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Transformation of the matrix structure of shrimp shells during bacterial deproteination and demineralization.

Xu Y, Bajaj M, Schneider R, Grage SL, Ulrich AS, Winter J, Gallert C - Microb. Cell Fact. (2013)

Bottom Line: The viscosity of biologically processed chitin was compared with chemically processed chitin.From transmission electron microscopy (TEM) overlays and electron energy loss spectroscopy (EELS) analysis, it was found that most protein was present in the exocuticle, whereas most chitin was present in the endocuticle.Bioprocessing of shrimp shell waste resulted in a chitin with high purity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biology for Engineers and Biotechnology of Wastewater, Karlsruhe Institute of Technology, Am Fasanengarten, Karlsruhe 76131, Germany. mini.bajaj@kit.edu.

ABSTRACT

Background: After cellulose and starch, chitin is the third-most abundant biopolymer on earth. Chitin or its deacetylated derivative chitosan is a valuable product with a number of applications. It is one of the main components of shrimp shells, a waste product of the fish industry. To obtain chitin from Penaeus monodon, wet and dried shrimp shells were deproteinated with two specifically enriched proteolytic cultures M1 and M2 and decalcified by in-situ lactic acid forming microorganisms. The viscosity of biologically processed chitin was compared with chemically processed chitin. The former was further investigated for purity, structure and elemental composition by several microscopic techniques and (13)C solid state NMR spectroscopy.

Results: About 95% of the protein of wet shrimp shells was removed by proteolytic enrichment culture M2 in 68 h. Subsequent decalcification by lactic acid bacteria (LAB) took 48 h. Deproteination of the same amount of dried shrimps that contained a 3 × higher solid content by the same culture was a little bit faster and was finished after 140 h. The viscosity of chitin was in the order of chemically processed chitin > bioprocessed chitin > commercially available chitin. Results revealed changes in fine structure and chemical composition of the epi-, exo- and endocuticle of chitin from shrimp shells during microbial deproteination and demineralization. From transmission electron microscopy (TEM) overlays and electron energy loss spectroscopy (EELS) analysis, it was found that most protein was present in the exocuticle, whereas most chitin was present in the endocuticle. The calcium content was higher in the endocuticle than in the exocuticle.13C solid state NMR spectra of different chitin confirmed < 3% impurities in the final product.

Conclusions: Bioprocessing of shrimp shell waste resulted in a chitin with high purity. Its viscosity was higher than that of commercially available chitin but lower than that of chemically prepared chitin in our lab. Nevertheless, the biologically processed chitin is a promising alternative for less viscous commercially available chitin. Highly viscous chitin could be generated by our chemical method. Comprehensive structural analyses revealed the distribution of the protein and Ca matrix within the shrimp shell cuticle which might be helpful in developing shrimp waste processing techniques.

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SEM and TEM of untreated waste shrimp shells. a) SEM micrograph of a thin section and b) TEM micrograph of different regions of a P. monodon shrimp-shell. A = Epicuticle (with a channel in Figure 3b), B = exocuticle, C = endocuticle and D = inner membrane of shrimp shells.
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Figure 3: SEM and TEM of untreated waste shrimp shells. a) SEM micrograph of a thin section and b) TEM micrograph of different regions of a P. monodon shrimp-shell. A = Epicuticle (with a channel in Figure 3b), B = exocuticle, C = endocuticle and D = inner membrane of shrimp shells.

Mentions: Viscosity is an important factor which indicates desired properties of chitin or chitosan. It is one of the major quality criteria for industrial production of chitosan and its applications. For this reason, the viscosity of chitin that was obtained by biological deproteination and decalcification was compared with chitin which was prepared by sodium hydroxide hydrolysis of proteins and subsequently, calcium was removed by hydrochloric acid. Chitin from chemical protein hydrolysis and decalcification had a much higher viscosity than the chitin that was prepared by bacterial deproteination and demineralization. Beaney et al. [9] also found that the quality of chemically processed chitin was better than the chitin processed with lactic acid fermentation from the shells of Nephrops norvegicus. In the present study, the chemically and biologically processed chitin batches were compared with a 99% pure chitin purchased from a chemicals company. The commercially available chitin had the lowest viscosity (Table 3). Therefore, a linear relationship between the chitin purity and viscosity may be ruled out and as evident from the results, protein and mineral contents are not the only parameters to be considered for the chitin quality. In our previous study [1], viscosities of chitin and chitosan extracted from shrimp shells were highly dependent on the method of preparation, even when the degree of deproteination and decalcification were similar in the end product. The SEM micrograph of chemically prepared chitin (Figure 2) clearly indicates that the shell cuticles were not intact anymore after harsh chemical treatment when compared to untreated shells (Figure 3A). Bade et al. [10] reported that after acid hydrolysis, the chitin structure no longer retained its native fine fibrous and covalently stabilized form, and it lost its resistance to chitinase activity of autochthonic bacteria. Structural disaggregation of chitin during defined chemical or biological purification might have led to a loosely packed chitin which could be dissolved in N-methyl-2-pyrrolidone (NMP)/LiCl solution and resulted in a liquid with a high viscosity. Although the commercially available chitin had the highest purity, it was least soluble in N-methyl-2-pyrrolidone (NMP)/LiCl solution and had the lowest viscosity. Molecular weight distribution after purification and the grade of polymerisation are apparently main factors that determine the viscosity of chitin. Different batches of biologically processed chitin from Penaeus monodon had molecular weights ranging from 0.3 - 1.8 MDa. Constant viscosities of chitin or chitosan solutions are required for application in the food and pharma industry [1,4] or for supply of chitin/gelatin membranes for tissue engineering [11].


Transformation of the matrix structure of shrimp shells during bacterial deproteination and demineralization.

Xu Y, Bajaj M, Schneider R, Grage SL, Ulrich AS, Winter J, Gallert C - Microb. Cell Fact. (2013)

SEM and TEM of untreated waste shrimp shells. a) SEM micrograph of a thin section and b) TEM micrograph of different regions of a P. monodon shrimp-shell. A = Epicuticle (with a channel in Figure 3b), B = exocuticle, C = endocuticle and D = inner membrane of shrimp shells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852495&req=5

Figure 3: SEM and TEM of untreated waste shrimp shells. a) SEM micrograph of a thin section and b) TEM micrograph of different regions of a P. monodon shrimp-shell. A = Epicuticle (with a channel in Figure 3b), B = exocuticle, C = endocuticle and D = inner membrane of shrimp shells.
Mentions: Viscosity is an important factor which indicates desired properties of chitin or chitosan. It is one of the major quality criteria for industrial production of chitosan and its applications. For this reason, the viscosity of chitin that was obtained by biological deproteination and decalcification was compared with chitin which was prepared by sodium hydroxide hydrolysis of proteins and subsequently, calcium was removed by hydrochloric acid. Chitin from chemical protein hydrolysis and decalcification had a much higher viscosity than the chitin that was prepared by bacterial deproteination and demineralization. Beaney et al. [9] also found that the quality of chemically processed chitin was better than the chitin processed with lactic acid fermentation from the shells of Nephrops norvegicus. In the present study, the chemically and biologically processed chitin batches were compared with a 99% pure chitin purchased from a chemicals company. The commercially available chitin had the lowest viscosity (Table 3). Therefore, a linear relationship between the chitin purity and viscosity may be ruled out and as evident from the results, protein and mineral contents are not the only parameters to be considered for the chitin quality. In our previous study [1], viscosities of chitin and chitosan extracted from shrimp shells were highly dependent on the method of preparation, even when the degree of deproteination and decalcification were similar in the end product. The SEM micrograph of chemically prepared chitin (Figure 2) clearly indicates that the shell cuticles were not intact anymore after harsh chemical treatment when compared to untreated shells (Figure 3A). Bade et al. [10] reported that after acid hydrolysis, the chitin structure no longer retained its native fine fibrous and covalently stabilized form, and it lost its resistance to chitinase activity of autochthonic bacteria. Structural disaggregation of chitin during defined chemical or biological purification might have led to a loosely packed chitin which could be dissolved in N-methyl-2-pyrrolidone (NMP)/LiCl solution and resulted in a liquid with a high viscosity. Although the commercially available chitin had the highest purity, it was least soluble in N-methyl-2-pyrrolidone (NMP)/LiCl solution and had the lowest viscosity. Molecular weight distribution after purification and the grade of polymerisation are apparently main factors that determine the viscosity of chitin. Different batches of biologically processed chitin from Penaeus monodon had molecular weights ranging from 0.3 - 1.8 MDa. Constant viscosities of chitin or chitosan solutions are required for application in the food and pharma industry [1,4] or for supply of chitin/gelatin membranes for tissue engineering [11].

Bottom Line: The viscosity of biologically processed chitin was compared with chemically processed chitin.From transmission electron microscopy (TEM) overlays and electron energy loss spectroscopy (EELS) analysis, it was found that most protein was present in the exocuticle, whereas most chitin was present in the endocuticle.Bioprocessing of shrimp shell waste resulted in a chitin with high purity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biology for Engineers and Biotechnology of Wastewater, Karlsruhe Institute of Technology, Am Fasanengarten, Karlsruhe 76131, Germany. mini.bajaj@kit.edu.

ABSTRACT

Background: After cellulose and starch, chitin is the third-most abundant biopolymer on earth. Chitin or its deacetylated derivative chitosan is a valuable product with a number of applications. It is one of the main components of shrimp shells, a waste product of the fish industry. To obtain chitin from Penaeus monodon, wet and dried shrimp shells were deproteinated with two specifically enriched proteolytic cultures M1 and M2 and decalcified by in-situ lactic acid forming microorganisms. The viscosity of biologically processed chitin was compared with chemically processed chitin. The former was further investigated for purity, structure and elemental composition by several microscopic techniques and (13)C solid state NMR spectroscopy.

Results: About 95% of the protein of wet shrimp shells was removed by proteolytic enrichment culture M2 in 68 h. Subsequent decalcification by lactic acid bacteria (LAB) took 48 h. Deproteination of the same amount of dried shrimps that contained a 3 × higher solid content by the same culture was a little bit faster and was finished after 140 h. The viscosity of chitin was in the order of chemically processed chitin > bioprocessed chitin > commercially available chitin. Results revealed changes in fine structure and chemical composition of the epi-, exo- and endocuticle of chitin from shrimp shells during microbial deproteination and demineralization. From transmission electron microscopy (TEM) overlays and electron energy loss spectroscopy (EELS) analysis, it was found that most protein was present in the exocuticle, whereas most chitin was present in the endocuticle. The calcium content was higher in the endocuticle than in the exocuticle.13C solid state NMR spectra of different chitin confirmed < 3% impurities in the final product.

Conclusions: Bioprocessing of shrimp shell waste resulted in a chitin with high purity. Its viscosity was higher than that of commercially available chitin but lower than that of chemically prepared chitin in our lab. Nevertheless, the biologically processed chitin is a promising alternative for less viscous commercially available chitin. Highly viscous chitin could be generated by our chemical method. Comprehensive structural analyses revealed the distribution of the protein and Ca matrix within the shrimp shell cuticle which might be helpful in developing shrimp waste processing techniques.

Show MeSH
Related in: MedlinePlus