Physicochemical Properties of Betacyclodextrin-Assisted Extracts of Green Rooibos (Aspalathus linearis)

Abstract

Betacyclodextrin (β-CD)-assisted extracts of green rooibos displayed elevated polyphenolic content and antioxidant activity compared with aqueous extracts. This study aimed to analyse the physicochemical properties of aqueous green rooibos and 15 Mm β-CD at 40 °C for 60 min. Sorption isotherms and colour (L*a*b*C*) were evaluated. Thermogravimetric analysis and Fourier transform infrared were conducted to verify encapsulation. Sorption isotherm studies revealed that β-CD reduced water uptake, resulting in a reduction in the monolayer value of GRE (7.90) to 6.40 for β-GRE. Betacyclodextrin contributed to increased lightness (L*) and decreased redness (a*) of green rooibos. However, storing extracts at varied water activity resulted in a reduction in L* and chroma (C*), with a higher reduction observed for GRE than β-GRE. Thermogravimetric analysis revealed that GRE degraded at 180 °C, followed by β-GRE at 260 °C and β-CD at 340–375 °C. Betacyclodextrin increased the thermal stability of green rooibos; as a result, β-GRE displayed a superposition of GRE and β-CD in its thermogram, confirming the formation of inclusion complexes. Fourier transform infrared spectra indicated the disappearance or shifting of characteristic peaks, with the formation of hydrogen bonds between GRE and β-CD at the 1255 cm⁻¹ band depicting C–O stretching of carboxylic acid.

1. Introduction

Recent trends in alleviating toxic compounds generated in foods during processing and storage focus on the utilisation of plant extracts. This fulfils the current trend in using natural ingredients as an alternative to synthetic additives [1]. Moreover, these polyphenolic-rich plant extracts are selected as food ingredients because of their known health-promoting properties. Aspalathus linearis infusions have received great attention in recent years because of their bio-functional properties such as antioxidant [2], anti-inflammatory [3], anti-carcinogenic [4], antimicrobial, anti-obesity, and hypoglycaemic [5] activities with the action of polyphenols responsible for the above-mentioned properties [6]. Green rooibos is of interest in the present study since it contains individual polyphenols that have been proven to inhibit non-enzymatic browning reactions in food and model systems.

For instance, quercetin and catechin at 50 µg.mL⁻¹ exhibited an inhibitory capacity of 40 and 36% against glycation of glucose-bovine serum albumin compared with 17% of aminoguadine [7]. Chlorogenic acid and epicatechin reduced arylamide in an asparagine–fructose model system and biscuit. In addition, 0.01 M of chlorogenic acid and epicatechin reduced the formation of furans in a glucose–glycine model system [1]. These polyphenols were found to exert their inhibitory capacities via forming adducts with NEB reactants or their intermediate products. Similar observations were made by authors who studied the anti-glycative effect of plant extracts, proving that these plant extracts exhibited considerably better anti-glycation than the identified individual polyphenols contained in them, attributing the superior inhibitory effect to the synergy between the different polyphenols [8]. Therefore, plant extracts and, in particular, green rooibos would be ideal as functional ingredients in food production. However, the instability of polyphenols during heat processing and storage may pose a challenge to using native plant extracts [9]. Consequently, the development of encapsulation methods and delivery systems containing specific compounds can improve some physicochemical properties of these extracts in food products. Encapsulation of sensitive compounds provides improved stability during processing and in the final product by preventing reactions with other components in food products such as oxygen or water [10]. The release of microparticle content at controlled rates can be triggered by the shearing, solubilisation, heating, pH, or enzyme activity of these agents, which are naturally present in certain plants, ensuring their stability. Common encapsulation material includes carbohydrates, protein and lipid polymers such as maltodextrin, inulin [11], soy protein isolates, sodium alginate, and cyclodextrins, to mention a few. The choice of polymer is crucial as it affects how the active compound is released. For instance, ref. Hidalgo et al. [12] reported elevated furosine levels in soy protein isolates applied to encapsulate beetroot pomace due to thermal treatments. Similarly, maltodextrin reacts with glycine to form browning compounds [13]. Grape skin phenolics encapsulated in sodium alginate beads interacted with alginate, resulting in reduced bioactivity. Therefore, the encapsulating polymer should not participate in any chemical reaction during processing or storage. Cyclodextrin were the obvious choice since they were proven not to participate in NEB reactions [13]. Cyclodextrins (CDs) are made up of cyclic oligosaccharides consisting of a number of α (1→4) linked D-glucose subunits. The most common forms, named alpha (α), beta (β), and lambda (γ-CDs), are composed of six, seven, and eight glucose units, respectively [10]. These molecules are widely used in the food, pharmaceutical, and chemical industries for their ability to form host–guest inclusion complexes with a wide range of bioactive compounds [10,14]. This results in the modification of the physicochemical properties of the encapsulated compound, leading to improved rheological and structural properties such as gelling, viscosity, solubility, and stability, as reviewed by [15]. Maraulo et al. [16] proved that β-CD enhanced the physical properties of olive pomace extracts via improved heat stability and reduced hydroscopicity, in addition to increased antioxidant activity.

Therefore, the aim of this study was to determine the impact of β-CD on the physicochemical properties of green rooibos extracts by determining the moisture content (MC) via sorption isotherms, water activity (a_w), colour (L*a*b* and C*), thermogravimetry analysis (TGA), and Fourier transform infrared spectroscopy (FTIR).

2. Materials and Methods

2.1. Green Rooibos and Reagents

Dry green rooibos was obtained from a major local producer (Rooibos Ltd., Clanwilliam, South Africa). Beta-cyclodextrin (β-CD) was purchased from Industrial Analytical (Kyalami, South Africa). Lithium chloride (LiCl), potassium acetate (KCOOCH₃•5H₂O), magnesium chloride (MgCl₂•6H₂O), potassium carbonate (K₂CO₃•2H₂O), and sodium chloride (NaCl) were purchased from Merck (Modderfontein, South Africa). The chemicals used in this study were of analytical grade, and chemical reagents were prepared according to standard analytical procedures. The water used was purified with the Milli-Q water purification system (Millipore, Microsep, Bellville, South Africa).

2.2. Solid–Liquid Extraction of Green Rooibos

Green rooibos, as received from Clanwilliam, was coarsely milled (Fritsch) using a sieve with a porosity of 0.2 mm. The extraction of the green rooibos plant was performed based on the method of [17,18] with slight modifications. Green rooibos plant and 0 and 15 mM β-CD aqueous solutions at a 1:10 (w/v) ratio of 10 g sample and 100 mL water/ β-CD solution were blended (Waring™) for two minutes at low power, followed by heating the mixture at 40 °C for 60 min. The extracts were cooled immediately and centrifuged at 5000 rpm for 15 min at 4 °C. The supernatant was transferred into pre-weighed aluminium dishes and the mass was taken, followed by freeze-drying using an SP (VirTis) Ultra 35L Pilot Lyophilizer (Pennsylvania, USA). The resulting powders from β-CD-assisted (β-GRE) and native (GRE) extracts were weighed to calculate the soluble constituent yield, followed by storage in an air-tight container at −20 °C until further analysis.

2.3. Moisture Content (MC), Water Activity (a_w), and Sorption Isotherms

The MC and a_w was determined using the gravimetric method described by [11]. A 2 g sample of green rooibos extract (β-GRE and GRE) was heated at 100 °C for 60 min using a Denver Instrument Infrared Moisture Analyser (Colarado, USA). The a_w was measured at 25 °C using a Novasina LabMaster-a_w meter (Lachen, Switzerland).

The water sorption isotherms were determined based on a gravimetric method described by [16]. The method was based on the use of saturated salt solutions to maintain a fixed relative humidity (RH). Five salts were chosen to achieve RH values in the range of 10–85%. Freeze-dried green rooibos extracts (β-GRE and GRE) were weighed (0.2 g) into 5 mL glass vials with rubber stopper caps and placed into desiccators containing saturated solutions of the following salts: LiCl, KCOOCH₃•5H₂O, MgCl₂•6H₂O, K₂CO₃•2H₂O, and NaCl, achieving water activities (a_w) values of 0.11, 0.22, 0.33, 0.43, and 0.75 at 25 ± 1 °C, respectively. The saturated salt solution water activity values (a_w) were confirmed using a Novasina LabMaster-a_w meter (Lachen, Switzerland). The samples in the desiccators were kept in an oven at 25 ± 1 °C for equilibration. After reaching the equilibrium condition (difference < 0.0005 g), the moisture content of the samples was determined by drying the samples in vials in a vacuum oven (735 mmHg) at 70 °C until constant weight. Analysis was conducted in triplicate, and the equilibrium moisture content of the samples was reported as g/100 g dry solids. The moisture content as a function of a_w was plotted for each sample, and the curves obtained were fitted with the Guggenheim–Anderson–de Boer (GAB) model.

Monolayer values were computed from the moisture sorption data by using mathematical modelling. The GAB model, as described by the following equation, is considered the best fitting model for a host of foodstuffs and reportedly provides accurate fittings over a wide range of water activity up to at least 0.9 and better evaluates the amount of water tightly bound to primary adsorption sites.

The GAB model is described mathematically by (Equation (1)):

m/m₀ = a_wGK/[(1 ― a_wK)(1 ― a_wK + a_wGK)]

(1)

where m and m₀, respectively, are the moisture content and the content of water tightly bound to primary adsorption sites as a monomolecular layer, a_w is the water activity, and G and K are constants related to the energies of interaction between the first and distant sorbed molecules at the individual sorption sites.

To calculate the monolayer values from moisture adsorption data, the GAB equation (Equation (1)) was transformed into the quadratic form (Equation (2))

a_w/m = Aa_w² + Ba_w² + C

(2)

to which the least-squares multiple linear regression method was applied to obtain the following regression coefficients in Equation (3) (A), Equation (4) (B), and Equation (5) (C).

A = (k/m₀)[1/G) ― 1]

(3)

B = (1/m₀)[1 ― (2G)]

(4)

C = 1/m₀GK

(5)

as well as the square of the multiple correlation coefficient (r²). The monolayer values were obtained from these coefficients according to (Equation (6)).

$$M_0={\displaystyle \frac{-b\pm \sqrt{b^2-4ac}}{2a}}$$

(6)

2.4. Colour Evaluation

The colour was determined following a modified method of [16] by measuring CIELab, L* (brightness, 100 = white, 0 = black), a* (+red; −green), and b* (+yellow; −blue) parameters by means of a spectrophotometer (CM-5, Konika Minolta, Japan) measuring the colour spectra using a D65 day light source, large viewing area, and the observer at a 10° angle. The colour measurement of freeze-dried green rooibos extracts (β-GRE and GRE) was performed on fresh powders and after exposure to different relative humidities using a portable colorimeter with an integrating sphere (model CM-700d, Konica Minolta, Japan). The a* and b* coordinates were used to calculate chroma (C*) using (Equation (7)).

$$C*=\sqrt{a{*}^2+b{*}^2}$$

(7)

where C* is the chroma parameter, a* and b* are the chromatic coordinates in CIELab colour space.

2.5. Thermogravimetric Analysis (TGA)

Thermogravimetric analysis (TGA) was performed using a Shimadzu DTG-60 instrument following the procedure described by [18] with slight modifications. Freeze-dried GRE and β-GRE (3–5 mg) were accurately weighed into open aluminium cells and heated from 25 to 800 °C at a heating rate of 20 °C/min, and a nitrogen gas purge rate of 35 mL.min⁻¹. The mass loss and heat flow in the sample were recorded as a function of temperature with reference to an empty pan.

2.6. Fourier Transform Infrared (FT-IR)

Fourier transform infrared spectroscopy analysis was performed to investigate possible β-CD-green rooibos interactions based on the modified method of [19]. The analysis was performed using a Perkin Elmer Fourier transform infrared spectroscope (FT-IR) equipped with a universal attenuated total reflectance (UATR) polarisation accessory for spectra. Prior to sample analysis, a background spectrum was collected, and finely ground (mortar and pestle) GRE and β-GRE were placed directly covering the surface of the ATR crystal. All spectra were acquired by co-addition of 32 scans at a resolution of 4 cm⁻¹ in the range of 400–4000 cm⁻¹. The UATR crystal was carefully cleaned with acetone to remove any residual sample prior to analysis.

2.7. Statistical Analysis

Statistical analysis was performed using SPSS 27.0 for Windows^®. Analysis of variance (ANOVA) was used to establish the significance of each dependent factor. Descriptive statistical analyses were used to determine the triplicates’ means and standard deviations (n = 3). Duncan’s multiple range tests were used to determine significant differences among means. The level of confidence required for significance was selected at 95%.

3. Results and Discussions

3.1. Moisture Content (MC) and Water Activity (a_w) of Green Rooibos Extracts

The MC and a_w of green rooibos extracts (GRE and β-GRE) are shown in

Table 1. Physicochemical properties and betacyclodextrin-encapsulated green rooibos extracts.

Sample	aw	MC	L*	a*	b*
GRE	0.181 ± 0.00 b	3.29 ± 0.18 a	47.95 ± 1.43 a	16.12 ± 0.51 b	23.01 ± 0.59 a
β-GRE	0.111 ± 0.01 a	2.45 ± 0.12 a	53.70 ± 0.80 b	15.96 ± 0.51 a	24.46 ± 1.01 a

Data presented as mean ± standard deviation (n = 3) of a_w—water activity, MC—moisture content, L*—lightness (0–100), a*—+red/−green, b*—+yellow/−blue of GRE—green rooibos extracts and β-GRE—betacyclodextrin-encapsulated green rooibos extract. ANOVA and t-tests were performed. ^ab Means with different letter superscripts in the same column denote significant differences (p < 0.05).

. The MC of 3.29% that was reported for GRE was not significantly different (p > 0.05) from the MC of 2.45% for β-GRE. The MC of β-GRE was in the range reported for aqueous extracts of green rooibos of 2.14% by [11] and 2.4% by [18]. Moreover, Miller et al. (2018) [18] also found no significant (p > 0.05) differences between the MCs of green rooibos vs. 1:1 green rooibos-inulin and green rooibos-maltodextrin encapsulates. Meanwhile, Human et al. (2020) [11] reported significant differences. Betacyclodextrin-encapsulated extracts are known to inhibit moisture uptake; however, in this case, our results contradict this notion. Therefore, β-CD’s inhibition of moisture uptake is better demonstrated via moisture sorption isotherms than a single point measurement of MC, as reported in this study.

Regarding water activity, the a_w of β-GRE at 0.111 was significantly lower (p < 0.05) than that of GRE at 0.181. Our results are in accordance with those reported by Miller et al. [18] but opposes the findings of Human et al. [11], where maltodextrin and chitosan encapsulates exhibited higher a_w values compared to aqueous green rooibos powdered extract. In solution, the nonpolar β-CD cavity is occupied by water molecules, because of energetically unfavoured interactions, and when another guest molecule is present, it readily substitutes the water as a guest molecule. The lower a_w exhibited by β-GRE is attributed to the interaction and binding of water by the hydrophilic sites of β-CD, resulting in reduced free water [20]. This contributes to the good storage stability of these powders when stored under dry conditions. This is an important factor considering the instability of aspalathin during storage [11,18].

3.2. Moisture Content as a Function of Water Activity via Sorption Isotherms

In addition, the moisture content at various relative humidities (water activity) was examined via sorption isotherms (

Table 2. Moisture adsorption data (g.100 g⁻¹ dry matter) of aqueous and betacyclodextrin-encapsulated green rooibos extracts at 25° C at varied water activities (a_w).

Sample	Sorption Isotherm					BET
	0.11	0.22	0.30	0.4	0.75	A	B	C	m0	aw
GRE	5.10 ± 0.20 a	6.00 ± 0.17 a	7.57 ± 1.06 b	10.98± 0.82 b	18.14 ± 0.62 b	−0.0910	−0.0222	0.0140	7.93 ± 0.11 b	0.289 ± 1.21 b
β-GRE	5.09 ± 0.01 a	5.82 ± 0.10 a	7.18 ± 0.42 a	8.90 ± 0.24 a	14.96 ± 1.24 a	−0.1168	−0.0144	0.0092	6.40 ± 0.52 a	0.226 ± 0.89 a

Data presented as mean ± standard deviation (n = 3) of sorption isotherm (moisture content as a function of relative humidity) of GRE—green rooibos extract and β-GRE—betacyclodextrin-encapsulated green rooibos extract. ANOVA and Duncan’s Post Hoc test. ^ab Means with different letter superscripts in the same column denote significant differences (p < 0.05). Guggenheim–Anderson–de Boer (GAB) model monolayer.

). The water sorption isotherms for GRE and β-GRE exhibited a Type II behaviour, which is synonymous with most food systems (Figure 1). The moisture content of the green rooibos extracts increased with increasing water activity, with both GRE and β-GRE exhibiting similar MCs (p > 0.05) at a lower a_w of 0.11 to 0.33. At an a_w of 0.44, GRE absorbed a higher quantity of moisture compared with β-GRE (p < 0.05), as can be seen in Figure 2. The GAB model exhibited an R² of 0.808 for GRE and 0.9611 for β-GRE, illustrating a good fit for both extract isotherms; thus, we can conclude by obtaining the characteristic parameters for a physical interpretation of the data (C, M₀, and K). The monolayer value (M₀) of the native GRE was 7.93 and decreased to 6.4 g H₂O. 100 g⁻¹ d.b. for encapsulated β-GRE. Maraulo et al. [16] also reported a 31% reduction in M_O of β-CD-olive pomace. The monolayer value is a critical parameter in the field of food science, particularly for food powders. A reduced monolayer reflects a reduction in available water-binding sites; in this instance, this could be attributed to complexation that resulted in fewer sites for water binding, thus improving the stability, protection, and shelf-life of the encapsulated bioactive compounds. The green rooibos extract water sorption behaviour determines the ideal storage conditions for dehydrated systems.

3.3. Colour (L*a*b*) of Green Rooibos Extracts

Regarding colour, the immediate colour of the GRE and β-GRE powders presented as L*, a*, and b* values are shown in Table 1. The β-GRE powder was lighter and less red (p < 0.05) compared with that of GRE, while the yellowness was similar (p > 0.05). The effect of encapsulation on the colour of green rooibos extracts was investigated by [18]. They reported an increase in L* and a decrease in the a* and b* of inulin and maltodextrin encapsulates, with the increase more pronounced on the latter encapsulant. Regarding the application of β-CD as an encapsulant, various authors reported different outcomes in terms of the colours of extracts. Tutunchi et al. [19] found that 1 and 5% w/v of aqueous β-CD had no significant effect on the lightness of red beet extract. However, using ethanol as a solvent at 1% β-CD increased the L* value, while no significant difference (p > 0.05) resulted when the concentration of β-CD in ethanol was increased to 5%. The latter concentration was too high, resulting in decreased solubility of β-CD. Besides the immediate colour analysis, in the present study, we also investigated the stability of colour as a function of water activity.

3.4. Colour (L*, a*, b*, and C*) of Green Rooibos Extracts as a Function of Water Activity

The colour of GRE and β-GRE fluctuated during the three weeks of storage at an a_w of 0.11 to 0.4, followed by a drastic decrease at 0.75 (

Table 3. The effect of relative humidity on the colour of aqueous and betacyclodextrin-encapsulated green rooibos extracts.

Sample	RH	L*	C*
GRE	0.11	45.57 ± 0.20 d	29.75 ± 0.53 f
GRE	0.22	32.69 ± 3.02 c	17.64 ±1.53 b
GRE	0.30	44.80 ± 0.35 d	28.03 ± 0.43 e
GRE	0.40	47.79 ± 0.11 e	23.23 ± 0.25 c
GRE	0.75	07.17 ± 0.35 a	06.91 ± 0.56 a
β-GRE	0.11	53.08 ± 0.43 f	28.03 ± 0.57 e
β-GRE	0.22	54.27 ± 0.48 f	30.22 ± 0.16 f
β-GRE	0.30	48.52 ± 0.36 e	30.55 ± 0.33 f
β-GRE	0.40	52.82 ± 0.63 f	31.80 ± 0.46 g
β-GRE	0.75	28.39 ± 0.83 b	26.37 ± 0.86 d

Data presented as mean ± standard deviation (n = 3) of a_w—water activity, MC—moisture content, L*—lightness (0–100) and C*—chroma of GRE—green rooibos extract and β-GRE—betacyclodextrin-encapsulated green rooibos extract. ANOVA and Duncan’s Post Hoc test. ^a–g Means with different letter superscripts in the same column denote significant differences (p < 0.05).

). An 80% decrease in the L* value was reported for GRE, while 45% was observed for β-GRE when comparing the initial colour and the day 21 a_w of 0.75 a_w (Figure 3). Similarly, the chroma (C*) of GRE decreased by 77%, whilst that of β-GRE remained almost constant at a 6% reduction, denoting stability imparted by encapsulation (Table 3). Maraulo et al. [16] also reported higher L* values of β-CD-assisted extracts of olive pomace compared with aqueous extracts. Moreover, they also reported the effect of varying a_w on the colour of extracts. They found that the darkening of aqueous extracts was increased as the a_w increased and that β-CD-assisted extracts maintained the lightness regardless of changes in a_w. This further proves the impact of β-CD encapsulation on plant extract colour stability during storage. Plant extracts are known to impart natural pigments into the food they are added to. The colour of plant extracts is important as it determines the suitability of their use as a food additive [12]. The colour of green rooibos extract is important, particularly for its intended use in another part of this study. The main aim was to apply β-GRE to inhibit browning in canned apples. The deep red colour of the extract may have an effect that contributes to visual browning; therefore, the lighter the colour of the extract with increased antioxidant activity, the better. Moreover, at high temperatures applied in processing, in this case, canning, GRE polyphenols might oxidise, resulting in browning [8,21]. Therefore β-CD encapsulated extracts are protected from direct exposure to heat. It is worth reiterating that in the studies by [2] and [16], β-CD-encapsulated extracts exhibited the highest antioxidant activity (AA) compared with their aqueous counterpart. The lighter colour, as well as increased AA, renders them suitable for application in inhibiting browning in canned apples.

3.5. Thermogravimetric Analysis (TGA)

Thermogravimetric analysis (TGA) was performed to confirm complex formation. This was achieved by determining thermal stabilities based on mass reduction as a function of increased temperature. The thermograms of GRE, β-GRE, and β-CD are illustrated in Figure 4. The thermal degradation process of pure β-CD underwent two major stages, with peaks appearing at approximately between 100 and 120 °C and 340 and 375 °C. The weight loss of 11.0% occurred in the first stage, a phenomenon associated with the evaporation of water absorbed on the surface of β-CD, as well as the water contained in the cavities. Li et al. [22] and Koteswara et al. [23] reported initial β-CD weight losses of 13% in the same region of 112 and 100 °C, respectively. Both these authors ascribed the weight loss to water evaporation from the β-CD cavities; Koteswara et al. [23] also mentioned the evaporation of water adsorbed on the surface of the β-CD molecule. In the second stage, further weight loss in β-CD of 67% was observed from 340 to 375 °C, which was due to the main thermal decomposition of β-CD. Once more, the main degradation of β-CD resulting in 76% weight loss was observed at 305 and 400 °C [22,23]. The result of this study lies within the ranges observed by these two authors.

Regarding the GRE samples, the initial sample weight was slightly higher, which can be linked to the hygroscopicity of the sample (based on visual observation). An initial weight loss of 2%, although not significant, was observed from regions as low as 40–120 °C. Miller et al. [18] reported similar results for aqueous extracts of green rooibos as a loss in surface moisture. The main degradation of GRE resulted in a 40% loss in weight and was seen from 180 °C, followed by a gradual decrease in weight as the temperature increased to 400 °C. This degradation pattern is synonymous with plant extract polyphenols, in particular, green rooibos. Li et al. [22] reported the degradation of Mulberry polyphenols at 138–600 °C, resulting in 59% mass loss. The second region of weight loss for green tea occurred at around 130 °C with 10–14% weight loss, which continued up to 900 °C.

The broad temperature range reported for crude plant extract degradation may be due to the versatile mixture of compounds, as the decomposition of resistant aromatic structures requires higher temperatures. The thermogram of β-GRE possessed characteristics of both GRE and β-CD; Li et al. [22] referred to this phenomenon as superposition. For instance, the initial phases resembled that of β-CD, where approximately 4% weight loss was seen at 60–100 °C, albeit lower for the former. The structural differences between β-GRE and β-CD lie in the fact that the latter’s truncated cavity was not occupied, therefore allowing more binding of water. The second-stage degradation for β-GRE was observed at 260 °C and resulted in a weight loss of 46%. Miller et al. [18] reported that green rooibos encapsulated with inulin and maltodextrin started to degrade at 190 and 220 °C, respectively. These results show that the thermal stability of GRE was improved by interaction with β-CD, thus confirming that complexation took place. Moreover, the thermal stability was greater than that found by other authors involved in green rooibos encapsulation.

3.6. Fourier Transforms Infrared (FT-IR)

FT-IR analysis was performed to confirm encapsulation via vibrational deviations formed when the host and guest molecules interact. The absorption spectra of β-CD, GRE, and β-GRE in the 400–4000 cm⁻¹ regions are depicted in Figure 5. The GRE and β-CD spectra resemble those reported by [22,24]. All samples displayed peaks located at 1024–1070 cm⁻¹ associated with the phenolic C–O bond, 1606–1646 cm⁻¹ C=C stretching of aromatic rings, 2929–2939 cm⁻¹ C–H stretching, and the broad absorption band at 3267–3300 cm⁻¹ representing the stretching vibration mode of O–H bond associated with the poly hydroxy groups of β-CD and polyphenols in GRE, in particular, the flavonoids being the most abundant polyphenols known to exhibit a broad absorption band between 3200 and 3600 cm⁻¹. Pure GRE showed specific bands at 1030–1070, 1255, 1606, 2939, and 3267 cm⁻¹. Meanwhile, the β-CD spectra showed strong bands at 578, 1024, 1078, 1153, 1646, 2946, and 3274 cm⁻¹. The spectra of the β-CD and β-GRE samples overlapped at certain regions and showed certain spectral differences in comparison with the aqueous extract (GRE). Similarities between GRE and β-GRE were observed at 578, 1025, and 1154 cm⁻¹. When the β-GRE inclusion complex formed, most characteristic peaks of GRE and β-CD disappeared, reappeared, or shifted bands took place in the newly formed complex. For instance, the 1255 cm⁻¹ band depicting the C–O stretching of carboxylic acid disappeared following the formation of hydrogen bonds with β-CD. The GRE 1030–1070, 1606, and 2939 cm⁻¹ bands shifted to 1025, 1615, and 2939 cm⁻¹ in the β-GRE complex, respectively. Paczkowska et al. [25] observed similar structural changes when forming a rutin–β-CD complex. The above results indicate that successful encapsulation took place, and a new compound with new characteristics was formed.

4. Conclusions

In this study, the physicochemical properties of β-CD-assisted extracts of green rooibos were characterised in order to ascertain the formation of inclusion complexes. The TGA thermogram and FT-IR spectrum revealed that green rooibos β-CD complexes were formed. This was based on the improvement in heat stability, as well as the formation of a new molecule, which was a superposition of β-CD and green rooibos. For future studies, we recommend the evaluation of sorption isotherm studies in order to determine the behaviour of the encapsulated extracts under various a_w values as opposed to single-value a_w and MC. In addition to studying sorption isotherms and colour changes during storage, we recommend studying the changes in polyphenolic content and antioxidant activity a result of storage at various a_w values.