## 3 Sample Preparation for Carotenoids Determination
### Sample Extraction Techniques
#### 3.1.3 Green Extraction Technologies  
Various green extraction methods have been developed to improve carotenoid recovery while reducing environmental impact. Figure 3 plots the number of methods published for each extraction method since 2015 to date. Among these, solvent extraction, UAE, and supercritical fluid extraction (SFE) are the most widely studied [62]. * **UAE** utilizes sound waves above 20 kHz to generate acoustic cavitation, which disrupts plant cell walls, enhancing solvent penetration and accelerating extraction. Studies have shown significant efficiency gains using UAE. [PERSON] et al. (2021) reported a 50% increase in carotenoid yield from sea buckthorn, while [PERSON] et al. (2021) observed that UAE extracted 151.41 ug/mL of \(\beta\)-carotene, compared to 96.74 ug/mL with NADEs [73; 74]. Similarly, [PERSON] et al. (2021) demonstrated that UAE combined with corn oil extracted 38.03 ug/g of carotenoids from pumpkin, nearly twice the amount obtained using hexane-isopropyl alcohol (19.21 ug/g) [71]. Extraction efficiency in the UAE depends on several parameters. Increasing ultrasound intensity produces a proportional rise in carotenoid yield [75]. Other influencing factors include extraction time, solvent-to-solid ratio, and temperature, with extraction time having the most significant effect, while temperature has minimal impact [76]. * **SFE** utilizes fluids in a supercritical state, characterized by solvent power as liquids and mass transfer properties as gas which accelerates extraction. Carbon dioxide is the most commonly used solvent due to its low critical temperature (31.1 \({}^{\circ}\)C) and pressure (7.38 MPa), making it suitable for heat-sensitive carotenoids [77]. SFE has demonstrated high extraction efficiencies. [PERSON] et al. (2019) optimized SFE for carotenoids in 15 vegetable and fruit waste matrices, achieving up to 96.2% recovery, while [PERSON]  
Figure 3: Trends in carotenoid extraction methods: number of published studies (2015–Present). SE, solvent extraction; UAE, ultrasound-assisted extraction; SFE, supercritical fluid extraction; MAE, microwave-assisted extraction; EAE, enzymatic-assisted extraction; PEF, pulsed electric field; HHPE, high-hydrostatic-pressure extraction; SWE, subcritical water extraction; OHE, ohmic heating extraction. The keyword “carotenoid” and each technique name and acronym were used for the Web of Science (WOS) database search. et al. (2018) reported a 98.6% recovery of astaxanthin from _H. pluvialis_[78; 79]. Despite its advantages, slow extraction kinetics limit SFE's efficiency. To address this, EAE and UAE can be combined with SFE to improve cell wall disruption and enhance carotenoid release [80]. * **MAE** applies microwave radiation to heat solvents through dipole rotation and ionic conduction, increasing cell permeability and accelerating extraction. It requires less solvent and shorter processing times but is unsuitable for thermolabile carotenoids. Comparisons between MAE and UAE show that while UAE achieved a higher yield (267 mg/100 g DW) at 200 W for 80 min, MAE still provided a notable improvement over conventional methods, yielding 262 mg/100 g DW at 120 W for 25 min. Additionally, UAE extracts exhibited higher antioxidant capacity and preserved more bioactive compounds, though they required significantly more energy consumption (229 kcal vs. 43 kcal for MAE). These findings highlight UAE's effectiveness for carotenoid recovery while underscoring the need for energy optimization in industrial applications. [81]. * **EAE** utilizes cell-wall-degrading enzymes (e.g., cellulases, pectinases, proteases) to enhance carotenoid release [82; 83]. [PERSON] et al. (2015) observed a 10-fold increase in lycopene and a 6-fold increase in total carotenoids from tomato paste following enzyme pretreatment [82]. Similarly, [PERSON] and [PERSON] (2008) reported a 20-fold rise in lycopene extraction from tomato waste using cellulase and pectinase [84].