Optimization of ultrasonic-assisted debittering of Ganoderma lucidum using response surface methodology, characterization, and evaluation of antioxidant activity

Fourier-transform infrared

The methodologies described in the references were modified to a limited extent (Cui, Siva & Lin, 2019; Hadian et al., 2023). Infrared spectroscopy (Nicolet 6700, Thermo Fisher Scientific, Waltham, MA, USA) was employed to obtain the infrared spectra of the samples at room temperature. The KBr was combined with 2-HP-β-CD, GLP, FDP and SDP in a 1:100 ratio for the purpose of tabletting. The wave number range was set at 400 to 4,000 cm⁻¹, with a scanning time of 32 s.

Nuclear magnetic resonance

A few minor alterations were made to the methodology outlined in the referenced article (Deshaware et al., 2018; Hadian et al., 2023). The nuclear magnetic resonance (NMR) experiments were conducted using a spectrometer (600 MHz, Bruker, Germany) with a proton frequency of 600.13 MHz and a five mm TXI probe at a temperature of 25 °C. Proton NMR spectra were obtained with a spectral width of 14,705.9 Hz, an acquisition time of 2.7525 s, and a relaxation delay of 1 s. Each sample was scanned eight times in total.

Moisture content

Moisture content was determined for both GLP, FDP and SDP samples using a moisture analyzer (Youke, Shanghai, China) at a temperature of 102 ±2 °C (Čulina et al., 2023).

Hygroscopicity

The study evaluated the moisture absorption of GLP, FDP and SDP using a modified version of the methodology described by (Čulina et al., 2023; Tonon, Brabet & Hubinger, 2008). Specifically, 1 g of powder was evenly distributed on a pre-weighed glass Petri dish and placed in a desiccator containing 100 mL of a saturated sodium chloride solution (74.3% humidity). Following a seven-day period, the powders were reweighed in order to ascertain their hygroscopicity, expressed as the quantity of water adsorbed per 100 g of powder (g/100 g). (2)${\displaystyle \text{Hygroscopicity}\left(g/100g\right)=\frac{m_7-m_0}{m_0}\times 100.}$ The variable m₇ represents the mass (in grams) of the powder after a storage period of seven days, while m₀ denotes the initial mass (in grams) of the powder prior to storage.

Solubility

The solubility of the MPs was determined using the method described in the reference (Čulina et al., 2023; Gawałek, 2022). Approximately 0.1 g of powder was added to 10 mL of water, and the resulting mixture was agitated (78-1, Jerrell, Jiangsu, China) at 700 r/min for 5 min and subsequently centrifuged at 4,000 r/min for 10 min. The resulting supernatant was collected, dried to a constant mass in a laboratory oven maintained at 55 °C, and used to calculate the percentage solubility using the provided equation. (3)${\displaystyle \text{Solubility}\left(\text{\%}\right)=\frac{m_1}{m_0}\times 100.}$ The variable m₁ represents the mass (in grams) obtained through desiccation of the liquid residue, while m₀ refers to the mass (in grams) of the substance utilised in the analysis.

Bulk density

The bulk densities of the MPs were determined using the method described in the referenced source (Čulina et al., 2023; Rodríguez-Cortina, Rodríguez-Cortina & Hernández-Carrión, 2022). In order to ensure uniform particle dispersion, the powders were transferred into a test tube and subjected to a 60 s vibration cycle on a vortex vibrator. The volume of powder was determined by positioning the tube on a stable surface, and the ratio of powder mass to its corresponding volume in millilitres (g/mL) was calculated to obtain the respective bulk densities.

Ferric reducing antioxidant power assay

The capacity of the sample to reduce the ferric tripyridyltriazine complex was evaluated using a slightly modified experimental method (Marčetić et al., 2022; Wang et al., 2023). Absorbance measurements were conducted at a wavelength of 590 nm. A calibration curve was constructed utilizing a standard FeSO₄ solution spanning a range of 1 to 5 µmol/mL, with α-Trolox employed as the positive control. The results are reported in µmol FeSO₄/mL.

Hydroxyl radical scavenging assay

Hydroxyl radicals are observed in the fenton reaction when it occurs in conjunction with reduced transition metal ions, such as Fe²⁺, and hydrogen peroxide (H₂O₂). A slightly modified version of the previously reported method (Xie et al., 2020) was employed to obtain data at 510 nm, with α-Trolox serving as the positive control. The results are presented as clearance expressed as a percentage.

DPPH radical scavenging capacity assay

The DPPH radical scavenging capabilities were evaluated using a previously outlined method, with certain modifications incorporated (Jeon et al., 2022). Absorbance measurements were taken at 517 nm with the objective of establishing a calibration curve, using Trolox (5–25 µg/mL) as the standard. The positive control was α-trolox. A reduction in the absorbance within the reaction mixture indicated an enhanced capacity for the scavenging of free radicals. The results are presented as a percentage clearance.

Model fitting

The statistical model demonstrated high accuracy with no significant lack of fit (p ≤ 0.05) for all variables listed in Table 3. A higher coeffcient of determination (R²) value indicates better alignment between the model and experimental data, while a lower R² value suggests insufficient explanation of behavioural variations (Li & Chiang, 2012; Mehmood et al., 2018). Our study demonstrated that the quadratic polynomial model accurately describes 2-HP-β-CD concentration (A), temperature (B), and host-guest ratio (C). The significance level of coefficients within this model was determined using ANOVA testing. Larger F-values and smaller p-values indicate highly significant effects on response variables (Mehmood, 2015; Mehmood et al., 2019).

The R² and the adjusted coefficient of determination (R²_adj) were both approximately equal to 1, indicating a high accuracy in predicting bitterness values, and a strong agreement between experimental and predicted values. Additionally, it is important to note that the coefficient of variation (CV) remained below 10%, indicating excellent precision and reproducibility within the model’s performance. The results indicate that the mathematical model accurately calculated bitterness values. Based on the F value, the bitterness value is influenced by the test conditions in the following order: A > C> B. The bitter taste of the liquid is primarily determined by variables A and C, which have significant linear (p < 0.0001), quadratic (p < 0.0001), and interactive (p < 0.05) effects. These results are summarized in Table 3. The quadratic model expressions below can be used to predict the bitterness value response. (4)${\displaystyle Y_{\mathrm{(bitternessvalue)}}=1.67-0.31A+0.016B-0.20C-2.500E-003AB+0.098AC-3.750E-003BC+0.25A^2-5.25E-003B^2+0.071C^2.}$

Response surface analysis

The study employed surface response plots, created using a quadratic polynomial model, to analyze the effect of independent variables on the dependent variable (Bukhari et al., 2022; Li & Chiang, 2012). Figures 1A, 1B demonstrates that the bitterness value initially decreased and then increased with the progressive increase in 2-HP-β-CD concentration. One potential explanation for this phenomenon is that as the concentration of 2-HP-β-CD increases, there is a greater probability of collisions between bitter molecules and 2-HP-β-CD molecules, which may result in a reduction in perceived bitterness. Nevertheless, a high concentration of the 2-HP-β-CD solution may result in intermolecular adhesion due to its physicochemical properties (Yinghui, 2021). This can result in a reduction in the proportion of 2-HP-β-CD molecules that encapsulate bitter molecules, which subsequently increases bitterness. The application of ultrasonication has been demonstrated to effectively reduce the intermolecular adhesion between 2-HP-β-CD molecules, thereby promoting homogeneity and enhancing the formation rate of ICs 48 (Mongenot, Charrier & Chalier, 2000). As illustrated in Figs. 1B, 1C, the perception of bitterness is more pronounced when the ratio of 2-HP-β-CD is less than that of the bitter substance. The relatively large number of bitter molecules causing collisions is responsible for the reduced entry of these molecules into the 2-HP-β-CD cavity. Additionally, due to a high presence of molecules in the solution system, smaller molecules may adsorb onto larger 2-HP-β-CD molecules through hydrogen bonding and intermolecular forces (Baoqiang, 2021). As shown in Table 3 and Figs. 1A, 1C temperature did not significantly influence the reaction, suggesting that it can be effectively carried out within a range of 20−40 °C. To ensure successful formation of ICs, it is important to avoid excessively high reaction temperatures. This is because the process involves an exothermic reaction (Ning, 2015), and as the temperature increases, molecular motion accelerates, reducing the collision between guest and host molecules and hindering encapsulation.

Fourier Transform Infrared Spectroscopy analysis

FTIR analysis is a commonly used technique in chemistry to investigate the chemical structure and interactions of compounds (Cui, Siva & Lin, 2019). The data obtained from FTIR can provide valuable information for establishing the formation of inclusion complexes based on the shift, appearance, or disappearance of characteristic peaks (Hadian et al., 2023). Figure 3 displays the FTIR spectra of 2-HP-β-CD, GLP, FDP and SDP in the wavelength range of 400–4,000 cm⁻¹. The absorption peaks at 3,385 cm⁻¹ and 2,926 cm⁻¹ correspond to O-H and C-H bond stretching vibrations, respectively (Liu et al., 2022b; Venuti et al., 2019). The peak at 1,644 cm⁻¹ indicates the stretching vibration of the C =O bond (Venuti et al., 2019). Additionally, symmetrical and asymmetrical stretching vibrations of C − O − C bonds were observed at 1,157 cm⁻¹ and 1,028 cm⁻¹, respectively (Liu et al., 2022a). Furthermore, the presence of an α-glycosidic linkage within the structure of 2-HP-β-CD was confirmed by a distinct band at 851 cm⁻¹ (Sun et al., 2022). FTIR spectrum of HP-β-CD agreed well with the literature results (Imam et al., 2020; Liu et al., 2022a; Venuti et al., 2019). The ICs spectrum was similar to that of β-CD, and compared to that of β-CD, the intensity peaks in the spectrum of the inclusion complex were decreased and shifted, which may be evidence of the successful formation of the inclusion complex.

Proton nuclear magnetic resonance spectra analysis

Nuclear magnetic resonance (NMR) spectroscopy is commonly used to analyze chemical structures and investigate interactions between 2-HP-β-CD and guest molecules in ICs (Celebioglu & Uyar, 2020; Hadian et al., 2023). By observing changes in chemical shift (Δδ) using ¹D NMR, the incorporation of the bitter substance into the cavity of 2-HP-β-CD can be confirmed (Deshaware et al., 2018). This can be calculated as Δδ = δ_(complex) -δ_(free). The results are presented in Table 5. The 2-HP-β-CD spectrum exhibited several prominent peaks ranging from 3.37 to 4.82 ppm. H1 (δ = 4.82 ppm), H2 (δ = 3.41 ppm), and H4 (δ = 3.37 ppm) were located on the outer region of the 2-HP-β-CD cavity, while H3 (δ = 3.74 ppm) and H5 (δ = 3.48 ppm) were inner protons within the 2-HP-β-CD cavity. These protons are crucial in investigating the interaction between guests and 2-HP-β-CD. H6 (δ = 3.52 ppm) is positioned along the edge of the cavity. The protons inside the 2-HP-β-CD (H3, H5 and H6) have greater mobility than those on its surface (H1, H2 and H4) when exposed to bitter compounds, as shown by their Δδ values. The H3, H5, and H6 signals exhibited a downfield shift of 0.04, 0.03, and 0.02 for FDP and 0.05, 0.01, and 0.02 for SDP, respectively. The change observed was consistent with the literature (Deshaware et al., 2018; Hadian et al., 2023). The shift in proton signals in the cavity suggests the formation of a partial IC.

Moisture content

The moisture level of the dried product is critical for its effectiveness, quality, range of applications, technological suitability, and stability during storage (Rattes & Oliveira, 2007). Klaypradit and Fredes’ research (Fredes et al., 2018; Klaypradit & Huang, 2008), suggests that dry food products with moisture contents between 3% and 10% exhibit excellent stability during storage. The results in Table 6 show that the moisture content of the microcapsules produced through SD and FD fell below the specified limits, indicating an extended shelf-life for these microcapsules. However, it is important to note that the moisture content was lower in the SDP (2.91 ± 0.12%) compared to the FDP (8.40 ± 0.75%). This finding is consistent with previous studies by Rezende, Nogueira & Narain (2018) and Rodríguez-Cortina, Rodríguez-Cortina & Hernández-Carrión (2022), which also reported lower moisture levels in samples subjected to SD compared to FD. Furthermore, the lower moisture levels in SD may be attributed to the impact of higher temperatures during the process.

Bulk density

The characteristics of powders, including their packaging, transportation, storage, flow, and shelf-life stability, can be influenced by their bulk and tapped densities (Yaman et al., 2023). It is generally unfavorable to have low bulk density due to the increased package volume and air entrapment between particles, which can cause oxidation and compromise storage stability (Čulina et al., 2023; Goula & Adamopoulos, 2004). Table 6 shows that the bulk densities of SDP and FDP were 0.26 ± 0.004 g/mL and 0.12 ± 0.001 g/mL, respectively. There are significant differences in the existence of the two (p < 0.05). The study’s findings are consistent with those reported by Yaman et al. (2023). They demonstrated that the bulk densities of FDP (0.19 ± 0.001 g/mL) were significantly lower than those of SDP (0.29 ± 0.001 g/mL). Franceschinis et al. (2013) found that the pore size distribution of microcapsules is affected by the structural disparity between SD and FD, with FD preserving the crystal size and structure while SD may result in wall material degradation, leading to a more compacted product.

Solubility

The solubility of the powdered product in water is affected by several factors, with particular emphasis on the feed composition and particle size (Rezende, Nogueira & Narain, 2018). There was no significant difference in solubility between the MPs prepared by the two methods (p > 0.05) (Table 6). Due to the small particle size and the addition of highly soluble encapsulating agents, the sample presented a high average solubility, indicating a high capacity of the powder to remain homogeneous with water (Valente et al., 2019). This is because smaller particle sizes provide a larger surface area for hydration or facilitate better particle dispersion (Valente et al., 2019). The 2-HP-β-CD encapsulation system is a popular choice for improving solubility studies of substances that are difficult to dissolve due to its high solubility (Venuti et al., 2019). Above characteristics make the powder suitable for various applications, including beverages, dairy products, and instant consumables.

Hygroscopicity

According to research findings Rodríguez-Cortina, Rodríguez-Cortina & Hernández-Carrión (2022), MPs are considered highly moisture-absorbent when the hygroscopicity value exceeds 20%. Excessive levels of hygroscopy can cause powders to become sticky, compromising their long-term shelf stability. It is important to maintain appropriate levels of hygroscopicity to ensure product quality. In this study, we were able to decrease the hygroscopicity of the MPs that was prepared, resulting in a noteworthy enhancement of its shelf-life and storage stability. As shown in Table 6, the hygroscopicity range was measured to be between 7.33 g/100 g and 8.33 g/100 g. These results demonstrate that the MPs has superior hygroscopic properties compared to GLP (37.00 ±  1.00 g/100 g) (p < 0.05). This distinction can be attributed to several factors, including composition, type, carrier concentration, and microcapsule size. These factors collectively influence the overall level of moisture absorption (Tontul & Topuz, 2017).