morphological characteristics of the spray-dried coacervates are depicted in Fig. 3. The image reveals a variety of particle sizes with smooth or shrunk surfaces, and the microcapsules show no signs of cracking or fissures, indicating good spray drying operation conditions and appropriate wall material selection (Rajabi et al., 2015). Furthermore, all samples exhibit the same morphological traits, which is expected given the same wall material blend and operating conditions applied. As shown in Fig. 2 (optical microscopic images), the coacervates had a narrow particle size distribution. However, after spray drying, a very size-heterogeneous system appeared in all spray-dried herbal extract samples. These observations could be attributed to the presence of a second wall material blend, i.e., MD and AG (Rajabi, Sedaghati, Rajabzadeh, & Sani, 2024). These wall materials seem to produce three types of microcapsules with different appearances: (1) The first type contains unencapsulated bioactive compounds from the herbal extract that were not entrapped during the complexation process, (2) The second type uses coacervates as the core, and (3) The third one is the hybrid core microparticle that contains both unencapsulated bioactive compounds and coacervates as the core (Fig. 4).

Fig. 3. SEM images of spray dried complex coacervates of herbal extracts. (A) Borage, (B) Saffron, (C) Balm, (D) rosa.

  • Fig. 4. Schematic representation of preparation of herbal extract microcapsules.

    Moreover, the compact-interconnected appearance of microcapsules in Fig. 3 could be due to interactions between those CH molecules that did not participate in the complexation process with AG that was added along with MD as the second wall material system. Accordingly, the amine groups of CH could interact with the carboxyl and hydroxyl groups of AG, leading to the formation of a dense-interconnected network.

    3.3. Nutraceutical dark chocolates characterization

    3.3.1. Color indices

    The color indices of chocolate are influenced by several factors, including the production process, recipe, and the crystal structure of fat (Saputro et al., 2017). The changes in the color of the NDCh samples were analyzed 24 h after production by computing different color indices (Table 4). The incorporation of microencapsulated herbal extract powders into the samples significantly altered the color profiles of the samples (p < 0.05). The level of lightness (L*) increased in correlation with the amount of spray-dried powders added. The lowest (26.15) and highest (32.11) L* values were recorded for the control and F2 samples (which contained the highest amount of microcapsules), respectively (p > 0.05). An increase in L* value or sample brightness enhances its visual quality. Therefore, it can be inferred that the incorporation of spray-dried powders, regardless of the type of herbal extract, significantly improved the color of dark chocolate samples, making them brighter. Similar results were reported by Hadnađev et al. (2023) and Agibert and Lannes (2018), who worked on fortifying dark chocolate using different microcapsules. The same trend was observed for a* (increased from 6.76 in the control sample to 9.86 in F2) and b* (increased from 4.09 in the control sample to 6.47 in F2). That is, as the amount of microencapsulated herbal extract in the recipe increased, these values significantly increased (p < 0.05), similar to the trend reported by Toker et al. (2018), who worked on fortifying chocolate with encapsulated phytosterols. Other researchers also reported that the addition of encapsulated bioactive compounds significantly changed the color indices of chocolate samples (Lončarević et al., 2018; Lončarević et al., 2019; Toker et al., 2018; Tolve et al., 2018).

    Table 4. Color indices of the NDCh samples.

    Chocolate sample code L* a* b* h C WI
    Control 26.15 ± 0.214 e 6.76 ± 0.163 f 4.09 ± 0.096 d 0.70 ± 0.01 c 7.90 ± 0.59 f 74.27 ± 1.21 a
    F1 28.93 ± 0.221 d 7.64 ± 0.099 e 4.88 ± 0.096 c 0.75 ± 0.05 b 9.06 ± 0.41 e 71.64 ± 0115 b
    F2 32.11 ± 0.124 a 9.86 ± 0.206 a 6.47 ± 0.092 a 0.79 ± 0.03 b 11.79 ± 0.62 a 68.90 ± 0.40 e
    F3 30.35 ± 0.154 bc 8.63 ± 0.184 c 6.01 ± 0.107 b 0.86 ± 0.02 a 10.52 ± 0.48 cd 70.43 ± 1.09 cd
    F4 29.97 ± 0.109 bc 8.21 ± 0.131 d 5.83 ± 0.124 b 0.89 ± 0.06 a 10.07 ± 0.74 d 70.75 ± 1.53 c
    F5 30.44 ± 0.099 b 8.77 ± 0.235 c 6.11 ± 0.112 b 0.86 ± 0.09 a 10.69 ± 0.45 c 70.37 ± 1.39 cd
    F6 29.22 ± 0.112 d 7.87 ± 0.228 d 4.95 ± 0.055 c 0.74 ± 0.06 bc 9.29 ± 0.58 e 70.17 ± 0.44 d
    F7 31.69 ± 0.107 a 9.26 ± 0.058 b 6.35 ± 0.218 a 0.84 ± 0.11 a 11.23 ± 0.74 b 69.23 ± 0.72 e
    F8 29.81 ± 0.087 c 8.11 ± 0.115 d 5.75 ± 0.111 b 0.88 ± 0.08 a 9.94 ± 0.29 d 70.89 ± 1.48 bc
    The values provided are the averages derived from three trials, with the standard deviation denoted as ±. In each column, values that are followed by distinct lowercase letters are statistically different from one another, with a significance level of less than 0.05.
    Three factors can be attributed to these observations:
    • 1.
      All the spray-dried powders had a surface color similar to the corresponding herbal extract, albeit with less intensity. Therefore, their incorporation into the formulation enhanced the color indices, especially lightness.
    • 2.
      The spray-dried powders had a small particle size and narrow particle size distribution. One of the most important factors influencing chocolate color is particle size.
    • 3.
      The presence of AG in the chocolate formulation, especially in the recipe containing the maximum amount of microencapsulated powder (F2 and F7), may have altered the surface properties of the samples, which directly affect the visual properties of chocolates.
    The phenomenon of fat blooming, which is considered a negative quality attribute, occurs when cocoa butter destabilizes, migrates through the chocolate matrix towards the surface, and recrystallizes, resulting in an off-white color. To track the occurrence and intensity of this phenomenon, the whiteness index (WI) is measured (Hadnađev et al., 2023). The WI of chocolate samples varied from 68.90 (F2) to 74.27 (control sample), indicating that the addition of microencapsulated herbal extract significantly decreased this index (p < 0.05). Despite the lowering effect of encapsulated powders on WI, it’s important to note that the appearance of chocolate samples did not show any signs of fat blooming (Fig. 5). The increase in WI could be attributed to the lighter color of encapsulated powders and their impact on enhancing cocoa butter recrystallization. Similar results were published about fortification of chocolate with microcapsules of fish oil (Hadnađev et al., 2023), microcapsules of peanut oil (Agibert & Lannes, 2018), and microcapsules of chia seed oil (Razavizadeh & Tabrizi, 2021).

Fig. 5. Nutraceutical dark chocolate samples.

Chroma (C) shows the degree of saturation or color intensity while hue (h) is an indicator of food luminance color. Both C and h were significantly influenced by the addition of microencapsulated herbal extract powders (p < 0.05), with both indices experiencing an increasing trend in parallel to increasing levels of fortification. Accordingly, the value of C varied from 7.90 (control sample) to 11.79 (F2) while h changed from 0.70 to 0.79 for control and F2 samples respectively. It has been shown that both these indices are matrix-dependent factors, i.e., their values significantly alter by changing particle size, particle size distribution, and product density (Afoakwa et al., 2008). Accordingly, a finer matrix scatters more light. Therefore, it can be concluded that adding microencapsulated powder due to effects on chocolate particle size pattern significantly changed the surface visual properties of samples, resulting in a lighter and more saturated appearance than control or those that had lower levels of fortification. These findings are in accordance with those reported by Toker et al. (2018) and Afoakwa et al. (2008).

3.3.2. Melting characteristics of chocolate samples

The thermal properties of chocolate are vitally important for product quality and consumer preferences. Accordingly, four essential thermal indices of NDCh samples were quantified using the DSC method (Table 5). Tonset, Tpeak, and Tend respectively varied in the range of 26.39–28.05 °C, 32.23–33.81 °C, and 34.19–35.28 °C, while enthalpy ranged from 28.19 (F3) to 29.03 (F7), with no significant changes (p > 0.05). Incorporation of microencapsulated powders into the chocolate formulation at the highest concentrations (845 and 885 mg/33 g chocolate) resulted in a significant enhancement of Tonset, Tpeak, and Tend. However, at lower levels of fortification (500, 540, 635, 655, 730, and 750 mg/33 g chocolate), these indices did not experience a pivotal change when compared with the control sample (p > 0.05). These observations could be attributed to the presence of AG, especially at higher levels of fortification, which resulted in increasing interactions between biopolymers and the formation of a more stable network. Furthermore, it was shown that by incorporating additional compounds into the chocolate recipe, its melting behavior was altered due to the eutectic effect (Zhang et al., 2020). These results were in accordance with findings by Hadnađev et al. (2023) and Toker et al. (2018), who respectively claimed that incorporating microencapsulated fish oil and eicosapentaenoic and docosahexaenoic acids into dark chocolate significantly changed thermal properties. On the other hand, the intensity of changes between these researches and the present study was different. For instance, in the research by Toker et al. (2018), Tend did not change significantly while others did. These differences could be ascribed to variations in the chocolate recipe (type of sweeteners, presence and type of bulking agent), processing conditions (duration and temperature of each step), type of fortificant (physicochemical properties of bioactive compounds), and level of fortification.

Table 5. Thermal properties of the NDCh samples.

Chocolate sample code Tonset Tpeak Tend ΔH
Control 26.39 ± 0.109 b 32.23 ± 0.154 b 34.19 ± 0.115 b 28.51 ± 0.115 a
F1 26.69 ± 0.109 b 32.38 ± 0.154 a 34.29 ± 0.115 b 28.34 ± 0.115 a
F2 28.05 ± 0.109 a 33.81 ± 0.154 a 35.28 ± 0.115 a 28.66 ± 0.115 a
F3 27.55 ± 0.109 b 32.82 ± 0.154 b 34.68 ± 0.115 b 28.19 ± 0.115 a
F4 27.13 ± 0.109 b 32.69 ± 0.154 b 34.54 ± 0.115 b 28.75 ± 0.115 a
F5 27.57 ± 0.109 b 32.87 ± 0.154 b 34.73 ± 0.115 b 29.12 ± 0.115 a
F6 26.68 ± 0.109 b 32.35 ± 0.154 b 34.28 ± 0.115 b 28.84 ± 0.115 a
F7 28.12 ± 0.109 a 33.74 ± 0.154 a 35.16 ± 0.115 a 29.03 ± 0.115 a
F8 27.11 ± 0.109 b 32.70 ± 0.154 b 34.51 ± 0.115 b 28.33 ± 0.115 a
The values provided are the averages derived from three trials, with the standard deviation denoted as ±. In each column, values that are followed by distinct lowercase letters are statistically different from one another, with a significance level of less than 0.05.

3.3.3. Rheological and textural characteristics of chocolate samples

The rheological behavior of NDCh samples was assessed by computing Casson’s yield stress and viscosity (Table 6). The viscosity of such a system is governed by particle size distribution, production process, and formulation (Afoakwa et al., 2008). As the former and latter were altered in this study, tracking the changes in rheological behavior is profoundly important as they are directly correlated with texture and consumer preferences. Incorporation of herbal extract microcapsules into the chocolate formulation at high levels of fortification had a significant effect on Casson viscosity (p < 0.05). The Casson viscosity ranged between 2.88 and 2.95 Pa s, respectively for control and F2 samples. A significant enhancement of Casson viscosity in two samples, F2 and F7, which contained 885 and 845 mg of microcapsules per 33 g of chocolate, may be due to the presence of a higher amount of AG than other formulations. AG can absorb water, interact with other biomolecules in the chocolate matrix, and consequently, increase the viscosity. In line with this, Hadnađev et al. (2023), Toker et al. (2018), and Agibert and Lannes (2018) reported that the inclusion of microcapsules into the chocolate recipe resulted in a significant increment of Casson plastic viscosity.

Table 6. Rheological and textural characteristics of the NDCh samples.

Chocolate sample code Rheological properties Textural properties
Casson plastic viscosity (Pas) Casson yield stress (Pa) Breaking force (N) Brittleness (mm)
Control 2.88 ± 0.028 b 6.85 ± 0.016 c 21.55 ± 0.404 c 69.33 ± 0.209 c
F1 2.89 ± 0.030 b 6.88 ± 0.024 c 21.63 ± 0.519 c 69.24 ± 0.221 c
F2 2.95 ± 0.025 a 7.22 ± 0.206 a 21.96 ± 0.196 a 67.21 ± 0.129 a
F3 2.90 ± 0.036 b 7.03 ± 0.029 b 21.69 ± 0.226 b 67.33 ± 0.154 b
F4 2.89 ± 0.031 b 6.90 ± 0.022 c 21.68 ± 0.617 c 69.75 ± 0.115 c
F5 2.91 ± 0.023 b 6.99 ± 0.016 b 21.73 ± 1.004 b 67.17 ± 0.199 b
F6 2.88 ± 0.032 b 6.89 ± 0.023 c 21.70 ± 0.251 c 69.50 ± 0.212 c
F7 2.94 ± 0.028 a 7.15 ± 0.026 a 21.98 ± 0.471 a 67.39 ± 0.159 a
F8 2.90 ± 0.022 b 6.91 ± 0.018 c 21.68 ± 1.055 c 69.7167 ± 0.221 c
The values provided are the averages derived from three trials, with the standard deviation denoted as ±. In each column, values that are followed by distinct lowercase letters are statistically different from one another, with a significance level of less than 0.05. In terms of Casson yield stress, the values ranged between 6.85 and 7.22 Pa, with the highest and lowest values recorded for the control and F2 samples, respectively. The addition of microencapsulated herbal extract at four levels of 730, 750, 845, and 885 mg per 33 g of chocolate significantly increased the yield stress (p < 0.05), while the change at lower levels of fortification was negligible (p > 0.05). This is in line with the findings of Lončarević et al. (2018) and Lončarević et al. (2019), who worked on fortifying chocolate with microcapsules of blackberry juice and green tea extract. The observed changes could be attributed to the effects of the second wall material system, i.e., AG. As mentioned before, GA, as a hydrophilic biopolymer, has the ability to increase the viscosity of food products and form a stable matrix. Consequently, NDCh samples fortified at high levels of microcapsules showed a network interconnected by the long chains of GA, which resisted movement and significantly increased yield stress. The inclusion of microcapsules of herbal extracts into the chocolate formulation had a greater impact on yield stress than viscosity, possibly because yield stress is more affected by the forces between particles (Hadnađev et al., 2023), which were enhanced through increasing the concentration of AG at high levels of fortification. The textural properties of chocolate samples were measured using a texture analyzer. The values of hardness and brittleness were found to lie between 21.55 and 21.98 N and 67-21-69.33 mm, respectively (Table 6). Fortification of chocolate samples at levels of 500, 540, 635, 655, 730, and 750 mg per 33 g of chocolate significantly increased both indices (p < 0.05), while the values of hardness and brittleness for the rest did not significantly differ from the control sample. The highest and lowest hardness were recorded for the sample containing the highest levels of fortificant and the control sample, respectively. A reverse trend was observed for brittleness, with the highest and lowest values found for the control sample and the sample containing the highest levels of fortificant (F2), respectively. Increasing brittleness (poorer snap), which means that the sample tends to bend instead of having a sharp snap, is considered a negative point (Beckett, 2019). Therefore, the addition of microencapsulated herbal extracts improved the textural properties. The enhancement of textural properties was in line with the results obtained for rheological characteristics, possibly due to the structural alteration caused by AG towards forming an interconnected network. It has been revealed that by increasing particle-particle interaction in chocolate, the textural features significantly change (Afoakwa et al., 2008). Moreover, AG’s ability to decrease the water activity of a food system could result in increased hardness and brittleness.

3.3.4. Stability of bioactive compounds in chocolate

The amounts of crocin and TA in the corresponding extract, spray-dried coacervates, and chocolate (F7) are shown in Table 7. Chocolate sample formulation F7 was selected based on the sensory analysis results (Table 8). The amounts of these compounds in the extract and spray-dried coacervates were almost the same (p > 0.05). Following the incorporation of spray-dried coacervates into the chocolate formulation, the amounts of crocin and TA significantly decreased (p < 0.05). Crocin decreased from 282 to 239 (∼15% loss), while the amount of TA decreased from 88.64 to 67.09 mg L−1 (∼24.5% loss). This loss was probably due to the heating during conching process (50 °C-15 min), as crocin and anthocyanin are both heat-sensitive compounds. Moreover, these results showed that the sensitivity of anthocyanin was greater than that of crocin, as it was destroyed 10% more. Despite the fact that about 15% and 24.5% of the bioactive compounds of borage and saffron were destroyed, respectively, it can be said that the chocolate is still an appropriate delivery system.

Table 7. The amounts of bioactive compounds in extract, spray dried coacervates, and chocolate sample.

Medicinal herb Extract Spray dried coacervates Chocolate (F7) Loss in chocolate (%)
Saffron (crocin,A1cm1%(λ440)) 286.96 ± 1.66 a 282.43 ± 0.39 a 239 ± 1.18 b 16.43
Borage (TA, mg L−1) 89.30 ± 0.47 a 88.64 ± 0.14 a 67.09 ± 0.52 b 24.87
Balm (TPC, GAE) 53.22 ± 0.71 a 52.77 ± 0.57 a – –
rosa (TPC, GAE) 241.65 ± 1.23 a 240.28 ± 2.11 a – –
Values represent the means ± standard deviation; n = 3. Values in the row followed by the different superscripts are significantly different (P < 0.05).

Table 8. Sensory properties of different NDCh samples.

Sensory attributes Control F1 F2 F3 F4 F5 F6 F7 F8
Appearance Brightness 5.89 ± 0.11 c 6.25 ± 0.25 b 6.76 ± 0.16 a 6.41 ± 1.12 b 6.24 ± 0.52 b 6.39 ± 0.48 b 6.25 ± 1.22 b 6.60 ± 0.77 a 6.20 ± 0.38 b
Color 6.87 ± 0.36 a 6.33 ± 0.63 b 5.84 ± 0.69 c 6.10 ± 0.58 b 6.35 ± 0.83 b 6.13 ± 0.55 b 6.35 ± 0.49 b 5.89 ± 0.69 c 6.30 ± 0.44 b
Texture Hardness 6.12 ± 0.59 b 6.20 ± 0.47 b 6.45 ± 1.05 a 6.11 ± 0.55 b 6.17 ± 0.63 b 6.25 ± 0.48 b 6.15 ± 0.26 b 6.48 ± 0.30 a 6.28 ± 0.37 b
Grittiness 1.85 ± 0.31 c 4.10 ± 0.18 b 5.17 ± 0.43 a 5.00 ± 0.28 a 4.45 ± 0.56 b 5.05 ± 0.29 a 4.15 ± 0.73 b 5.15 ± 0.44 a 4.22 ± 0.19 b
Melting 2.15 ± 0.49 b 2.55 ± 0.82 b 3.88 ± 0.70 a 2.40 ± 0.35 b 2.67 ± 0.49 b 2.39 ± 0.51 b 2.71 ± 0.22 b 3.95 ± 0.35 a 2.25 ± 0.88 b
Taste and aroma Herbal 1.21 ± 0.21 c 2.59 ± 0.39 b 3.62 ± 0.17 a 3.56 ± 0.93 a 2.16 ± 0.78 b 3.74 ± 0.89 a 2.38 ± 0.18 b 3.66 ± 0.49 a 2.30 ± 0.65 b
Astringency 1.12 ± 0.44 c 2.64 ± 0.76 b 3.87 ± 0.26 a 3.88 ± 0.73 a 2.45 ± 0.66 b 3.79 ± 0.17 a 2.54 ± 0.59 b 3.90 ± 0.83 a 2.21 ± 0.70 b
The values provided are the averages derived from three trials, with the standard deviation denoted as ±. In each row, values that are followed by distinct lowercase letters are statistically different from one another, with a significance level of less than 0.05.

3.3.5. Sensory analysis of chocolate samples

The sensory attributes of NDCh samples were analyzed and the results are presented in Table 8. The panelists were able to perceive the presence of microencapsulated herbal extract at high levels of fortification. Accordingly, the samples containing 885 and 845 mg per 33 g of chocolate were found to have the highest brightness intensity (p < 0.05). The intensity of the brown color was also significantly affected by the fortification process, with the control sample having the highest intensity (p < 0.05), which aligns with the color analysis results (Table 4). The intensity of sandiness or grittiness in NDCh samples significantly increased with the amount of microcapsules, consistent with findings reported by Lončarević et al. (2019) who worked on fortifying white chocolate with green tea extract microcapsules. Similarly, as the amount of microcapsules increased, so did the herbal intensity. Finally, the astringency intensity in NDCh sampels was significantly higher than that of the control sample, especially in samples with the highest amount of balm extract. This is in line with Sik et al. (2021), who reported that the astringency of dark chocolate fortified with balm extract was significantly higher than that of the control sample. It can be concluded from Table 7 that the samples fortified with minimal amounts of balm and borage microcapsules, and maximal levels of saffron and rosa, received higher scores than even the control sample.

4. Conclusion

A dark chocolate was fortified by adding extracts of four medicinal herbs, which were double encapsulated using CH, AG, and MD. The fortified chocolate exhibited significantly higher hardness, brightness, and melting temperature, especially at the highest levels of fortification. The Casson’s viscosity and yield stress of the chocolate samples increased in parallel with the increase in AG concentration, which was attributed to the interaction of functional groups of gum with those of the other ingredients. The snap of the chocolate was improved by the addition of microcapsules, while some sensory attributes such as astringency were negatively influenced at the highest levels of concentration. This study presents an effective strategy for safeguarding the bioactive compounds found in medicinal herbs, while also offering a worthwhile delivery system for these health-enhancing compounds. The findings have the potential to significantly improve the mental and physical well-being of diverse communities by paving the way for the creation of a range of functional foods enriched with these protected bioactive compounds.

CRediT authorship contribution statement

Hamid Rajabi: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft. Samineh Sedaghati: Formal analysis, Methodology, Validation, Visualization, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This work was financially supported by the Ario Rad Mehr Torshiz Co.- Mirmohannay (Project number: RD2023-ICGAU-14077).

Data availability

Data will be made available on request.

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