- Open Access
Marine organisms as source of bioactive molecules applied in restoration projects
© Barresi et al.; licensee Springer. 2016
- Received: 30 May 2014
- Accepted: 28 April 2015
- Published: 25 May 2015
In recent decades research in the conservation and restoration field has provided sustainable alternatives to traditional procedures for cleaning or controlling the microbial colonization of works of art. In the present study, for the first time novel bioactive molecules extracted from marine invertebrate organisms (Anthozoa) were tested instead of chemical compounds for removing protein layers or as a biocide for controlling fungal or bacterial colonization. In particular, Bioactive Molecules with Protease activity (BMP), acting in a temperature range of 4- 30°C, were tested for the hydrolysis of protein layers on laboratory specimens. The cleaning protocol provides a selective procedure to avoid damage to the original materials constituting the heritage object.
Concurrently, enzymatic cleaning was also performed using commercial Protease from Aspergillus sojae (Type XIX), in order to compare their hydrolytic activities. Bioactive Molecules with Antimicrobial activity (BMA1, BMA2) were tested to control bacterial (Bacillus, Micrococcus) or fungal (Aspergillus, Penicillium) growth, previously isolated from colonized canvas samples and characterized by an integrated approach based on in vitro culture, microscopy and molecular investigations. These molecules were tested to define the Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal/ Fungicidal Concentration (MBC/MFC). Specifically, BMAs were used to control fungal growth during the relining of the painting (laboratory specimens), carried out using a canvas support, and glue paste as binder.
In our hypothesis, these molecules provide an important contribution to the development of innovative protocols for biocleaning or microbial growth control, based on fast and easy application, operator friendly and environmentally sustainable molecules.
- Marine invertebrate
- Protein layer
- Antimicrobial peptides
- Biodegradation control
Great progress has recently been made in the application of bioactive molecules isolated from marine organisms such as sponges, jellyfish, sea-anemones, shellfish (Blue-Biotechnology) representing an important resource useful to the health, food and processing or preservation industries [1-3]. The peculiarities of these molecules are stability, activity at low-temperature and specificity of action. For these reasons new molecules from the body of invertebrate marine organisms (Anthozoa)  were extracted and applied for biocleaning or controlling microbial growth on heritage objects.
The earliest biocleaning attempts date back to 1970, initially performed to remove animal glue layers from paper, canvas and polychrome artefacts, and later to hydrolyze glue paste or protein/oily binder [5,6]. Another available alternative to enzymatic cleaning (stones) is the use of sulphate-reducing bacteria (Desulfovibrio spp.), nitrate-reducing bacteria (Pseudomonas stutzeri) and others [7-9]. Combining the metabolic activity of viable Pseudomonas stutzeri with enzymatic action (Protease) protein layers were removed from the surface of frescoes .
In this study, Bioactive Molecules (BMs) with Proteolytic (BMPs) or Antimicrobial (BMAs) activity were utilized for the biocleaning of casein layers, or to control the microbial growth on glue paste-canvas substrates. BMs were extracted from the body of marine organisms by homogenization (Ultra-Turrax-5 minutes in ice) in TBS (150 mM NaCl/10 mM Tris–HCl pH 7.4) buffer, centrifuged (20’-21,000 g-4°C) and the supernatant recovered. Proteins were analyzed by SDS–polyacrylamide gels, using 5% (w/v) stacking −15% (w/v) separating gel. After running (190 V-45 minutes) the gel was stained in Coomassie solution (2 gr C-Brillant-Blue/500 ml methanol/100 ml acetic acid/400 ml d-water) and de-stained (10% acetic acid/40% methanol/50% d-water). Protein content was estimated by the Bradford method (standard = Bovine Serium Albumine) .
The BMPs showed a high gelatinolytic activity that disappears after adding 1.10-Phenanthroline (metalloproteinase-inhibitor), suggesting that it is a metalloproteinase . Casein layers were stratified on specimens surfaces in order to mimic the removal of overflowing, disfiguring or simply an altered repainting, frequently performed during the lifetime of the painting.
Cleaning tests were carried out on a 2 cm2 casein layer on oil painting specimens laid on linen canvas: i) a preparatory ground (CaCO3/ linseed oil/ochre pigment); ii) a first paint layer (linseed oil/dark green pigment); iii) a second paint layer (casein medium binder/yellow ochre), on which the enzymes act. The first green (for 2,000 hours) and the second casein (for 2,220 hours) layers were artificially aged (UV-A 300–400 nm; T = 22 ± 5°C; RH = 60-65%).
Colour values, were calculated through the CIE 1976 L*, a*, b* (CIELAB) coordinate
1) Green pigment layer
2) Yellow casein layer
After removal of yellow casein layer
3) Green layer
Antimicrobial activity of BMA1 and BMA2 was tested against these microbial taxa, establishing the MIC (Minimal Inhibitory Concentration) and the MBC/MFC, Minimal Bactericidal/Fungicidal Concentration [18-20]. Laboratory tests were performed on two different sets of twelve glue paste – canvas specimens. BMAs or Nipagin-M solutions (final concentration of 1.4 mg/ml) were added in 2.5 ml of glue paste, followed by its deposition on linen or synthetic canvas.
We demonstrate that these bioactive molecules can be utilized in cultural heritage field, by implementing the efficiency of applicative protocols, according to the conservative restoration procedure. This study focalizes on sustainable restoration alternatives which are both operator and environment-friendly, reducing costs and operating times.
This study was developed within the research project It@cha, “Ricerca e Competitività 2007-2013”, PON 01_00625 (FP) and partially supported by FFR-UNIPA research grant (MC). A special acknowledgement to Laboratorio U.O. VIII di Fisica - Centro Regionale per la Conservazione e il Restauro – Palermo for spectrophotometer measurement and S. Schiavone for processing the colorimetric values. Thanks also to Carmela Di Liberto for SEM analysis.
- Salomone M, Cuttitta A, Seidita G, Mazzola S, Bertuzzi F, Ricordi C, et al. Characterization of collagenolytic/proteolytic marine enzymes. Chem Eng Trans. 2012;27:1–6.Google Scholar
- Otero-Gonzàlez AJ, Magalhañes BS, Garcia-Villarino M, Lòpez-Abarrategui C, AmaroSousa D, Campos SD, et al. Antimicrobial peptides from marine invertebrates as a new frontier for microbial infection control. FASEB J. 2010;24:1320–34.View ArticleGoogle Scholar
- Smith VJ, Desbois AP, Dyrynda EA. Conventional and unconventional antimicrobials from fish, marine invertebrates and micro-algae. Mar Drugs. 2010;8:1213–62.View ArticleGoogle Scholar
- Li H, Parisi MG, Parrinello N, Cammarata M, Roch P. Molluscan antimicrobial peptides, a review from activity-based evidences to computer-assisted sequences. Invertebr Surviv J. 2011;8:85–97.Google Scholar
- Wendelbo O, Fosse B. A restoring procedure applied on paper. Restaurator. 1970;1:245–8.Google Scholar
- Cremonesi P. Gli enzimi nella pulitura di opere policrome. Padova: Il Prato; 1999.Google Scholar
- Cappitelli F, Toniolo L, Sansonetti A, Gulotta D, Ranalli G, Zanardini E, et al. Advantages of using microbial technology over traditional chemical technology in removing of black crusts from stone surfaces of historical monuments. Appl Environ Microbiol. 2007;72:5671–5.View ArticleGoogle Scholar
- Bosch-Roig P, Ranalli G. Biocleaning of animal glue on wall paintings by Pseudomonas stutzeri. Chemistry Today. 2013;31(1):50–3.Google Scholar
- Bosch-Roig P, Regidor-Ros JL, Montes-Estellés RM. Biocleaning of nitrate alterations on wall paintings by Pseudomonas stutzeri. Int Biodeter Biodegr. 2013;84:266–74.View ArticleGoogle Scholar
- Ranalli G, Belli C, Alfano G, Lustrato G, Colombini MP, Bonaduce I, et al. Biotechnology applied to cultural heritage: biorestoration of frescos using viable bacterial cells and enzymes. J Appl Microbiol. 2005;98:73–83.View ArticleGoogle Scholar
- Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.View ArticleGoogle Scholar
- Mackay AR, Ballin M, Pelina MD, Farina AR, Nason AM, Hartzler JL, et al. Effect of phorbol ester and cytokines on matrix metalloproteinase and tissue inhibitor of metalloproteinase expression in tumor and normal cell lines. Invasion Metastasis. 1992;12:168–84.Google Scholar
- CIE 15: 2004. Colorimetry, 3rd edition, 2004.Google Scholar
- Hackney S: Paintings on Canvas, Lining and Alternatives. TATE Papers, 2013: 1–12.Google Scholar
- Palla F. Analytical techniques. In Science and Conservation for Museum Collections. Edited by Fabbri B, Nardini: Firenze; 2012: 459–470Google Scholar
- Palla F, Billeci N, Mancuso FP, Pellegrino L, Lorusso LC. Microscopy and molecular biology techniques for study biocenosis diversity in semi-confined environments. Conservation Science in Cultural Heritage. 2010;10:185–94.Google Scholar
- Palla F, Barresi G, Di Carlo E. Identification of bacterial taxa in archaeological waterlogged wood. Conservation Science in Cultural Heritage. 2014;14:247–62.Google Scholar
- Klare I, Konstabel C, Muller-Berting S, Reissbrodt R, Huys G, Vancanneyt M, et al. Evaluation of new broth media for microdilution antibiotic susceptibility testing of lactobacilli, pediococci, lactococci and bifidobacteria. Appl Environ Microbiol. 2005;7:8982–898.View ArticleGoogle Scholar
- Domig KJ, Mayrhoter S, Zitz U, Mair C, Petersson A, Amtmann E. Antibiotic susceptibility testing of Bifidobacterium thermophilum and Bifidobacterium pseudolonum: Broth microdiluition vs. agar disc diffusion assay. Int J Food Microbiol. 2007;120:191–5.View ArticleGoogle Scholar
- National Committee for Clinical Laboratory Standards. Reference method for both dilution antifungal susceptibility testing of yeasts; Approved standard - Second edition. NCCLS document M27-A2. Wayne; PA 22, 15; 2002.Google Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.