10.6092/issn.1973-9494/10347
Damage to buildings and monuments by microbiological growth is a cause of serious concern. Due to the necessity of treatment being assessed for in situ application, it is important to select a safe and effective strategy approach that safeguards both the environment and human beings. In the past decade the most frequently used and effective biocides have been banned due to their environmental and health hazards. This paper reports the development of remediation actions based on environmentally innocuous alternatives derived from active compounds produced by Bacillus sp. in conjugation with natural plant products from Brazilian Cerrado plant extracts.
application/pdf
1973-9494
10347
en
Ana Teresa Caldeira
Conservation Science in Cultural Heritage
antifungal activity
Green Bioactive Compounds: Mitigation Strategies for Cultural Heritage
Text.Serial.Journal
https://conservation-science.unibo.it/
19
Articles
2020-03-18
2020-01-10
2020-03-18
2020-03-18
133-142
CSCH
Ana Teresa Caldeira
2019
10.6092/issn.1973-9494/10347
133
HERCULES Laboratory, University of Évora, Évora, Portugal; 
Department of Chemistry, School of Sciences and Technology
University of Évora, Évora
1973-9494
antifungal activity
en
142
Green Bioactive Compounds: Mitigation Strategies for Cultural Heritage
https://conservation-science.unibo.it/article/download/10347/10603
19
G     REEN BIOACTIVE COMPOUNDS: MITIGATION
      STRATEGIES FOR CULTURAL HERITAGE

   Mara Silva, Tânia Rosado
   HERCULES Laboratory, University of Évora, Évora, Portugal

   Zita Lopes da Silva
   HERCULES Laboratory, University of Évora, Évora, Portugal; Department of Phar-
   maceutical Sciences, School of Health Sciences University of Brasilia, Brasilia-DF,
   Brazil

   Yanna Nóbrega, Dâmaris Silveira
   Department of Pharmaceutical Sciences, School of Health Sciences University of
   Brasilia, Brasilia-DF, Brazil

   António Candeias, Ana Teresa Caldeira*1
   HERCULES Laboratory, University of Évora, Évora, Portugal; Department of Chem-
   istry, School of Sciences and Technology University of Évora, Évora, Portugal


   Keywords: biodegradation, Bacillus sp., Pouteria ramiflora, antifungal activity


   1. Introduction

    The preservation of historic monuments and buildings constitutes a high societal prior-
ity in order for future generations to continue to witness their ancestors’ achievements [1].
    Biodeterioration phenomena represent a combination of physical and chemical altera-
tion processes due to microorganisms in various materials, such as those constituting the
objects that represent our cultural heritage [2]. Despite the involvement of several microor-
ganisms in the deterioration process, the specific role of fungi is particularly central due to
their high level of proliferation that can induce severe chemical attack in the materials [3-4].
Only the identification of the microbial communities associated with the different materials
and the understanding of the role of such communities in the biodeterioration processes will
enable the prevention and/or remediation of the problems related to bio-decay [5]. Several
authors have reported biocide characteristics to eliminate biodeteriogenic organisms on
stone monuments [6-8]. However, in recent years many of the most effective biocides have
been banned due to their environmental and health hazards [7, 9].
    Thus, the development of accurate remediation actions for microbiologically contami-
nated historic materials, based on environmentally safe solutions, is of vital importance.
    Bacillus species are emerging as a promising alternative for built heritage treatment and re-
habilitation due to their capacity to produce a great diversity of secondary metabolites with bio-
logical activity [10-11] such as surfactin, fengycin and iturin. The nontoxic mechanisms of action
of these amphiphilic cyclic biosurfactants are therefore directly related, having unique features,
high biodegradability, and non-harmful and environmentally friendly characteristics [12].

*1 Corresponding author: atc@uevora.pt
                                                                                                    Another alternative are medicinal plants, which have been widely used against
                                                                                                 pathogenic microorganisms in many parts of the world due to the effective anti-
                                                                                                 microbial activity of their extracts, such as essential oils. Plants of the Brazilian
                                                                                                 Cerrado, a tropical highland savanna in the midwestern region of Brazil have been
                                                                                                 broadly used in popular medicine. This region is characterized by an enormous
                                                                                                 range of extracts and essential oils with proven potential antifungal activity [13-
                                                                                                 15]. Several plants found in this biome belonging to different families, e.g. Apocyn-
                                                                                                 aceae, Sapotaceae, Fabaceae, are known for their antimicrobial, anti-inflammato-
                                                                                                 ry and antifungal activity [16-17]. One of them, Pouteria ramiflora (Sapotaceae) is
                                                                                                 known for its edible fruits and medicinal properties and is used in the treatment of
                                                                                                 obesity, helminthiasis, dysentery or inflammation. The literature reports different
                                                                                                 biological activities for this plant; however, few studies about its antifungal proper-
A.T. Caldeira et al. - Green bioactive compounds: mitigation strategies for cultural heritage




                                                                                                 ties are described.
                                                                                                    This work combines the potential of these two natural sources of green active
                                                                                                 compounds and intends to develop a natural green alternative to chemical bioc-
                                                                                                 ides, thus contributing to a sustainable and environmentally safe conservation-
                                                                                                 restoration process for built cultural heritage.


                                                                                                   2. Materials and methods


                                                                                                   2.1. Bacillus bioactive compounds antifungal activity assessment

                                                                                                    The Bacillus sp. CCLBH 1053 (Genbank accession number AY785774) were
                                                                                                 inoculated in 100 mL NB (Nutrient Broth, HIMEDIA) media for 72 hours at 30ºC in
                                                                                                 an orbital shaker at 150 rpm. After 48 hours (stationary-phase) of culture growth,
                                                                                                 the bacterial cells were removed from the culture by centrifugation and the sec-
                                                                                                 ondary metabolites with antifungal potential (BEVOTECH 3, BEVOTECH 4, BEV-
                                                                                                 OTECH 5) were maintained at -20ºC for further analysis [18, 11]. The biodeterio-
                                                                                                 genic strains isolated from biodegraded mural paintings, Penicillium glandicola
                                                                                                 CCLBH-MP101, Alternaria sp. CCLBH-MP401, Fusarium oxysporum CCLBH-I302
                                                                                                 and Mucor sp. CCLBH-AA501 and belonging to the laboratory collection (HERCU-
                                                                                                 LES Biotech laboratory, Évora University) were used to perform antifungal tests
                                                                                                 to evaluate their potential to inhibit fungal proliferation. These assays were con-
                                                                                                 ducted according to previous study [11].


                                                                                                   2.2. Pouteria ramiflora extracts antifungal activity assessment

                                                                                                     2.2.1. Toxicity assay in Artemia salina
                                                                                                     The Pouteria ramiflora extracts used in this work were provided by the research
                                                                                                 group of the Laboratory of Natural Products, Faculty of Health Sciences, Universi-
                                                                                                 ty of Brasilia (UnB). The aqueous (AE) and ethanolic extracts (EE) used in antifun-
                                                                                                 gal assays were prepared by drying and powdering the Pouteria ramiflora leaves
                                                                                                 at room temperature. The ethanol crude extract was obtained by the maceration of
                                                                                                 plant material (40 g) at room temperature for seven days, with ethanol (2 L). After
                                                                                                 filtration, the solvents were removed under reduced pressure. The aqueous crude


                                                                                           134
extract was obtained by infusion. After the filtration procedure, water was removed
by lyophilization [19].
    The toxicity of the EE and AE was evaluated using the Artemia salina test
kit (Artoxkit MTM, Microbiotest). The Pouteria ramiflora extracts were tested in a
range of concentrations 50-3125 mg/L to establish lethal concentrations (LC50 –
lethal concentration 50%) [20, 9].

   2.2.2. Antifungal activity assay
   The evaluation of the antifungal activity of Pouteria ramiflora extracts against
biodeterogenic fungi isolated from artworks, Cladosporium sp. CCLBH-MP601,
Ulocladium sp. CCLBH-EP701, Fusarium tricinotum CCLBH-MP303, Penicillium
sp. CCLBH-EP102 and Aspergillus niger CCLBH-MP202, was carried out using
antifungal disc diffusion assay [10, 9]. The results of this assay were represented
according to a qualitative classification: +/− not determined (inhibition halo >12.7
mm), + positive test (inhibition halo = 12.7 mm), ++ (inhibition halo <12.7 mm).
The microdilution method was conducted according to (Clinical Laboratory Stand-
ards Institute (CLSI) standard M27-A3) [21], using a biodeteriogenic yeast iso-
lated from a mural painting, Rhodotorula sp. CCLBH-YMP502,


  3. Results and discussion

   The methodologies and products used for the conservation of artworks must be
chosen bearing in mind the microbial population and the environmental impact,
avoiding negative effects on/in the materials. Many strains are known to suppress
fungal growth due to the production of antifungal antibiotics [22-27] especially cy-
clic lipopeptides that show a great potential for biotechnological, biopharmaceuti-
cal and agricultural applications.




                                                                                             CONSERVATION SCIENCE IN CULTURAL HERITAGE
   The antifungal capacity of bioactive compounds produced by Bacillus strains
was confirmed by means of antifungal tests to verify its efficiency as a green bio-
cide capable of suppressing the proliferation of biodeteriogenic fungi on heritage.
   Three formulations of bioactive compounds produced by Bacillus were tested
against Penicillium glandicola CCLBH-MP101, Alternaria sp. CCLBH-MP401,
Fusarium oxysporum CCLBH-I302 and Mucor sp. CCLBH-AA501. Figure 1 shows
the antifungal activity results.




                                                                                       135
A.T. Caldeira et al. - Green bioactive compounds: mitigation strategies for cultural heritage




                                                                                                 Figure 1. Antifungal activity of BEVOTECH 3, BEVOTECH 4, BEVOTECH 5 against biodeteriogenic
                                                                                                 fungi. Penicillium sp.; Alternaria sp.; Mucor sp.; Fusarium oxysporium.



                                                                                                     The antifungal assay, using the Bacillus bioactive compounds BEVOTECH 3,
                                                                                                 BEVOTECH 4, BEVOTECH 5, showed a high inhibition level, independently of the
                                                                                                 fungal isolate tested, confirming previous studies that show the potential of BEVO-
                                                                                                 TECH to be applied in the rehabilitation of cultural heritage and moreover, the ab-
                                                                                                 sence of toxicity [11, 28, 9].
                                                                                                     To find new, natural and effective solutions for treating biodegradation and broaden-
                                                                                                 ing the spectrum of BEVOTECH’s action, Pouteria ramiflora plant extracts from the
                                                                                                 Brazilian Cerrado which in previous studies showed a strong antifungal capacity in a
                                                                                                 medical context, were also tested [19, 29].
                                                                                                     To evidence the non-toxicity of Pouteria ramiflora, both an ethanolic extract (EE)
                                                                                                 and aqueous extract (AE) were analysed using brine shrimp Artemia salina for the
                                                                                                 determination of LC50. The results are presented in Figure 2.
                                                                                                     The results show that with a concentration of 50 mg/mL the AE presents less toxic-
                                                                                                 ity than the EE and does not cause the death of any brine shrimp. However, for the
                                                                                                 highest concentration tested (3125 mg/L) both the EE and AE caused similar mortality
                                                                                                 (65% for AE and 63% for EE).
                                                                                                     For both the EE and AE concentrations tested, none of the extracts caused
                                                                                                 100 % mortality. The AE fraction showed a LC50 of 1806 ± 1.03 mg / L and for the
                                                                                                 EE, the LC50 is close to 1560 mg / L. Both extracts proved to be considerably less
                                                                                                 toxic than chemical synthesis biocides used in the treatment and conservation of
                                                                                                 artworks, mainly AE [9], proving it to possess green characteristics and its poten-
                                                                                                 tial to provide a valid alternative to the toxic chemical synthesis biocides usually
                                                                                                 applied.



                                                                                           136
Figure 2. Toxicity of AE and EE at concentrations of 50 and 3125 mg/L in Artemia salina. (    AE;
    EE).



    In order to evidence the antifungal potential of both the EE and AE obtained from
Pouteria ramiflora, in the context of cultural heritage, an antifungal assay was accom-
plished using five different filamentous fungi isolated from biodegraded artworks Cla-
dosporium sp. CCLBH-MP601, Ulocladium sp. CCLBH-EP701, Fusarium tricinotum
CCLBH-MP303, Penicillium sp. CCLBH-EP102 and Aspergillus niger CCLBH-MP202.

Table 1. Inhibition growth capacity of Ethanolic extract (EE) and Aqueous extract (AE) against
biodeteriogenic fungi.




                                                                                                          CONSERVATION SCIENCE IN CULTURAL HERITAGE
                                                       EE                         AE
Biodeteriogenic fungi
                                             12.5      25       50      12.5      25         50
Cladosporium sp. CCLBH-MP601                   +        +       ++       +/-      +/-        +
Ulocladium sp. CCLBH-EP701                    +/-      +/-      +/-      +/-       +         +
Fusarium tricinotum CCLBH-MP303               ++       ++       ++       ++       ++         ++
Penicillium sp. CCLBH-EP102                   +/-      ++       ++       +/-      ++         ++
Aspergillus niger CCLBH-MP202                 +/-       +       ++       +/-      +/-        +

+/− not determined (inhibition halo >12.7 mm; + positive test (inhibition halo = 12.7 mm), ++
(inhibition halo <12.7 mm)



   The results show that Fusarium tricinotum CCLBH-MP303 was the most inhib-
ited strain by the EE and AE (Table 1). Mostly, concentrations of 50 and 25 mg/
mL for both extracts had an inhibitory action higher than 12.5 mg/mL. In the case
of EE, the results showed a greater inhibitory capacity compared with AE. From
a future perspective, a combination of EE and BEVOTECH may be considered to
enhance the antifungal effect of both and thus create a totally green biocide with
real effectiveness against fungal biodeteriogenic strains.


                                                                                                    137
                                                                                                     Additionally, the minimum inhibitory concentration (MIC) of the two crude extracts
                                                                                                 tested against biodeteriogenic yeast isolated from contaminated mortars Rhodoto-
                                                                                                 rula sp. CCLBH-YMP502 were also determined. The assay shows that both the EE
                                                                                                 and AE from Pouteria ramiflora present a MIC value of 6.25 mg/mL (Figure 3), a sig-
                                                                                                 nificantly low inhibitory concentration that evidences the potential of these extracts to
                                                                                                 be used to prevent the proliferation of this biodeteriogenic yeast.
A.T. Caldeira et al. - Green bioactive compounds: mitigation strategies for cultural heritage




                                                                                                 Figure 3. Minimum inhibitory concentration (MIC) of AE and EE against Rhodotorula sp. PC-
                                                                                                 Positive control; SC-Solvent control; A- Aqueous extract; B- Ethanolic extract



                                                                                                    The results reveal the promising characteristic of the Pouteria ramiflora extracts to
                                                                                                 be used as an antifungal agent simultaneously against biodeteriogenic yeast and fila-
                                                                                                 mentous fungi, as its action is able to increase the effect of the Bevotech compounds.
                                                                                                    Therefore, the antifungal activity of both natural products from Pouteria ramiflora
                                                                                                 and the bioactive compounds produced by Bacillus strains have shown they are ef-
                                                                                                 fective against a set of filamentous fungi and yeast in a wide range of concentrations,
                                                                                                 thus making them prominent alternatives to be used separately or in a combined form
                                                                                                 as new additives for novel green biocides for cultural heritage. In conclusion, the tested
                                                                                                 products amplify the spectra of natural alternatives, paving the way to a new environ-
                                                                                                 mentally friendly biocide as an alternative to common chemical synthetized biocides,
                                                                                                 which can represent a serious risk for the environment, the artwork itself or even the
                                                                                                 conservator-restorer who handles it.


                                                                                                   Acknowledgements

                                                                                                    The authors gratefully acknowledge the funding source: Project “MEDUSA-Micro-
                                                                                                 organisms Monitoring and Mitigation–Developing and Unlocking Novel Sustainable
                                                                                                 Approaches” ref. ALT20-03-0145-FEDER-000015, co-financed by the European Un-
                                                                                                 ion through the European Regional Development Fund ALENTEJO 2020 (Regional
                                                                                                 Operational Programme of the Alentejo), and National Council for the Improvement of
                                                                                                 Higher Education (CAPES) for the Zita Lopes da Silva research scholarship (Capes/
                                                                                                 PDSE/ Process nº 88881.133369/2016-01).


                                                                                           138
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[29] Lopes Da Silva, Z., (2018). Avaliação da capacidade antifungica de extractos de
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  Biographical notes

  Mara Silva, PhD in Biochemistry. HERCULES Laboratory, University of Évora.

  Zita Lopes da Silva, PhD in Pharmaceutical Sciences at Department of Pharma-
ceutical Sciences, School of Health Sciences, University of Brasilia.

  Yanna Nóbrega is Full Professor in Pharmacy Department at University of Brasília.
She does research in the field of Natural Products Chemistry.

  Dâmaris Silveira is professor in Pharmacy Department at University of Brasília.
She does research in the field of Natural Products Chemistry.

   Tânia Rosado, PhD in Biochemistry. HERCULES Laboratory, University of Évora.
She is a Postdoctoral Researcher who has contributed to the development and imple-
mentation of innovative approaches on materials characterisation, biodecay studies of
cultural heritage assets (mural paintings, easel paintings, wood materials, paper, mor-
tars, rock art painting, rock materials buildings and stone monuments), identification
of biocolonisers and their role on the biodegradation/biodeterioration processes. She
has also been involved in developing and testing the effectiveness and safety of green
alternatives for biocolonisation cleaning, treatment and prevention.

    António Candeias is specialized in chemistry applied to cultural heritage and sur-




                                                                                                  CONSERVATION SCIENCE IN CULTURAL HERITAGE
face chemistry. He is Vice-rector for Research and Development and professor in the
Chemistry Department of the University of Évora, Portugal. In terms of research activ-
ity, his main interests have focused on the study of mortars and renders, the study and
characterization of cultural assets and archaeological materials and the optimization
and application of analytical techniques for the study of cultural heritage, with special
emphasis on micro-analysis and in-situ analysis.

   Ana Teresa Caldeira is professor in the Department of Chemistry at the Univer-
sity of Évora, specialized in biochemical analysis and microbial physiology. She is
the coordinator of the Biotechnology and Biodegradation Unit of the HERCULES
Lab. Her main areas of research focus on new strategies for diagnosis, monitoring
and mitigation applied to cultural heritage including new methods for measuring bio-
degradation, development of new green biocides and methodologies for the identifi-
cation of biomolecules.


    Summary
    Damage to buildings and monuments by microbiological growth is a cause of seri-
ous concern. Due to the necessity of treatment being assessed for in situ application,
it is important to select a safe and effective strategy approach that safeguards both


                                                                                            141
                                                                                                 the environment and human beings. In the past decade the most frequently used and
                                                                                                 effective biocides have been banned due to their environmental and health hazards.
                                                                                                 This paper reports the development of remediation actions based on environmentally
                                                                                                 innocuous alternatives derived from active compounds produced by Bacillus sp. in
                                                                                                 conjugation with natural plant products from Brazilian Cerrado plant extracts.


                                                                                                    Riassunto
                                                                                                    I danni a edifici e monumenti, dovuti alla crescita microbiologica, sono motivo di
                                                                                                 grave preoccupazione. Per valutare il trattamento più efficace da applicare in situ, è
                                                                                                 importante individuare un approccio strategico, sicuro ed efficace che salvaguardi sia
                                                                                                 l’ambiente che gli esseri umani. Nell’ultimo decennio i biocidi più efficaci e frequente-
A.T. Caldeira et al. - Green bioactive compounds: mitigation strategies for cultural heritage




                                                                                                 mente usati sono stati banditi a causa dei loro rischi ambientali e sanitari. Questo la-
                                                                                                 voro riporta lo sviluppo di azioni di bonifica basati su prodotti, innocui dal punto di vista
                                                                                                 ambientale, derivati da composti attivi del Bacillus sp. coniugati con estratti vegetali
                                                                                                 naturali di piante provenienti dal Cerrado brasiliano.




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