Evaluating Methods for Optimal Phycocyanin Extraction and Purification

 Introduction: 

     In the previous week, a two factor DOE was started in order to determine statistical significance of activated charcoal and two percent chitosan solution for purification of C-PC crude extract. Eight tubes, each with one gram of Spirulina plantesis, 0.25 grams of glass beads, and ten milliliters of deionized water were all sonicated in an ultrasonic bath and centrifuged twice to be read for initial purity values which could be compared to post purification results. The degradation of phycocyanin once isolated from spirulina has been an ongoing issue for accurate testing. It has been found that predominantly exposure to light and unfavorable pH values accelerated phycocyanin degradation,. Minimal light exposure and an environmental pH between 5.0 and 6.0 are ideal conditions for optimal stability (Wu et al., 2016).  It should be assumed going forward in this DOE that tubes were wrapped in aluminum foil tp prevent light exposure whenever possible, unless otherwise specified. 

Methods:

    All tubes were removed from freezer and set out to thaw at room temperature. Once liquid, each tube was tested for purity values in order to test for any changes from previous results. Tubes 1A and 1B were stored in a refrigerator to be tested later as the control group. Tubes 2A and 2B were put in a PD-10 column with 0.7 grams of charcoal which had been ground with a pestle and mortar, and surgical cotton previously hydrated with approximately one centimeter of deionized water. The outside of the column was wrapped in aluminum foil, and seven test tubes (a, b, c, d, e, f, and g) were used to evaluate the concentration of phycocyanin being filtered throughout the process. A ten ml syringe was used to apply gradual pressure in order to promote successful filtration. One ml of two percent chitosan solution was added to tubes 3A, 3B, 4A, and 4B to allow for thirty minutes of sedimentation. Tubes 3A and 3B were filtered in a column with cotton, no charcoal, aided by the syringe. Tubes 4A and 4B were put in a column identical to tubes 2A and 2B and filtered. Absorbance values were tested with a nanodrop. 

Results:

    Figure one is the initial recorded results of all tubes prior to purification once thawed completely. The purity value of 2B (3.316) was much higher than all other tubes. Every tube declined in purity as compared to the week prior, with the tubes with the most significant difference being tubes 1A (1.425), 2A (1.237), and 4B (2.035). Figure two is a re-evaluation of Figure one's results due to a lack of confidence in accuracy, which shows three individual purity values gathered for each tube and averaged. The highest purity value was then tube 4B (2.563), and the lowest being 4A(1.564). Figure three is the results of each tube post purification. The highest purity values were seen from tubes 1A (1.777) and 1B (2.139), and the lowest being 3B (0.612) and 4B (0.438). 

Tube

A620

A280

A652

Purity (A620/A280)

1A

2.349

1.648

2.439

1.425

1B

2.364

1.358

2.394

1.741

2A

2.195

1.407

2.350

1.237

2B

1.658

0.500

2.160

3.316

3A

2.975

1.751

2.250

1.699

3B

2.634

1.402

2.191

1.879

4A

2.408

1.278

3.311

1.884

4B

2.395

1.177

2.884

2.035

Figure 1


Tube

P1

P2

P3

PAVG

2A

1.706

2.046

2.694

2.149

2B

2.517

2.012

2.141

2.223

3A

2.077

2.06

1.661

1.933

3B

1.589

2.048

1.493

1.709

4A

1.636

1.712

1.344

1.564

4B

2.897

2.847

1.944

2.563

Figure 2


Tube

A620

A280

A652

Purity (A620/A280)

1A

2.447

1.377

2.275

1.777

1B

2.604

1.217

2.279

2.139

2A

2.233

2.174

1.236

1.027

2B

2.256

2.593

1.294

0.870

3A

2.347

2.693

1.252

0.872

3B

1.317

2.153

0.658

0.612

4A

2.932

2.679

1.379

1.094

4B

1.061

2.420

0.585

0.438

Figure 3

Conclusion:

    The data suggests multiple issues with this protocol for purification. The variety of results recorded from identical tubes, or even the same tube, suggests that test results are not accurate. Sediment may be present before and after purification, which is skewing the results. Further tests should be done by centrifuging samples in order to ensure sediments are not still present in these stages. It is also concerning that either charcoal or chitosan, and the use of both, significantly decreased purity levels when the intended goal was the opposite effect. The absorbance values at 280 nm seem to increase and 620 and 652 values decrease in general, and there is no current reason that can be deduced for these results. It should be further investigated whether or not these substances are actually effective for purification purposes, as well as the use of other items such as a frit or filter paper in comparison to surgical cotton. The adaptability of such processes should also be considered for future large scale extractions. 

References:

1. Hua-Lian Wu, Guang-Hua Wang, Wen-Zhou Xiang, Tao Li & Hui He (2016) Stability and Antioxidant Activity of Food-Grade Phycocyanin Isolated from Spirulina platensis, International Journal of Food Properties, 19:10, 2349-2362, DOI:10.1080/10942912.2015.103856

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