Controlled Release of D-Limonene from Biodegradable Films with Enzymatic Treatment

Abstract

The instability of many volatile organic compounds (VOCs) limits their usage in different fragrance carriers and products. In scratch-and-sniff applications, VOCs are bound so strongly that release cannot happen without an external trigger. On the other hand, other fixatives like cyclodextrins release unstable volatile molecules too rapidly. We engineered biodegradable gelatin films whose release profile can be tuned by glycerol plasticization and alkaline protease degradation. Digitalized VOC release profiles acquired with the described near-real-time analysis toolkit are digital twins that replicate the behavior of the evaluated films in silico. Seven formulations were cast from 10% gelatin containing D-limonene, glycerol (5%, 20%), protease-C 30 kU mL^-1, and samples with additional water to establish a higher hydromodule for protease catalytic activity. Release profiles were monitored for nine days at 23 ± 2 °C in parallel by metal-oxide semiconductor (MOS) e-noses, gravimetric weight loss, and near-infrared measurements (NIR). These continuous measurements were cross-checked with gel electrophoresis, FTIR spectroscopy, hardness tests, and sensory intensity ratings. Results showed acceleration of VOC release by enzymatic treatment during the first days, as well as overall impact on the release profile. Differences in low and high glycerol films were observed, and principal component analysis of NIR spectra separated low and high glycerol groups, mirroring the MOS and FTIR data. Usability of MOS data was explored in comparison to more biased and subjective intensity results from sensory panel evaluation. Overall, the created toolkit showed good cross-checked results and enabled the possibility for close to real-time analysis for bio-based VOC carriers.

Keywords: D-limonene; MOS sensor; VOC; carriers; controlled release; digital twin; gelatin; protease.

Figure 1

Optical images of films, scale bar: 100 µm, (a) L@Gel, (b) L@Gel/0.5Gl, (c) L@Gel/0.5Gl/PT, (d) L@Gel/0.5Gl/PTxW, (e) L@Gel/2Gl, (f) L@Gel/2Gl/PT, (g) L@Gel/2Gl/PTxW. Samples (c,d,f,g) containing protease-C might exhibit a more pronounced yellow color due to the enzyme’s impact.

Figure 2

D-limonene diffusion plot (in ppb) measured with the MOS sensor by hours, values are normalized by reducing the base by the 10th percentile on a 5 s time window, and after being averaged per hour (in the corner chart are original raw values from the sensor). The bottom chart’s yellow line shows a non-loaded sensor.

Figure 3

Impact of protease-C load into soft gelatin capsule (samples (b,c): 0.3 mL protease-C 3 K U mL⁻¹:0.5 mL D-limonene and 0.5 mL protease-C 3 K U mL⁻¹:0.3 mL D-limonene) versus solo D-limonene in gelatin capsule (a) and solo water in gelatin capsule (d).

Figure 4

FTIR spectra for films and separately glycerol and D-limonene, taken on the 3rd day after film casting.

Figure 5

FTIR wavelength windows based on identified regions of impact: (a) range O-H/N-H, 3300–3280 cm⁻¹ (b) glycerol, stretch at 1030 cm⁻¹. Glycerol and D-limonene are added for reference and are raw materials.

Figure 6

FTIR wavelength windows based on identified regions of impact: (a) C-H 2913 cm⁻¹, (b) =C-H at 887 cm⁻¹, (c) 1643 cm⁻¹ for D-limonene identification, (d) 1404 cm⁻¹ COO⁻ range. Glycerol and D-limonene are added for reference and are raw materials.

Figure 7

FTIR peak aggregated data for films with protease-C and without, the grey dashed line highlights spectra differences.

Figure 8

Gel-electrophoresis of films in order from left to right (lanes 1–7): L@Gel, L@Gel/0.5Gl, L@Gel/0.5Gl/PT, L@Gel/0.5Gl/PTxW, L@Gel/2Gl, L@Gel/2Gl/PT, L@Gel/2Gl/PTxW, lane 8—albumin with molecular weight 66.5–67 kDa.

Figure 9

Plot (a) shows the full range of overall normalized TVOC, ppb (normalization approach described with Figure 2), Plot (b) same films, data is zoomed in to be in the range of 0–15 normalized TVOC ppb values.

Figure 10

NIR charts: (a) daily values for NIR measurements of 810 and 860 nm over 9 days for films L@Gel, L@Gel/0.5Gl, L@Gel/0.5Gl/PT, L@Gel/0.5Gl/PTxW put on the same chart. (b) pairwise significance of difference between films on 810 nm (smaller value, larger difference).

Figure 11

PCA analysis of NIR data for samples: L@Gel, L@Gel/0.5Gl, L@Gel/0.5Gl/PT, L@Gel/0.5Gl/PTxW, L@Gel/2Gl, L@Gel/2Gl/PT, L@Gel/2Gl/PTxW.

Figure 12

Sensory panel mean intensity values of 3 batches of measurement over 10 days with indoor conditions between 20–25 °C: L@Gel, L@Gel/0.5Gl, L@Gel/0.5Gl/PT, L@Gel/0.5Gl/PTxW, L@Gel/2Gl, L@Gel/2Gl/PT, L@Gel/2Gl/PTxW.