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. 2020 Jun 10;12(6):1328.
doi: 10.3390/polym12061328.

Microbial Reclamation of Chitin and Protein-Containing Marine By-Products for the Production of Prodigiosin and the Evaluation of Its Bioactivities

Van Bon Nguyen  1   2 , Dai Nam Nguyen  3 , San-Lang Wang  4   5
Affiliations

Microbial Reclamation of Chitin and Protein-Containing Marine By-Products for the Production of Prodigiosin and the Evaluation of Its Bioactivities

Van Bon Nguyen et al. Polymers (Basel). .

Abstract

Chitin and protein-containing marine by-products (CPCMBPs), including crab shells, squid pens, and shrimp shells, were investigated as the sole carbon/nitrogen (C/N) source for prodigiosin (PG) production by Serratia marcescens TNU01 in a 250 mL Erlenmeyer flask and a 10 L bioreactor system. Among the used C/N source of CPCMBPs, squid pens powder (SPP) showed the most optimum PG productivity. Different ratios of chitin/protein combination were also used as the C/N sources for PG production. With a similar chitin/protein ratio (4/6) of squid pens, a significant PG productivity was achieved when the chitin/protein ratios were controlled in the range of 3/7-4/6. Maximum PG yield (3450 mg/L) by S. marcescens TNU01 was achieved in the bioreactor system containing 3 L medium of 1.75% SPP, 0.03% K2HPO4, and 0.05% MgSO4 at 25 °C for 12 h in dark. The results of in vitro bioassays reveal that the purified PG possesses acetylcholinesterase inhibitory activity and antioxidant as well as anticancer activities. This study suggests that squid pens may have the potential to be used for cost effective production of bioactive PG at a large-scale.

Keywords: AChE inhibitor; anticancer; bioconversion; bioreactor; prodigiosin; protein; β-chitin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PG production by different S. marcescens strains, including S. marcescens TNU01, S. marcescens TNU02, S. marcescens CC17 and S. marcescens TKU011 (a), and the use of different chitin and protein–containing marine by–products for PG production by S. marcescens TNU01 (b). SPP: squid pens powder, SHP: shrimp head powder, deCSP: demineralized crab shells powder, deSSP: demineralized shrimp shells powder.
Figure 2
Figure 2
The influence of chitin/protein ratio (a) and various carbon sources (b) on PG biosynthesis by S. marcescens TNU01. A carbohydrate/protein ratio of 3/7 or 4/6 was used for fermentation.
Figure 3
Figure 3
The effect of sulfate salts (a), added MgSO4 (b), phosphate salts (c), and added K2HPO4 (d), initial pH of the medium (e), cultivation temperature (f), shaking speed (g), fermentation in light (no cover the flask) or in dark (cover the flask) (h), the volume of air headspace percentage (i), and cultivation time (j) on PG production by S. marcescens TNU01.
Figure 4
Figure 4
PG production by S. marcescens TNU01 in 10 L bioreactor systems and in a 100 mL-flask. An amount of 300 mL of seed bacteria was prepared in a flask for 1.5 days and injected in 10L bioreactor systems containing 3 L of liquid medium with other optimized compositions of other parameters as obtained from Section 3.2. The medium was sampled, and PG was detected from 4 to 16 h of fermentation. PG production also by S. marcescens TNU01 in optimal conditions in a 100 mL flask.
Figure 5
Figure 5
The process of PG purification. The liquid culture medium fermented by S. marcescens TNU01 under optimal condition (a) was centrifuged to obtain the supernatant containing PG, which was primarily extracted by ethyl acetate (b). The crude PG containing in the ethyl acetate layer was next separated on a silica gel column (c) and finally, isolated as a pure compound by TLC separation (d).
Figure 6
Figure 6
Mass of purified PG was detected by MALDI-TOF MS spectrum. A matrix, including 2,5-dihydroxybenzoic acid in TFA-H2O-CAN (0.1/50/50%, v/v/v, respectively) solution was used to prepare the sample. The prepared sample was analyzed by MALDI-TOF using a nitrogen laser generator emitting at 337 nm in a linear mode. For each spectrum, the data of 30–50 laser shots were acquired and analyzed.
Figure 7
Figure 7
The spectrum of UV absorption of purified PG produced by S. marcescens TNU01 under optimal condition.
Figure 8
Figure 8
Biological activities of PG, including anticancer activity (a,b), antioxidant activity (c), enzyme inhibitory activity target in anti-diabetes (d), and AChE inhibitory activity: Acetylcholinesterase inhibitory activity (e).
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