Evaluation of Cell Rupture Techniques for the Extraction of Proteins from the Microalgae Tetradesmus obliquus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Tetradesmus obliquus Biomass Processing
2.2. Composition of Tetradesmus obliquus Microalgae
2.3. Separation and Identification of Proteins in the Microalgae Tetradesmus obliquus
- Molecular mass profile
- Identification by mass spectrometry (MALDI-TOF)
2.4. Biomass Cell Disruption
- Mechanical disruption by ball mill: Freeze-dried biomass (10 g) was mechanically disrupted in a cylindrical ball mill constructed using AISI-304 stainless steel with an internal volume of 0.235 L (Tecnal Equipamentos Científicos, model R-TE-350, Piracicaba, São Paulo, Brazil), following Vieira et al. [17]. The mill balls (0.635 cm in external diameter) and the biomass swung up and down vertically in batches for 25 min, approximately 10 times per second and 617 strokes per minute. The disrupted biomass was kept at 20 °C until use;
- Mechanical disruption by ultrasound: Freeze-dried biomass was mechanically disrupted by ultrasound after resuspension in distilled water (10.0% w/v) following the optimal conditions determined by Silva et al. [21]. The suspensions were mechanically stirred (IKA, RW20 digital, Staufen, Germany) at 25.0 °C overnight and disrupted in a tip sonicator (Sonics, VCX 500, Newtown, CT, USA) at a frequency of 20 kHz and 98% amplitude for 6 min of ultrasonication. The cells were disrupted by cooling in an ice bath to avoid overheating the system. After disruption, the cell suspensions were frozen, freeze-dried (Terroni, LS 3000, São Carlos, São Paulo, Brazil), and stored at 20 °C until use;
- Mechanical disruption by high-pressure homogenization: Microalgal biomass was suspended in distilled water (1.5% w/v) and processed in a homogenizer (Alitec, A100, Pindamonhangaba, São Paulo, Brazil) at 350 bar according to the optimal conditions determined by Shene et al. [30], with modifications. Twenty-five passes of the suspensions in the homogenizer were used, and the suspension was cooled to avoid compound degradation due to an increase in temperature. Based on the results of preliminary tests, we observed that a total of 25 passes promoted greater protein recovery. However, biomass overheating was observed in tests with more than 25 passes. The homogenized samples were collected, frozen, and freeze-dried (Terroni, LS 3000, São Carlos, São Paulo, Brazil) and stored at 20 °C until use.
2.5. Cell Disruption Indicators
- Evaluation of cell disruption level through cell counting
- Evaluation of cell disruption level through the amount of extracted soluble protein
2.6. Statistical Analysis
3. Results
4. Discussion
4.1. Composition of T. obliquus Biomass
4.2. Identification of Main Proteins in T. obliquus Protein Extract
4.3. Cell Rupture and Protein Extraction from T. obliquus
- Evaluation of the cell disruption level through cell counting
- Evaluation of the cell disruption level through the amount of extracted soluble protein
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Characteristics | Ball Mill | High-Pressure Homogenization | Ultrasound |
---|---|---|---|
Protein mass yield (g/100 g) | 16.1 ± 1.3 a | 17.1 ± 1.4 b | 20.8 ± 0.8 a |
% Protein content (g/100 g) | 29.7 ± 1.2 a | 29.1 ± 0.6 a | 30.7 ± 0.4 b |
L* | 46.4 ± 0.46 a | 52.0 ± 0.76 c | 46.1 ± 0.26 a |
A | 4.73 ± 0.11 a | 5.53 ± 0.17 c | 4.98 ± 0.096 b |
B | 13.6 ± 0.40 a | 17.6 ± 0.30 c | 11.8 ± 0.29 b |
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da Silva, C.A.S.; Sibaja, K.V.M.; de Ramos Cizilio, S.; Miranda Júnior, J.R.; de Castro Santana, R.; Martins, M.A.; de Oliveira Leite, M.; de Oliveira, E.B.; Coimbra, J.S.d.R. Evaluation of Cell Rupture Techniques for the Extraction of Proteins from the Microalgae Tetradesmus obliquus. Phycology 2024, 4, 87-103. https://doi.org/10.3390/phycology4010005
da Silva CAS, Sibaja KVM, de Ramos Cizilio S, Miranda Júnior JR, de Castro Santana R, Martins MA, de Oliveira Leite M, de Oliveira EB, Coimbra JSdR. Evaluation of Cell Rupture Techniques for the Extraction of Proteins from the Microalgae Tetradesmus obliquus. Phycology. 2024; 4(1):87-103. https://doi.org/10.3390/phycology4010005
Chicago/Turabian Styleda Silva, César Augusto Sodré, Karen Vanessa Marimón Sibaja, Sabrina de Ramos Cizilio, José Roberto Miranda Júnior, Rejane de Castro Santana, Marcio Arêdes Martins, Maurício de Oliveira Leite, Eduardo Basílio de Oliveira, and Jane Sélia dos Reis Coimbra. 2024. "Evaluation of Cell Rupture Techniques for the Extraction of Proteins from the Microalgae Tetradesmus obliquus" Phycology 4, no. 1: 87-103. https://doi.org/10.3390/phycology4010005