Table 3.
Description of the extraction methods used for disrupting the phycocyanin cell membrane, their conditions, advantages, and limitations.
| Cell Disruption Method | Purity * | Yield (mg/g) | Conditions | Advantages | Limitations | Ref. |
|---|---|---|---|---|---|---|
| Freeze/thaw cycles | 0.56–0.66- | 73.73–74.51 | 6 cycles, −40 °C/4 h + room temperature/1 h, 0.1 M phosphate buffer pH (6.8), 1:6, 1:8 and 1:10 S/L ratios | Continuous damage to the plasmatic membrane, easy to perform, availability | Time and energy consuming, and often require high solvent, leading to an increase in the production costs. Not suitable for industrial scale | [141] |
| 0.4 | ND | 25 °C/4 h, distilled water, 1:25 S/L ratio | [142] | |||
| 0.77 | 217.18 | 3 cycles, −20 °C + room temperature/24 h, 20 mM of sodium acetate and 50 mM of NaCl buffer (pH 5.1) 1:20 S/L ratio | [143] | |||
| 2.10 | 41.90 | 4 cycles, −20 °C/4 h + room temperature/1.5 h, sodium hydroxide, (pH 6.8), 1:20 S/L ratio | [144] | |||
| Mixing and homogenization | 0.6 | 52.11 | 25,200× g/10 min, 0.1 M phosphate buffer (pH 6.8), 1:6 S/L ratio | Simple, availability, reproducibility |
Increased temperatures during the process, time consuming, not suitable for industrial scale, cell debris released | [141] |
| ND | ND | Rotary shaker at 30 °C, 10 mM sodium phosphate buffer (pH 7.0), 10 mM sodium acetate buffer (pH 5.0), NaCl 0.15 M and CaCl2 10 g/L, 1:25 S/L ratio | [142] | |||
| ND | 67.61 | 1.71% biomass/solvent ratio, 6237.66 homogenization rate, 15 min extraction time | [145] | |||
| 0.67 | 103.07 | Oven-dried biomass preparation, 70 °C/4 h, extraction at 25 °C/24 h by 0.01 M phosphate buffer using homogenization assisted method at 0.02 g/mL biomass concentration | [146] | |||
| Bead milling | 0.46 | 217.14 | Low-density beads for low viscosity media, 80–85% degree of bead filling |
Low time, high biomass disruption, low energy | Time very dependent of the type of bead, low purities, cell debris, additional purification steps needed | [144] |
| ND | 119.48 mg/g | Bead diameter 0.3 mm, glass beads at a speed of 3580 rpm. 4 cycles of milling each 25 s and subsequent cooling at 4 °C | [147] | |||
| ND | 90% recovery | Dakot Zirconia beads (0.5–1.4 mm), 330 rpm agitation, 8 h under 0.5 M Ca (II) in a 0.35 M acetate buffer (pH 6.8) | [148] | |||
| 0.21 | ND | Bead Beater, diameter 0.1 mm, agitation of 4800 rpm, 10 cycles of 10 s. Following each cycle, samples cool down in water at 0 °C |
[149] | |||
| ND | 94.90 | Glass beads 0.25–0.5 mm of diameter) in 2 mL flasks, 4 cycles of 25 s at 30 Hz of vibrational frequency |
[150] | |||
| Ultrasound | 0.62 | 51.51 | Pre-soaked for 120 min, ultrasonication amplitude of 50%, 2.5 min, 1:6 S/L | High purities, reproducibility, suitable for industrial scale, temperature could be controlled | Increased temperatures during the process, complex process, expensive, specific equipment | [141] |
| ND | 98.84 | 1% biomass/solvent ratio, 60% amplitude, 16.23 min extraction time | [145] | |||
| 0.67–0.93 | 90.00 | Frequencies of 20–100 kHz, power intensities >1 W/cm2, PBS soaking | [146] | |||
| 0.65 | 18.20 | Power 60 W, extraction for 10 s, 30 cycles in total, in ice bath | [151] | |||
| Electric field | 2.45 | 143.33 | Freeze/thaw and pulsed electric field maximum charging voltage of 30 kV, square bipolar pulses with a variable pulse width of 4–32 μs and a pulse frequency up to 300 Hz. | Increased permeability of the membrane | Long time to optimization, complex equipment, intracellular compounds might not be completely released | [141] |
| 0.51 | 151.94 | 40 °C, 25 kV/cm, 150 μs | [149] | |||
| ND | ND | 50 to 200 pulses at 20 kV | [152] | |||
| High-pressure homogenization | ND | ND | 3.5 min with pressures between 50 and 600 MPa, distilled water ratio of 6% (wt%) | Simpler, scalable for industrial application, environmentally friendly, high recovery | Expensive, not useful in extracting dry biomass, can lead to protein denaturation | [152] |
| ND | 291.90 | 100 mM Na-phosphate solvent (pH 7), 1400 bar | [153] | |||
| 1.2–1.4 | 90% recovery | 300 MPa for 10 min, deionized water or phosphate buffer (pH 6.8), 1/20 (w/v) ratio |
[154] | |||
| Enzyme-assisted | 0.80–0.90 | 20–25 | 1 mg/mL lysozyme, high pressure homogenizer D-15M at 10–12,000 p.s.i., 4–8 °C | Stable, efficient, eco-friendly | More efficient when combined with other methods | [153] |
| 1.19 | 82.07 | 1.0% enzyme concentration, 16 h incubation time, 1:6 S/L ratio | [155] | |||
| 1.09 | 92.73 | 2.5 min Ultrasonication at 50% Amplitude, 0.6% enzyme concentration, 16 h incubation, 1:6 S/L ratio |
* Phycocyanin purity index: A620/A280 > 0.7—food grade, >1.5—cosmetic grade, >3.9—reagent grade, >4.0—analytical grade [41]. ND: Not determined.