Insights into bacterial cellulose for adsorption and sustained-release mechanism of flavors

Abstract

The stabilities and sustained-release properties of citral are significant for foods. Herein, bacterial cellulose (BC) was innovatively reported for adsorption and sustained-release of citral via gas-phase adsorption technique, and the adsorption mechanism was disclosed. BC was prepared from tobacco stem waste extract (TSWE), and better adsorption capacity (124.98 mg/g) was obtained through response surface optimization. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Flourier transform Infrared Spectroscopy (FTIR), and Brunauer-Emmett-Teller (BET) were utilized to verify the successful adsorption. Thermo-gravimetry (TG) analysis showed that the release of citral was delayed. Temperature responsiveness indicated the release of citral was controlled by internal diffusion. Density functional theory (DFT) calculations indicated the interactions between BC and citral was mainly composed of van der Waals forces and hydrogen bonds. BC-Citral also exhibited excellent antibacterial capability. This work provided a new approach for constructing controlled-release materials of citral, which offered good application prospects in food industry.

Highlights

• •BC (Bacterial cellulose) was innovatively prepared from tobacco stem waste extract medium.
• •BC was firstly used to adsorb flavors by gas-phase adsorption technique.
• •BC-Citral could realize stable and slow release of citral at specific temperature.
• •Preliminary exploration was conducted on the adsorption and release mechanisms.

1. Introduction

Flavors play a crucial role in various industries and products, enhancing the sensory experience and overall appeal of food, beverages, cosmetics, and pharmaceuticals. Citral, one of the typical flavors, widely used in food additives due to a strong lemon aroma, which commonly found in plants and fruits such as lemon, citrus, and lemon grass (Fahad et al., 2018; Horn et al., 2012; Idrees et al., 2019). Citral has significant bactericidal effects on different bacteria and fungi, including gram-negative bacteria, gram-positive bacteria, and fungi (Mokarizadeh et al., 2017; Yoplac et al., 2021). In addition, the properties of anti-inflammatory, antioxidant activity and free radical scavenging were presented on the citral (Boukhatem et al., 2014; Fahad et al., 2018; Xu et al., 2022). However, citral is chemically unstable because of the C C bonds and aldehyde groups in the structure. This results in poor aroma stability of citral, which is prone to oxidation, cyclization, and rearrangement reactions. Losing fresh lemon flavor and accompanying by troublesome off-flavor, hurdle the application of citral (Chen et al., 2021; Kimura et al., 1983; Song et al., 2022; Ueno et al., 2004; Yin et al., 2021). Consequently, in recent years, studies have proposed encapsulation or adsorption of citral, that can weaken the impact of external adverse environmental factors on citral without changing its physical and chemical properties, so as to achieve the goals of reducing degradation rate and improving the bioavailability and stability of citral (Kim et al., 2019). Actually, a number of researchers had been studied the sustained and controlled release of citral. Han et al. (2023) developed the double cross-linked acrylic acid/bagasse cellulose porous hydrogels by using cold plasma technology instead of chemical initiators, which can realize the controlled release of citral. Feng et al. (2023) compared the effects of three different preparation methods (dispersion, ultrasonication and microfluidization) on the encapsulation of citral in nanostructured lipid carrier (NLC), which indicated that illustrated that the sustainable release of citral might be achieved by controlling the energy input of the preparation method. However, the method is subject to its high costs and high equipment requirements. Additionally, traditional encapsulation methods have the problems of preparation conditions, safety, and encapsulation quantities (Feng et al., 2023; Ma et al., 2020; Xu et al., 2022; Yang et al., 2022). Liu et al. (2023) compared the stabilizing effects of β-cyclodextrin-based Pickering emulsions loaded with lemon essential oil, which can achieve the regulated release of flavors in emulsions. Du et al. (2024) prepared the citral nanoemulsion by ultrasonic method, then the chitosan solution loaded with nanoemulsion was assembled on the gelatin film, and the uniform and smooth gelatin-chitosan bilayer film was successfully prepared, which had efficient and durable antibacterial activity, and realized the specific control of the release of citral through pH and gelatinase. Nevertheless, the method of Pickering emulsions has the disadvantage of limited application range and smaller loading amounts of flavors (Stasse et al., 2020). The adsorption method has various advantages such as simpler operation, larger adsorption capacity, and lower cost than above methods (Chen et al., 2009; Gao et al., 2021). Hence, using adsorption method to load citral is extremely considerable.

Bacterial cellulose is a water-soluble extracellular polysaccharide produced by bacterial metabolism, which is composed of glucose molecules β-1,4-glycosidic bonds polymerize to form a three-dimensional mesh through intramolecular and intermolecular hydrogen bonds, which possessed high tensile strength and elasticity, good biocompatibility and degradability. (Zhang et al., 2019; Liu, Sun, et al., 2024). Being a dietary fiber, BC is considered “generally recognized as safe” (GRAS) by the Food and Drug Administration (FDA), which indicates BC possesses manifold potentialities in food industries (Azeredo et al., 2019; Shi et al., 2014). For example, one of the uses of BC is the manufacturing of a Filipino traditional dessert with a smooth mouth feel that is called “nata de coco”, which is now quickly spreading worldwide in the form of a dessert used in milky tea or beverage (Klemm et al., 2018). Furthermore, BC is widely used as new materials in vegetarian meat and heat processed foods as well (Ullah et al., 2016). The production cost of BC is high, requiring a large amount of chemical reagents such as glucose, yeast extract, and peptone, which hinders the large-scale production of BC. Industrial waste and by-products from agriculture, processing industries, etc. mostly contain various valuable enzymes and biochemical components, which can become low-cost alternative carbon sources. Moreover, it contains protein, carbohydrates, and trace elements, which are also the main raw materials for producing BC. The recycling and reuse of agricultural or industrial waste to produce BC is a more sustainable and eco-friendly solution (Fathiyah et al., 2021; Liu et al., 2015; Mathivanan et al., 2024). Tobacco is widely planted across the world especially in China, which means that a large amount of tobacco waste (tobacco stem and discarded tobacco leaf) needs to be treated, which is harmful to the environment and is generated during the cultivation and manufacturing processes (Pasaribu et al., 2024; Shen et al., 2024). Most tobacco stems are burned or directly buried in traditional ways, which not only pollutes the ecological environment, increases treatment costs, but also wastes a huge amount of existing biomass resources (Ye et al., 2019). In recent years, more and more researchers have utilized the low-cost substrates include various waste biomass by-product such as industrial residues, fruit wastes, and agricultural waste as carbon sources of medium to obtain BC (Kumar et al., 2024; Singhania et al., 2022). For instance, Cheng et al. (Cheng et al., 2017) synthesized the BC by acetobacter xylinum via organic acid pre-hydrolysis liquor of agricultural corn stalk used as carbon source, which demonstrated that the combination detoxification treatment was efficient, and the highest BC production of 2.86 g/L was achieved under the optimized pretreatment and detoxification conditions. Lin's group (Lin et al., 2022) studied that sugarcane bagasse (SB) was hydrolyzed with sulfuric acid, and atmospheric cold plasma (ACP) was used to remove the toxic inhibitors. The detoxified SB hydrolysate was used as alternative nutrients for BC production. Wu and his group (Wu et al., 2021) obtained the BC by Chinese medicinal herb residues acid hydrolysate with gellan gum adding, which could cause an increasing yield of 47 %–59 %, and the gellan gum loading showed a little influence on the crystalline structure and thermal degradation behaviors of bacterial cellulose samples. However, though the above treatment methods can to some extent recycle waste, the use of chemical reagents such as sulfuric acid to acidify substrates in the pretreatment process not only possessed dangerous in operation, but also may cause environmental pollution.

BC was presently known as one of the slimmest fibers, featuring a porous structure, significant specific surface area, and numerous pores, predicting its exceptional adsorption capacity. Meanwhile, BC is also considered a new type of natural biological nanomaterial with excellent performance (Singhania et al., 2022; Ahmed et al., 2022; Ullah et al., 2016; Chen et al., 2009). The utilization of BC as a foundation for adsorption and the development of metamaterials represents a novel area of investigation (Badshah et al., 2018; Mohite & Patil, 2014). Zhao's group (Zhao et al., 2024) developed an innovative method involving in situ introduction of amide bonds and Schiff base reaction crosslinking to produce BC-bentonite@ Polyethyleneimine (BCB@PEI) multifunctional composite membranes for water treatment, demonstrating excellent adsorption performance. Barjasteh and his co-workers (Barjasteh et al., 2024) designed a polymeric composite based on a chitosan/bacterial cellulose (CS/BC) matrix filled with MIL-100(Fe) particles and utilized as an efficient adsorbent for removing dacarbazine (DTIC) from wastewater. Ha et al. studied the adsorption of bacterial cellulose on dyes, and the results showed that bacterial cellulose exhibited high adsorption capacity for cationic dyes such as methylene blue and malachite green (Ha et al., 2023). Arooj et al. reported that bacterial cellulose exhibits excellent adsorption performance for N₂, CO₂, O₂ (Arooj et al., 2023). And the unique three-dimensional network nanostructure makes BC widely used in the biomedical field and drug delivery field, such as being used as a wound dressing material and delivering various drugs (Badshah et al., 2018; Li et al., 2021; Liu, Liu, et al., 2024; Liu, Sun, et al., 2024; Liu, Wang, et al., 2024; Tangsatianpan et al., 2020; Villalva et al., 2024; Ye et al., 2024), which can prove that BC has an important role in sustained-release. The above studies indicate that BC is feasible as an adsorbent and the internal porous structures can provide good loading and controlled release environment (Wu et al., 2024; Blanco Parte et al., 2020; Kontturi et al., 2018). However, to the best of our knowledge, there are no relevant reports using tobacco stem waste extract as a substrate for producing BC and then utilizing it as absorbent for the absorption of flavors, and the adsorption mechanism as well as release mechanism were not definite.

The aiming of this article was to construct a BC-citral controlled release material by using gas-phase adsorption technology, and revealed the adsorption and release mechanisms between BC and citral. The BC prepared from TSWE medium was used as an adsorbent material to adsorb citral through gas-phase adsorption technique, and the optimal preparation conditions were obtained through response surface methodology. SEM, XRD, FTIR and BET was performed to verify the synthesis of BC-Citral. TG, release kinetics, and exothermic kinetics simulations were used to investigate the thermal stability and release characteristics of BC-citral. The DFT calculations was performed to explore the adsorption mechanism of citral on BC and its effect on sustained release performance. Moreover, the antibacterial performance of BC-Citral on E. coli and S. aureus were also studied. It provided a theoretical basis for the application of BC in the field of controlled and sustained release of flavors and fragrances, and provided valuable insights for the food industry.

2. Materials and methods

2.1. Materials

All the chemical reagents used in this study were obtained commercially and used without further purification. Strain Komagataeibacter xylinus AX-1, Escherichia coli (E. coli), and Staphylococcus aureu (S. aureus) were stored in our laboratory. Tobacco stem waste was collected from China Tobacco Hebei Industrial Co., Ltd.

2.2. Medium

The tobacco stem waste extract (TSWE) medium was made at 80 °C for 2.5 h with a solid-liquid ratio of 1:15. After cooling, the liquid was filtered and assembled with gauze. Then, the pH of liquid was adjusted to neutral. The medium was obtained by sterilizing the liquid at 115 °C for 20 min.

2.3. Preparation of BC and BC-Citral

The BC was prepared and purified according to the previous method (Suneetha et al., 2024). Firstly, the activated bacterial strains were inoculated into the seed culture medium, oscillated at 160 r/min and 30 °C for 18 h, and then transferred to the TSWE fermentation medium at 30 °C for a specific duration with an inoculation percentage of 10 % (v/v). Afterwards, the BC membrane was removed, washed with a large amount of deionized water, and boiled with NaOH solution at 80 °C for 2 h to remove residual culture medium and bacterial cells. During this period, we replaced the alkaline solution once, and finally washed it repeatedly with deionized water until the membrane was neutral. And then, the processed BC membrane was freeze-dried for 36 h to a constant weight. The citral was adsorbed directly by BC materials via gas-phase adsorption. And the processes were as follows: The freeze-dried BC membrane and citral were placed respectively into an airtight container (90 mm × 90 mm × 25 mm). Then the container was kept in an oven at 60 °C for 6 h to fully adsorb, resulting in a composite material of BC and citral, called BC-Citral (Scheme 1).

Scheme 1.

Illustration of preparation of BC and BC-Citral.

2.4. Establishment of standard curve and detection of the adsorption capacity

The gas chromatography–mass spectrometry (GC–MS, Agilent 7890B-5977 A, USA) was used to detect the content of citral (Paudel et al., 2018). Chromatographic conditions: DB-5MS column (30 m × 250 μm, 0.25 μm), injection port temperature of 250 °C. Temperature ramping program: initial temperature 50 °C, ramped to 250 °C at 10 °C/min, held for 5 min. Carrier gas: helium, flow rate: 1.0 mL/min, injection volume: 1 μL, split ratio: 10:1. Mass spectrometry conditions: utilizing an EI ionization source, electron energy at 70 eV, ion source temperature at 230 °C, quadrupole temperature at 150 °C, transfer line temperature at 250 °C, solvent delay of 3.9 min, data collection in full scan mode, mass range of 30–500 m/z. Qualitative analysis was performed using the NIST 2017 spectral library and mass spectra, while semi-quantitative analysis was conducted using peak area normalization, expressed as relative content. Firstly, the standard curve of citral was drew by diluting it with anhydrous ethanol to a constant volume. Subsequently, a certain amount of the BC-Citral was weighed and anhydrous ethanol was added. The citral of as-prepared samples was thoroughly extracted by sonication at 40 °C for 20 min, followed by centrifugation at a speed of 7500 × G for 5 min. The supernatant was filtered and placed in a headspace bottle. And the results were substituted into the standard curve and adsorption formula to calculate the adsorption capacity (Carvalho Da Silva et al., 2017). The details of standard curve can be found in Supporting Information named Fig. S1.

2.5. Optimization of adsorption conditions

At the outset of the investigation, the adsorption capacity was used as an indicator to evaluate aromatic dosage (100 μL, 200 μL, 300 μL, 400 μL, 500 μL), adsorption temperature (30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C), and adsorption time (1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h). The results of single factor experiment were shown in Supporting Information (Fig. S2-S4, Table S1-S3). Then, three independent variables were used to investigate the influence on adsorption capacity: aromatic dose (A), adsorption temperature (B), and adsorption duration (C). The adsorption capacity (Y) was utilized as the response value, and the components were encoded. A response surface analysis experiment was conducted on a total of 17 experimental points with three factors and three levels to further optimize the preparation process (Table S4-S5). The values of factors and levels were shown in Table S6.

2.6. Structure characterization

2.6.1. SEM

The SEM (Zeiss Sigma 300, Aalen, Germany) were performed according to a previously described method (Zhang et al., 2021). BC and BC-Citral were gold-plated in a vacuum environment for 90 s. Observation of acceleration voltage was from 10 kV to 15 kV.

2.6.2. XRD

XRD was used to investigate the crystal structure of samples on X-ray powder diffractometer (Rigaku SmartLab SE, Japan) with Cu Kα radiation operating at 40 kV and 200 mA. All detections were performed over the 2θ range of 5–90°, with ramping at 10° min⁻¹. The crystallinity index (CrI) was calculated according to the following expression:

$$\mathrm{CrI}\left(\%\right)=\frac{{\mathrm{I}}_{200}-{\mathrm{I}}_{\mathrm{am}}}{{\mathrm{I}}_{200}}\times 100\%$$ (1)

where “I₂₀₀” was the maximum intensity of the crystalline peak lattice diffraction (at about 2θ = 22.5° for cellulose I) and “I_am” was the intensity of amorphous part diffraction (at about 2θ = 18° for cellulose I) (Segal et al., 1959).

2.6.3. FTIR

The FTIR analysis (Thermo Scientific Nicolet iS20, USA) was determined according to a previously described method (Zhang et al., 2021). BC and BC-Citral were fully freeze-dried and mixed with potassium bromide (KBr) in a mass ratio of 1:60. The scanning frequency was 2 cm⁻¹ with a range of 400–4000 cm⁻¹.

2.6.4. N₂ adsorption-desorption experiment

N₂ adsorption-desorption studies were carried out at −196 °C to determine the BET surface area (Micromeritics 3Flex, USA). The specific surface area, pore size distribution, and pore volume were obtained by BET and Barrett-Joyner-Halenda (BJH) methods (Li et al., 2020).

2.7. Temperature response performance

2.7.1. TG

The TG analysis (Netzsch STA 449 F3, Germany) of BC, citral, and BC-Citral was determined according to a previously described method (Liu, Liu, et al., 2024; Liu, Sun, et al., 2024; Liu, Wang, et al., 2024). Approximately 3 mg of each sample was placed in an alumina crucible to start the program. The determination conditions were as follows: nitrogen flow rate 20 mL/min, temperature range 30–800 °C with increasing rate of 10 °C /min.

2.7.2. Sustained release performance analysis

0.200 g of BC-Citral samples were equilibrated at 100 °C for 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min. The content of citral was detected by static headspace gas chromatography to indicate the release characteristics of the BC-Citral. Headspace injection conditions: The instrument is Agilent headspace sampler (7694E) from the United States, with a gas-liquid equilibrium temperature of 60 °C, a gas-liquid equilibrium time of 30 min, and a quantitative loop of 1.0 mL (Li et al., 2024). The Formula (2) for calculating the release rate is as follows:

$$\text{Release rate}\left(\%\right)=\frac{A_1}{A_2}\times 100\%$$ (2)

where “A₁” represented the area of essence released from sample, “A₂” referred to the area of maximum peak of essence released.

The release kinetics of BC-Citral and citral were investigated using two mathematical models, including first-order kinetic equation, and Peppas kinetic equation (Hu et al., 2024).

2.8. Mechanism of thermal decomposition kinetics analysis

The pyrolysis kinetics experiment was conducted by TG using non isothermal methods. In a normal pressure air atmosphere, the protective gas of the thermal balance was high-purity nitrogen gas, and 17 mg of the sample was weighed and placed at the bottom of a 70 μL alumina crucible. It was then placed on the balance tray of the thermogravimetric analyzer, and the sample was heated according to the set rate. The test was performed at different heating rates of 5, 10, 20, 40, and 80 °C/min, and an air flow rate of 20 mL/min (Zhou et al., 2020).

2.9. Computational details

All electronic structure calculations were performed using the ORCA (version 5.0.4) software package (Neese, 2022). ORCA is a versatile quantum chemistry program capable of performing various types of calculations, including density functional theory (DFT), Hartree-Fock (HF), and post-HF methods.

Geometry optimizations were carried out using the B97—3C functional with the embedded basis set. Single-point energy calculations were performed using RI-B3LYP-D3(BJ) function with the larger def2-TZVP basis set to obtain accurate electronic energies. And the Hirshfeld charge analysis was carried out in Multiwfn code to study the charge transfer between the molecules (Lu, 2024).

To account for dispersion interactions, the DFT-D3 correlation correction was included in the calculations. This correction accounts for the long-range dispersion forces that are not adequately captured by standard DFT functionals. The D3 correction was applied with the Becke-Johnson damping scheme (Grimme et al., 2010).

The independent gradient model based on Hirshfeld partition (IGMH) calculations were performed to study the weak interactions between the molecules. These calculations were carried out based on the wavefunctions obtained from ORCA in B3LYP-D3(BJ) function with def2-TZVP basis set (Lu & Chen, 2022).

2.10. Antibacterial performance of BC-Citral

By using a spread plate and serial dilution, the antibacterial activity of the samples against S. aureus and E. coli was assessed (Xu et al., 2022). Particularly, S. aureus and E. coil were incubated at 37 °C with constant shaking to obtain the activated bacterial solution (10⁵ CFU/mL). 0.5 g of the as-prepared samples were cut into small pieces, and then sterilized under a UV lamp for 3 h, respectively. Initially, the activated bacterial solution was diluted 100 times with liquid culture medium, and then diluted it 100 times with PBS buffer. Afterwards, the sterilized membrane samples were added into a conical flask containing 50 mL of diluent and shaken continuously at 180 rpm for 18 h. Diluting the shaken bacterial solution with sterile water in a 10-fold gradient to obtain the required bacterial suspension for the experiment. Then, 100 μL of bacterial suspension was evenly spread onto solid culture medium. Three parallel samples were set for each gradient. Finally, the coated solid culture medium was placed upside down in a constant temperature incubator at 37 °C for a certain period of time and then took photos for counting. The matching solvents were used as controls for the cultures of S. aureus and E. coli. Formula (3) was used to determine the bacterial viability based on the number of colonies of S. aureus and E. coli following incubation:

$$\text{Antibacterial ratio}\left(\%\right)=\frac{N_{\text{control}}-N_{\text{experiment}}}{N_{\text{control}}}\times 100\%$$ (3)

where “N_control” and “N_{expenrimental}” represent the number of the bacteria of the control sample and the experimental samples (BC, Citral, BC-Citral), respectively.

3. Results and discussions

3.1. Optimization and validation of response surface methodology (RSM)

Based on the results of the single factor experiment (Fig. S2-S4), three factors, aromatic dosage (A), adsorption temperature (B), and adsorption time (C), significantly affecting the adsorption capacity, were selected as independent variables. The BC adsorption capacity (Y) was taken as the response value, and a response surface analysis experiment was set up for a total of 17 experimental points with three factors and three levels to further optimize the preparation process. The response surface test results were shown in Table S4. Design Expert software was used for regression analysis on the results of each treatment of the response surface. From Table S4, it was noticeable that the highest adsorption capacity 129 mg/g corresponding to the adsorption experiment carried out with the aromatic dosage of 400 μL, adsorption temperature of 70 °C and adsorption time of 6 h (Table S4, entry 11). When the aromatic dosage of 300 μL, adsorption temperature of 70 °C and adsorption time of 7 h, the adsorption capacity was only 83 mg/g (Table S4, entry 7). After regression fitting, the regression eq. Y = 126.6 + 7.87 A + 1.12B-1.50C + 7.00AB + 3.75 AC-0.25 BCE-16.8A²–8.8B²–13.05C² was obtained for each factor.

Residual analysis is an important part of assessing the adequacy of quadratic regression models. The externally studentized residual points in Fig. 1a, which were defined as residuals (deviations between experimental and predicted values), strongly concentrated around the straight line. The performance of normality hypothesis was tested by constructing a normal probability map of externally studentized regression. The remaining points of RSM were quite near to the straight line, indicating the experimental data followed a normal distribution. As shown in Fig. 1b, the externally studentized residual points were randomly dispersed, illustrating that the variance of the original observation values is constant and independent of the response values.

Fig. 1.

Residual plots: (a) Normal probability plot of adsorption capacity studentized residuals; (b) Plot of the studentized residuals versus the predicted adsorption capacity; (c1-e2) Response surface diagram of pairwise interaction of various factors.

The results of analysis of variance (ANOVA) for the adsorption model were provided in Table S5. The established model had a F-value (40.78) with a p-value less than 0.001, while the mismatched term had a p-value of 0.1927, which was not significant. The results indicated that the quadratic model had a good fitting effect on the experimental data with minimal experimental errors and reliably fitted the effects of aromatic dosage, adsorption temperature, and adsorption time on adsorption capacity. Each element had a distinct impact on the adsorption capacity within the range of chosen parameters. And the linear effect A, AB, A², B² and C² had an extremely significant influence on the test results (p-value <0.01). Based on the results, it could be inferred that the quadratic regression model provided a sufficient description of the BC adsorption process. In addition, the influence of three factors on the adsorption capacity of adsorbent was A > C > B, that was, aromatic dosage > adsorption time > adsorption temperature.

According to the regression equation, the interaction between aromatic dosage (A), adsorption temperature (B), and adsorption time (C) was analyzed. The response surface curves of the interaction between each factor were shown in Fig. 1c1-e2. It was obviously observed that the contour maps of the response surfaces for the pairwise interaction of the three factors were all elliptical, indicating a significant interaction between the aromatic dosage, adsorption temperature, and adsorption time.

The extremum point of the model was obtained by the first partial derivative of the model equation. The optimal preparation process for BC was aromatic dosage of 426.78 μL, adsorption temperature of 71.70 °C, and adsorption time of 5.98 h. Under these conditions, the predicted adsorption capacity of citral by the model was 127.77 mg/g. Considering the actual operation, the final preparation process was set as aromatic dosage of 430 μL, adsorption temperature of 70 °C, and adsorption time of 6 h for three validation experiments. The average actual adsorption capacity was 124.98 mg/g, in which the fitting degree was 97.82 % with the predicted adsorption capacity by the model. The optimization on preparation process of citral adsorption via RSM was viable.

3.2. Characterization of morphology and structure

3.2.1. Morphological characteristics of different samples

Fig. 2 showed the morphologies of BC and BC-Citral samples. As illustrated in Fig. 2a, the pure BC prepared with TSWE exhibited interconnected three-dimensional nano- and microfiber networks structure. It was crucial to mention that the interior of BC exhibited an abundant porous structure, which was consistent with previous reports (Li et al., 2014; Zhang, Dong, & Qi, 2022; Zhang, Luo, et al., 2022). The internal porous structures could provide a good loading and controlled release environment, which could explain some properties of BC such as the ability of adsorbing and releasing. After the adsorption process, fewer cavities of BC were observed, manifesting a high adsorption capacity of citral (Fig. 2b).

Fig. 2.

SEM images of (a) BC and (b) BC-Citral.

3.2.2. Structural characteristics of different samples

The XRD pattern was displayed in Fig. 3a. The BC showed obvious diffraction peaks at around 14.5°, 16.6° and 22.7°, corresponding to (10$\overline{1}$), (101), and (200) reflection planes, respectively, (Lee et al., 2015) belonging to the typical cellulose I crystal form. Meanwhile, the citral sample exhibited a broad peak around 18°, indicating that it was amorphous material. The crystallinity index for both BC and BC-Citral stood at around 99.3 % and 89.5 %, respectively. The decrease of crystallinity index may due to the intermolecular reaction between BC and citral, which causes BC molecular chains difficult to move. (Kim et al., 2010; Liu et al., 2019). As shown in Fig. 3b, the structures of BC, citral, and BC-Citral were investigated by FTIR spectrophotometer. The BC produced with TSWE showcased distinctive cellulose molecule peaks. There was a band observed at 3342 cm⁻¹ in the BC spectrum, corresponding to the -OH group stretching mode in cellulose. Additionally, the absorption peak around 2897 cm⁻¹ originated from the C—H group stretching mode in cellulose. The band at 1059 cm⁻¹ was regarded as the stretching vibration of the C—O group from the pyran ring in cellulose molecules. The band at wave numbers of 900 cm⁻¹ was the characteristic absorption peak of glycosidic in cellulose molecules (Heru et al., 2023). The above bands conformed to the structure of the cellulose standard peak, indicating that the product was bacterial cellulose. There were multiple stretching vibration peaks of CH₂ and CH₃ near 2800–3000 cm⁻¹ in citral. The band at wave numbers of 1670 cm⁻¹ was attributed to the stretching vibration of the -CHO group in citral. For BC-Citral, a band at 1047 cm⁻¹ was attributed to the stretching vibration of the C—O group from the pyran ring from BC. Compared to BC, a new band at 1670 cm⁻¹ was attributed to the stretching vibration peak of the conjugated -CHO group in citral, which indicated the successful preparation of BC-Citral. Besides, the peak intensity of O—H was weakened compared to that in the curve of BC, speculating that citral was adsorbed by some -OH group. The above results indicated that BC showed excellent adsorption capacity of citral.

Fig. 3.

(a) XRD images of three samples; (b) FTIR images of three samples; N₂ adsorption-desorption isotherms of (c) BC and (d) BC-Citral.

In order to gain a deeper comprehension of the pore structure of samples, N₂ adsorption-desorption experiment was conducted. Fig. 3c-d showed the N₂ adsorption-desorption isotherms and pore size distribution curves of the BC (Fig. 3c) and BC-Citral (Fig. 3d), which suggested that the networks were basically mesoporous with pore sizes mostly < 50 nm. The adsorption amount of N₂ for samples increased gradually in the region of 0 < P/P₀ < 0.5, and in this region the adsorption/desorption curves were overlapped, revealing the presence of small micropores with monolayer adsorption. Then, N₂ adsorption-desorption isotherm was typical IV curve with obvious hysteresis loop, indicating that there were abundant mesopores structures in BC, which were beneficial for the diffusion of gas molecules. The obtained results were also evident from the BJH pore size distribution presented, which was similar to BC's BET analysis reported previously by Hassan et al. and Mani et al. (Hassan et al., 2019; Mani et al., 2020). The increases in the number and size of the surface pores resulted in the extension of the surface area (Guo et al., 2011). As depicted in Table S7, after the adsorption of citral on the BC, the gas molecules of citral may gradually occupy the micropores and mesopores on the membrane, which caused the specific surface area and pore volume of BC-Citral decreased. The pore volume and surface area of the BC-Citral decreased because of absorbed citral.

3.3. Temperature response performance

3.3.1. TG analysis of different samples

The thermogravimetric analysis results were shown in Fig. 4a. Citral had an extremely fast weight loss rate, starting at around 60 °C, with the fastest weight loss rate at 129.1 °C and a mass loss of 84.61 % at 260 °C, indicating that citral had poor stability (high volatility) and was difficult to store. Therefore, certain methods need to be taken to delay its volatilization rate. The pyrolysis mass loss of BC consisted of three stages. The first stage was at about 30–110 °C, which was mainly due to the release of moisture within the biomass and the breaking of hydrogen bonds between BC, where the mass loss was about 1.71 % (Zhang, Dong, & Qi, 2022; Zhang, Luo, et al., 2022). The second stage occurred between 110 and 430 °C. It was mainly due to the pyrolysis temperatures of cellulose within the range of 200–350 °C. In the interval, the chain of macromolecule carbohydrates was destroyed to produce smaller molecules, such as CO₂, CO, CH₄, where the mass loss was about 93.95 % (Jia et al., 2020). The third stage occurred after 430 °C. The mass loss was caused by the cracked of trace amount of cellulose, with the mass loss became small and the quality was almost constant. There were three primary weight reduction intervals for BC-Citral. The initial range involved a loss of weight between 34 °C and 110 °C caused by moisture evaporation and citral absorption on the BC surface, leading to a decrease in mass of 1.11 %. The second stage occurred between 110 and 393 °C, which was mainly due to the pyrolysis of BC and the release of the citral adsorbed to the inner side of the membrane. The maximum decomposition rate was reached at 299 °C, resulting in a mass loss of 62.63 %. The third stage occurred after 393 °C. The quality was almost constant. Compared with citral, the maximum decomposition rate of BC-Citral was 299 °C and the weight loss temperature shifted to the right, manifesting that BC-Citral releases citral slowly and improved the thermal stability.

Fig. 4.

(a) Thermogravimetric analysis of three samples; (b) Release characteristics of three samples; (c) Release kinetics fitting curve: first-order kinetic equation; (d) Korsmeyer-Peppas kinetic equation. Note: *** Indicating a highly significant difference, p < 0.001.

3.3.2. Sustained-release performance analysis

Considering that BC-Citral may have the potential applications in heat processed foods and instant foods, and most instant foods dissolve at 100 °C. Therefore, the heat release of BC-Citral at 100 °C in 120 min was investigated (Fig. 4b). Under the condition of 100 °C, the release rate of citral and BC-Citral sharply increased and then tended to a plateau. The release rate of BC-Citral for the first 70 min reached 52.6 %, while the citral released 69.34 % in 70 min. Subsequently, the release rate of samples was slowed down, and then tended to be gentle, with a final release rate of 75.4 % and 57.3 %. The citral adsorbed on the surface of BC was released firstly during the release process at 100 °C, and then the citral adsorbed to the inner side of BC was evaporated. And the above results indicated that the citral adsorbed by BC can be stably and slowly released at specific temperature.

3.3.3. Sustained-release kinetics analysis

As shown in Fig. 4c-d, the release data of citral and BC-Citral at different time under the same temperature fitted the first-order kinetic equation (y = a*(1-e^-kx)) and Korsmeyer-Peppas kinetic equation (y = K*xⁿ). The correlation coefficients of the first-order kinetic equation and Korsmeyer-Peppas kinetic equation for citral under 100 °C were 0.9895 and 0.9796, respectively. Meanwhile, the correlation coefficients of two equations for BC-Citral under 100 °C were 0.9915 and 0.9806, respectively. Therefore, the first-order and Korsmeyer-Peppas models could describe the citral release kinetics of the BC-Citral well under 100 °C. Among them, the first-order release model represented the best fitting, indicating that citral was mainly adsorbed to the inner side of the pores. Furthermore, the obtained results indicated that the release of citral from BC-Citral was mainly controlled by internal diffusion, which conformed to the release properties and regulars of sustained-release materials (Zhang et al., 2019).

3.3.4. Heat release kinetics analysis

Fig. 5 showed the TG curves of BC-Citral at different heating rates. It can be seen that the weight of the BC-Citral changed slowly before 200 °C, which was mainly due to the evaporation of moisture and the citral adsorbed on the surface of BC-Citral. As the temperature rose, the release of citral entered a pronounced stage of mass decline. Afterwards, the mass remained constant. In addition, the increased heating rate led to temperature difference between the inside and outside the sample, which made the sample reach to release temperature faster and caused the TG curve to shift towards a high-temperature end. This might be due to the increased heating rate accelerated the reaction rate between BC-Citral and oxygen, resulting to an increase in the maximum weight loss rate of the sample. The heat release characteristic parameters of the BC-Citral were shown in Table 1. T_i and T_f were the initial and final release temperatures of the samples obtained by the TG and DTG (Derivative thermos-gravimetry) tangent method, respectively. CRI was the comprehensive release index of the samples calculated by Eq. S1, which was used to reflect the thermal release characteristics of BC-Citral. It was clearly observed that T_i and T_max all increased along with the heating rate, but the increase amplitude was lower than that of heating rate, indicating that the intensification of thermal hysteresis was not linearly related to the heating rate. The DTG_max increased from 8.00 %/min to 174.31 %/min significantly, and the CRI increased from 0.01748 % to 0.47976 %, demonstrating that a faster heating rate enhanced the heat release rate of BC-Citral, which was beneficial for the heat release reaction of BC-Citral. And the DTG_max had a good linear relationship with heating rate β. The specific linear equation relationship was y = 0.4524× + 1.6422 (R² = 0.9763).

Fig. 5.

TG curves of BC-Citral at different heating rates.

Table 1.

Heat release characteristic parameters of BC-Citral under different heating rates.

β/ (°C/min)	Ti/°C	Tmax/°C	DTGmax/ (%/min)	Tf/°C	Tf−Ti	Tmax*(Tf−Ti)	CRI
5	261.4	286.7	8.00	421.0	159.6	45,757.32	0.01748 %
10	273.2	299.8	15.73	411.1	137.9	41,342.42	0.03805 %
20	284.6	311.6	55.62	336.3	51.7	16,109.72	0.34526 %
40	296.9	325.1	70.82	415.3	118.4	38,491.84	0.18399 %
80	308.9	328.8	174.31	419.4	110.5	36,332.4	0.47976 %

According to the TG analysis, the heating rate had a significant impact on the heat release behavior of BC-Citral. The Kissinger-Akahira-Sunose (KAS) method (Eq. S2) based on multiple heating rates was used to calculate the activation energy of BC-Citral with a heat rate between 0.1 and 0.7. Fig. 6 showed the KAS normal fitting curves for different heat release rates, in which the linear curve was well fitting with a coefficient of determination R² between 0.9440 and 0.9937. The fitting equation and activation energy assignment for different heat release rates were shown in Table 2. The overall activation energy ranged from 102.24 to 169.46 kJ/mol.

Fig. 6.

KAS fitting curve of BC-Citral under heat rate of 10 % ∼ 70 %.

Table 2.

Kinetic fitting results for the heat release of BC-Citral based on KAS model.

Conversion rate	Fitted equation	R2	E/(KJ/mol)
0.1	y = −12,297x + 14.006	0.9801	102.24
0.2	y = −16,601x + 20.278	0.9937	138.02
0.3	y = −17,470x + 21.134	0.9926	145.25
0.4	y = −18,722x + 22.853	0.9879	155.65
0.5	y = −19,003x + 22.955	0.9803	157.99
0.6	y = −19,731x + 23.838	0.9674	164.04
0.7	y = −20,383x + 24.479	0.9440	169.46

In order to investigate the release process of BC-Citral, a kinetic reaction model was built, based on the coefficient of determination using Eq. S3 and S4 via the Coats-Redfern method. The enthalpy change (ΔH), Gibbs free energy (ΔG), and entropy change (ΔS) corresponding to the reaction system during the heat release process were calculated. The fitting calculation results were shown in Table 3. Common expression of reaction mechanism function was listed in the Table S8. The second-order reaction model D1 can well describe the heat release process of BC-Citral at various heating rates, and the apparent activation energy E of the heat release process at each heating rate ranges from 109.06 to 210.07 kJ/mol. Fig. 7 showed the fitting curves of the D1 reaction model function for BC-Citral under different heating rates. There was a good linear relationship within the range of 10 %–70 % heat release conversion rate, and the determination coefficients R² were all greater than 0.9.

Table 3.

Thermodynamic parameters for the thermal release of BC-Citral under different heating rates.

β/ (°C/min)	Fitted equation	R2	E/ (kJ/mol)	A/ (min−1)	△H/ ((kJ/mol)	△G/ ((kJ/mol)	△S/ ((kJ/mol/K)
5	y = −13,118x + 9.3457	0.9653	109.06	1.50 × 109	106.68	179.19	−0.2529
10	y = −15,417x + 12.77	0.9809	128.18	5.42 × 1010	125.68	201.62	−0.2533
20	y = −21,432x + 22.877	0.9984	178.19	7.38716 × 1015	175.43	259.72	−0.2541
40	y = −19,025x + 17.676	0.9957	158.17	3.61388 × 1013	155.47	238.03	−0.2539
80	y = −25,267x + 27.632	0.9920	210.07	1.01167 × 1018	207.34	290.86	−0.2540

Fig. 7.

Coats-Redfern fitting curve of BC-Citral at different heating rates.

3.4. Analysis of interaction mechanism

The adsorption configuration of interacting BC with citral was displayed in Fig. 8a. The molecule of citral was located in the vicinity of-OH groups on BC. The calculated adsorption energy was a negative value (−12.454 kJ/mol), indicating the formation of stable complexes as well as spontaneous adsorption of citral on BC (Hosseini & Mousavi, 2020). To quantify the charge transfer, the Mulliken charge transfer was performed for BC-Citral. The charges in the composite reflected the charge transfer after the interaction was formed (Cuautli et al., 2023). The direction of charge transfer was from BC to citral, which possessed the number of charge transfer with 0.216|e| after calculation.

Fig. 8.

(a) The adsorption configuration of citral on BC; (b) Sign(λ₂) ρ colored IGMH scatter maps of BC-Citral.

In order to gain a more reliable and deeper insight into the nature of interaction between citral molecule and BC, the IGMH was performed, which exhibited better graphical effect than independent gradient model (Lu & Chen, 2022). IGMH showcased the subtle connections between molecules within a system in an intuitive manner. This method illustrated the regions of interaction within a simulation model, facilitating a rapid comprehension of diverse interaction types. It not only visualized the interaction area but also delineated the strength and categories of interactions. Various interaction types and schemes are projected onto the isosurface of IGMH using color variations, where the size of the isosurface indicates the force's intensity. Blue accentuated the interaction‑hydrogen bonding, correlating with the quantity of intermolecular hydrogen bonds, while red signified repulsion, typically attributed to steric effects. Green symbolized intermediate interactions, encompassing van der Waals forces (Huang et al., 2021). As depicted in the Fig. 8b, the interactions between -OH group of BC with -CHO group of citral was predominantly shaded in green and blue. The peaks exhibited the strong van der Waals forces and weak H-bonds interaction. The conclusions manifested that the weak interactions between BC and citral was mainly composed of van der Waals forces and hydrogen bonds.

3.5. Antibacterial performance analysis

Bacterial infection usually occurs in food spoilage during storage and usage, which deteriorates quality and may be harmful to human health. The citral exhibited strong antibacterial action by rupturing the cell membrane structures of microorganisms, resulting in a loss of integrity and modification of activities (Xu et al., 2022; Yoplac et al., 2021). In this work, the antibacterial activity of samples were investigated using E. coli and S.aureus as the gram-negative and gram-positive model, respectively. The results of photographs of the bacterial culture plates could be seen in Fig. S5. As depicted in Table S9, when BC, citral and BC-Citral were added to E. coli bacterial solution separately, the antibacterial rate of citral was the highest, reaching 93.85 %. BC had no antibacterial effect with an antibacterial rate of solely 2.77 %. After adsorbing citral, the antibacterial rate of BC-Citral reached to 80.85 % when compared to BC. Similar trend was observed with S.aureus that citral outperformed BC and BC-Citral complex, reaching 98.21 %. The antibacterial rate of BC was only 5.74 %. The antibacterial rate of BC-Citral reached 89.16 %. The findings confirmed citral's potent capacity to hinder bacterial proliferation. This capacity was minimally compromised by BC adsorption, indicating that BC-Citral maintained some resilience against E. coli and S. aureus.

4. Conclusion

In summary, BC membrane prepared by using TSWE as medium is feasible. BC-Citral adsorbent was fabricated from BC through gas-phase adsorption technique. The optimal preparation conditions were obtained through RSM. The optimum preparation process was set as aromatic dosage of 430 μL, adsorption temperature of 70 °C, and adsorption time of 6 h. Under this condition, the adsorption capacity reached 124.98 mg/g. The SEM, FTIR, XRD and BET results indicated that BC had successfully adsorbed citral. TG analysis showed that citral in BC-Citral sample released slowly and the BC improved the thermal stability of citral.

At the same time, the preliminary exploration of adsorption and release mechanisms of citral on BC were also studied. Through release performance analysis, the citral adsorbed by BC can be stably and slowly released at specific temperatures, and the releases of both BC-Citral and citral under 100 °C were more in line with the first-order kinetic, indicating that citral was mainly adsorbed to the inner side of the pores, and its release from BC-Citral was mainly controlled by internal diffusion, conforming to the release properties and regulars of sustained-release materials. A satisfactory linear fitting curve was achieved when analyzing the heat release kinetics at various heating rates. The adsorption mechanism of citral molecule on BC was discussed in terms of Mulliken charge transfer, adsorption energy and IGMH, which not only proved the formation of BC-Citral originated from the spontaneous adsorption of citral on BC, but indicated the interactions between BC and citral was mainly composed of van der Waals forces and hydrogen bonds.

Besides, BC-Citral had excellent ability to resist E. coli and S.aureus, compared with BC. We expect the design of BC-Citral could unlock a novel method of heat processed foods and food additives, and widen the multifunctionality. Meanwhile, the adsorption mechanism can be helpful to explore novel approaches for BC to adsorb flavors through gas-phase method in highly efficient ways.

CRediT authorship contribution statement

Jingyi Hu: Writing – original draft, Formal analysis, Data curation. Longfei Wang: Writing – original draft, Formal analysis, Data curation. Menglan Xiao: Methodology, Investigation, Formal analysis. Weihua Chen: Software, Data curation. Meng Zhou: Software, Data curation. Yihan Hu: Validation, Data curation. Yujie Zhang: Validation, Data curation. Miao Lai: Supervision, Writing – review & editing. Aimin He: Writing – review & editing, Supervision. Mingqin Zhao: Writing – review & editing, Supervision.

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.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.