Saponaria officinalis L. extract: Surface active properties and impact on environmental bacterial strains

Colloids and Surfaces B: Biointerfaces 150 (2017) 209–215

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Saponaria officinalis L. extract: Surface active properties and impact on environmental bacterial strains Wojciech Smułek a , Agata Zdarta a , Amanda Pacholak a , Agnieszka Zgoła-Grze´skowiak b , d,e ˛ Łukasz Marczak c , Maciej Jarzebski , Ewa Kaczorek a,∗ a

Institute of Chemical Technology and Engineering, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland Institute of Chemistry and Technical Electrochemistry, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland c European Centre for Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland d NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland e Department of Physical Chemistry and Physicochemical Basis of Environmental Engineering Institute of Environmental Engineering Off-Campus Faculty of Low and Social Sciences in Stalowa Wola Catholic University of Lublin, Kwiatkowskiego 3A, 37-450 Stalowa Wola, Poland b

a r t i c l e

i n f o

Article history: Received 4 September 2016 Received in revised form 3 November 2016 Accepted 25 November 2016 Available online 27 November 2016 Keyword: Biodegradation Hydrophobicity Saponaria officinalis L. Surfactant

a b s t r a c t Plant-derived surfactants are characterised by low toxicity, high biodegradability and environmental compatibility. They therefore have many applications; for instance, they can be used in bioremediation to accelerate biodegradation processes, especially of hydrophobic pollutants. This paper analyses the properties of an extract from Saponaria officinalis L. containing saponins and its impact on bacterial strains isolated from soil, as well as its potential for application in hydrocarbon bioremediation. The tested extract from Saponaria officinalis L. contains gypsogenin, hederagenin, hydroxyhederagenin and quillaic acid aglycone structures and demonstrates good emulsification properties. Contact with the extract led to modification of bacterial cell surface properties. A decrease in cell surface hydrophobicity and an increase in membrane permeability were recorded in the experiments. An increase of up to 63% in diesel oil biodegradation was also recorded for Pseudomonas putida DA1 on addition of 1 g L−1 of saponins from Saponaria officinalis L. Saponaria extract showed no toxic impact on the tested environmental bacterial strains at the concentration used in the biodegradation process. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Saponaria officinalis L., also known as soapwort or fuller’s herb, is a well-known perennial plant in the family Caryophyllaceae [1]. It grows naturally from Europe to Central Asia in various habitats, usually along roadsides, in hedges and close to water [2–4]. In many countries it is also cultivated as a horticultural plant. Saponaria officinalis L. is characterised by a high content of saponins [5–7]. Because the highest concentration of these compounds is found in the rhizomes, it is that part of the plant that is usually used in investigations [8,9]. Saponins from Saponaria officinalis L. have a wide range of applications. The rhizomes of the plant have been used as a detergent since ancient times. In traditional phytomedicine, extracts of Saponaria officinalis L. are still used to treat bronchitis, skin

∗ Corresponding author. E-mail address: [email protected] (E. Kaczorek). http://dx.doi.org/10.1016/j.colsurfb.2016.11.035 0927-7765/© 2016 Elsevier B.V. All rights reserved.

ailments and rheumatic disorders [3,4]. Soapwort saponins are active ingredients of many cosmetics and medical products [10]. Saponaria officinalis L. is also known for the fact that its saporins, plant enzymes belonging to the class of ribosome-inactivating proteins (RIPs) − can be applied in cancer therapy [11–14]. One recent publication reports on the antifungal activity of saponins from S. officinalis L. against Candida albicans, the most common opportunistic fungal pathogen of humans [10]. Saponins have also been used as adjuvant compounds in vaccines [15–19]. Due to their foaming properties, saponins have also found applications in the food industry. They are used to remove cholesterol from dairy products, as preservatives, flavour modifiers, and as foaming agents in beverages [5,20,21]. However, the pharmaceutical and cosmetic industries are not the only possible users of saponins. These compounds may also find applications in other sectors [5,20]. One of these is protection of the natural environment. Saponins, as natural surface active agents, can be a significant factor in the biodegradation of hazardous compounds. The addition of surfactants can change the surface

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properties of bacterial cells, which consequently can improve the rate of biodegradation processes [22,23]. The influence of saponins on biodegradation has been studied by many authors. The investigation of Tang et al. [24] has shown that tea saponin significantly increases the bioavailability of decabromodiphenyl ether, a flame retardant and toxic pollutant, and its biodegradation by Brevibacillus brevis. Decabromodiphenyl ether is a worldwide environmental pollutant. It belongs to the group of polybrominated diphenyl ethers, which are considered highly persistent and resistant to biodegradation. Huang et al. [25] have studied the biodegradation of triphenyltin and its metabolites by Bacillus thuringiensis in the presence of saponins. The studies of Kobayashi et al. [26] have demonstrated that saponins significantly increase the degradation of pyrene by Sphingomonas sp. Filipkowska et al. [27] have investigated the adsorption of Cd(II), Zn(II) and Cu(II) ions and the desorption of those ions from biosorbent using saponins and other surfactants. The desorption efficiency was found to be highest in the presence of saponin. Wang et al. [28] and Zhu et al. [29] have reported that saponins can enhance the phytoremediation process of soils co-contaminated with organic compounds and heavy metals. Budan et al. [30] found that saponins from Saponaria officinalis L. reduce methane production in the rumen, and therefore have potential application as a feed additive for ruminants. Moreover, Yucca and Quillaja saponins are used as feed additives for ruminants to improve rumen functions [31,32]. The aim of the present study was to obtain a saponin-rich extract from roots of S. officinalis L. and to evaluate its surface active properties as a natural surfactant. Liquid chromatography coupled with mass spectrometry was used to identify the main components of the extract. For that purpose measurements were made of the surface tension of aqueous extract solutions at different concentrations. The critical micelle concentration and adsorption parameters were calculated. Then, the particle size distribution was established and emulsification tests were carried out. To evaluate the impact of the S. officinalis L. extract on the environmental microcosm, a test of its toxicity on soil bacteria strains was conducted. Additionally, modifications of cell surface properties caused by the presence of the extract were described. Measurements of changes in microbial adhesion to hydrocarbons and cell membrane permeability were performed. It was concluded that the S. officinalis L. extract has potential for use as an alternative to synthetic surfactants. 2. Materials and methods 2.1. Plant material and chemicals Saponaria officinalis L. rhizomes were supplied as a commercial herbal raw material (Flos Sp. z o.o., Poland). All chemicals were of analytical grade. All media and aqueous solutions were prepared using deionised and ultrapurified Mili-Q water. Fine chemicals, e.g. hydrocarbons, used in the experiments all had purities of 98% or greater. They were purchased from Sigma-Aldrich. 2.2. Plant material extraction Crushed roots of S. officinalis L. underwent extraction in a Soxhlet apparatus for 8 h at 65 ◦ C using 10 mL methanol per gram of plant material. After solvent evaporation, the solid extract was freezedried using an Alpha 1–2 LD Plus freeze-dryer (Christ, Germany). 2.3. Extract analysis The preliminary testing of the S. officinalis L. extract included FT-IR (Vertex 70, Bruker, Germany) and UV (UV–vis Spectropho-

tometer V–650, Jasco Inc., Japan) analysis. Observation of the surface morphology of the freeze-dried extract was carried out using a scanning electron microscope (SEM) (Hitachi S-3400N, Japan) coupled with an energy dispersive spectrometer (EDS) (Thermo Electron Corp., model No. 4481B-1UES-SN, with NSS Spectral Imaging System software). 2.3.1. LC–MS/MS analysis The lyophilised Saponaria officinalis L. extract was redissolved in water before use in biodegradation experiments. Identification of surfactants in the extract was performed by LC–MS/MS analysis. The dissolved extract was purified using an octadecylsilica SPE sorbent, from which saponins were eluted with 30% methanol. Screening for saponins was carried out using the Ultimate 3000 HPLC system from Dionex (Sunnyvale, CA, USA) coupled with a QTRAP 4000 mass spectrometer from ABSciex (Foster City, CA, USA) operating in positive ion mode for identification of sodium adducts. A Gemini-NX C18 column (100 mm × 2.0 mm I.D.; 3 ␮m) from Phenomenex (Torrance, CA, USA) was used with a mobile phase flow rate of 0.4 mL min−1 . The mobile phase consisted of 0.1% formic acid in water and acetonitrile. Exact mass measurements were made using the Acquity UPLC system from Waters (Milford, MA, USA) coupled with a high resolution micrOTOF-Q mass spectrometer from Bruker (Bremen, Germany) operating in negative ion mode. A Zorbax Eclipse XDB-C18 column (2.1 × 150 mm, 3.5 ␮m) from Agilent (Palo Alto, CA, USA) was used at a 0.6 mL min−1 mobile phase flow rate. The mobile phase consisted of solvent A (0.05% (v/v) formic acid in water) and solvent B (0.05% (v/v) formic acid in acetonitrile). 2.3.2. Extract adsorption parameters Evaluation of the adsorption parameters of the S. officinalis L. extract was conducted according to Kaczorek et al. [33]. Briefly, the equilibrium surface tension of aqueous solutions of the extract was measured at 21 ± 1 ◦ C using the du Nouy ring technique with an Easy Dyne K20 tensiometer (Krüss, Germany) with a platinum ring. Each measurement was repeated ten times. The surface tension data can be fitted by adsorption equations. The Szyszkowski equation [34] was identified as the most suitable. Moreover, the influence of pH of the extract solution was tested. To provide different stable pH values, citrate-phosphate-boric acid buffers were prepared [35]. 2.3.3. Particle size measurements The average hydrodynamic diameters of the S. officinalis L. extract solutions were obtained by DLS (dynamic light scattering) and NTA (nanoparticle tracking analysis) using a NanoSight system with 2.3 NTA software. A detailed description of the theoretical aspects of the measurement of hydrodynamic diameter (d) ´ et al. [36]. Before the experiby DLS and NTA is given by Sliwa ments, each sample was diluted with water and filtered through a Millex-GV 0.22 ␮m syringe filter. The laser beam wavelength in NTA was 405 nm. The viscosity of the measured sample was set automatically according to the temperature. The minimum track length (TL) of the particle was adjusted to 30 frames. The DLS and NTA measurements were performed for 60 and 215 s respectively. 2.3.4. Emulsification test The emulsification properties of the plant extract were determined using the method described by Sneha et al. [37]. In 35 mL screw-capped test tubes, 6 mL of extract solution in Mili-Q water at a concentration of 2.0, 1.0, 0.5, 0.2 or 0.1 g L−1 and 0.02 mL of hexadecane were mixed using a vortex mixer. The optical density at 550 nm was then measured. The control was a sample with Mili-Q water and hexadecane only.

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2.4. Bacterial strains and culture conditions To investigate the influence of the obtained extract on environmental microorganisms, two bacterial strains, Achromobacter sp. SA1 and Pseudomonas putida DA1, were selected. The conditions for genetic identification of tested strains were described previously [38]. The 16S rRNA gene sequences of the tested strains have been deposited in the NCBI GeneBank database under accession numbers KP096514.1 (Achromobacter sp. SA1) and KP096520.1 (Pseudomonas putida DA1). They are Gram-negative, rod-shaped, flagellated bacteria commonly present in soil ecosystems, and were isolated from soil samples collected in northern Poland. The bacterial strains, stored on Petri dishes with Mueller–Hinton agar, were cultivated in a liquid mineral salts medium, prepared according to ® Kaczorek et al. [33]. The cultures were grown in 250 mL Duran laboratory glass bottles (Schott, Mainz, Germany) containing 50 mL of medium inoculated by adding a loopful of cells from the agar plate. After approximately 24 h, ca. 5 mL of this liquid culture was used for inoculation of the final cultures, to obtain an initial cell concentration of 108 cells per mL (OD600nm ≈ 0.1). 2.5. Toxicity tests The toxicity of the obtained extract was determined using the MTT cell proliferation assay. This method is based on the transformation of the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) to its formazan. The process is catalysed by cellular reductases, the activity of which may be used for the evaluation of cell viability. The method of Jafarirad et al. [39] was applied with some modifications. Briefly, 0.1 mL of the MTT solution (5 g L−1 ) and 0.5 mL of a bacteria cell suspension in the culture mineral medium were mixed in 1.5 mL Eppendorf tubes. The samples were quenched up to 1.0 mL with the culture mineral medium. The control was a sample without the addition of surfactant. The samples were incubated 24 h at 30 ◦ C and shaken gently. Thereafter, the samples were centrifuged (15000g, 5 min), the supernatant was removed and the precipitate was dissolved in 1 mL of 1-propanol. The samples were vortexed and centrifuged (15000g, 5 min) again. Finally, the absorbance of the supernatant at 560 nm was measured. All measurements were referred to the value obtained for the control. 2.6. Microbial adhesion to hydrocarbons To determine the cell surface hydrophobicity, the modification of the microbial adhesion to hydrocarbon (MATH) method [40] was used. The carbon and energy sources in the bacterial cultures were: diesel oil, hexadecane, glucose, sodium succinate and S. officinalis L. extract at different concentrations (0.5 g L−1 , 1.0 g L−1 , 3.0 g L−1 ). The biomass from 7-day cultures was centrifuged (8000g, 5 min, 10 ◦ C) and washed twice in the mineral salts medium to remove residual surfactant and carbon sources. Afterwards, the cells were re-suspended in the culture medium and the optical density at 550 nm (OD550 ) was fitted to ca. 1.0. For all measurements a V-650 UV–vis spectrophotometer was used. Next, 0.5 mL hexadecane was added to 5 mL of cell suspension and vortexed for 2 min. After separation of the organic and aqueous phases, the OD550 of the aqueous phase was measured. 2.7. Cell membrane permeability Changes in inner membrane permeability were determined using the colorimetric method described by Zhang et al. [41] in a modified version. Bacterial cells from 7-day liquid cultures were centrifuged at 5000g for 5 min. Then, the biomass was re-suspended in the mineral salts medium. Next, 500 ␮L o-nitrophenyl-ˇ-d-

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galactopyranoside (ONPG) at concentration 30 mmol·L−1 was added to 5 mL of cell suspension (OD550 = 1.0). The samples were incubated for 2 h at 30 ◦ C. After this time the suspension was centrifuged at 5000g for 5 min and the absorbance of the supernatant at 415 nm was determined using a V-650 UV-Vis Spectrophotometer. 2.8. Hydrocarbons biodegradation Tests of the biodegradation of hydrocarbons were conducted ® in 250 mL Duran bottles (Schott, Germany) containing 50 mL of the mineral salts medium. Diesel oil (1% w/v) was used in the cultures as the sole carbon and energy source. When used, S. officinalis L. extract at concentration 0.5, 1.0 or 3.0 g L−1 was added to the medium. The cultures were incubated for 7 days at 30 ◦ C and shaken at 120 rpm. Then 20 mL of saturated NaCl solution and 0.5 mL of 38% hydrochloric acid were added to the culture. The aqueous phase was then extracted twice with 25 mL diethyl ether. After evaporation of the organic phase, the residue was measured as the quantity of hydrocarbon after biodegradation [42]. The final results were calculated with reference to blank samples. The results have an absolute (100%) quantitative value. 2.9. Statistical analysis All of the measurements were performed in triplicate, and the mean values and statistical error were calculated. Statistical analyses, according to the ANOVA method, were performed using Statistica 10.0 software. 3. Results and discussion 3.1. Extract composition The FT-IR spectrum analysis of Saponaria officinalis L. extract shows the presence of specific functional groups of saponins (Fig. 1). The intense and broad O H stretching absorption in the region 3350–3420 cm−1 indicates hydroxyl groups of acids and alcohols. Alkanes are characterised by stretching absorption of the C H bond in the range 2960–2920 cm−1 and bending vibrations in the region 1440–1430 cm−1 . A narrow band of a carbonyl moiety in the region 1660–1650 cm−1 is also visible on the spectra. Moreover, the presence of C O stretching vibrations at a wavenumber above 1050 cm−1 was noted. The signal of polyphenols is not visible on the UV/Vis spectra (data not shown). Moreover, the extract from Saponaria officinalis L. was analysed using the LC–MS/MS system with a low resolution mass spectrometer. The extracted ion chromatogram at selected m/z values is presented in Fig. S2a, and the mass spectrum from the peak at retention time 13.3 min in Fig. S2b. The pseudomolecular ion of the sodium adduct at m/z = 849.4 is clearly visible. The loss of two sugar units and formation of ions at m/z = 647.4 and m/z = 511.4 is also shown. The results were obtained after screening among the recorded mass to charge signals. The selected compounds were studied using a high-resolution mass spectrometer, in which deprotonated ions were monitored. For the sodium adduct ions presented in Fig. S2b, the corresponding deprotonated ions are shown in Fig. S2c, i.e. the pseudomolecular ion at m/z = 825.4256 and two important fragmentation ions at m/z = 663.3715 and m/z = 487.3388. A number of saponins were identified in the Saponaria officinalis L. extract, containing gypsogenin, hederagenin, hydroxyhederagenin and quillaic acid aglycone structures as given in Fig. 2. The osidic chain in the R1 position contains from 1 to 3 sugar units including glucuronic acid (GlcA), glucose (Glc) and xylose (Xyl). Cie´slak et al. [43] found using UPLC–MS analysis that the main components of S. officinalis L. root extracts were saponariosides, vaccarioside and dianchineo-

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Fig. 1. FT-IR spectrum of freeze dried extract from Saponaria officinalis L.

Table 1 The dependency of surface tension of S. officinalis extracts solutions on pH. pH

2.7 4.2 6.0 7.7 9.2

Fig. 2. Structures of saponins identified in the Saponaria officinalis L. extract.

side, while some amounts of glycosides of quillaic, gypsogenic and 16␣-hydroxygypsogenic acids were also identified. Lu et al. [44] analysed an extract from the same source and found it to be mainly composed of quillaic acid and gypsogenin bisdesmosides. These results differ from those obtained in this study; however, this can be explained by the different extraction method used. The SEM image (Fig. S1) of the lyophilised extract from Saponaria officinalis L. shows its homogeneous structure. The surface is smooth without crystalline regions, and there are no visible pores. 3.2. Surface activity of the extract To characterise the surface activity of the obtained extract, measurements were made of the surface tension of solutions of the extract at different concentrations. These made it possible to calculate the adsorption parameters of the fitted adsorption isotherm, which are given in Table S1. The adsorption parameters were cal-

Surface tension (mN m−1 ) 0.25 CMC

0.75 CMC

38.2 ± 0.2 34.6 ± 0.2 29.7 ± 0.2 34.6 ± 0.3 34.0 ± 0.3

36.3 ± 0.2 34.7 ± 0.3 32.1 ± 0.2 38.1 ± 0.3 37.5 ± 0.2

culated from the Szyszkowski equation. The obtained value of the surface excess at the saturated interface (∞ ) was equal to 2.20·10−6 mol m−2 . The minimum surface occupied by the statistical molecule (Amin ) in the adsorption layer and the free energy of adsorption (-Gads ) were calculated at 7.55·10−19 m2 and −29.7 kJ mol−1 respectively. Comparing these results with those found for an extract from Sapindus mukorossi [45], the extract investigated here exhibits a higher surface excess at the saturated interface. On the other hand, the S. mukorossi extract has higher Amin and −Gads values. This indicates the higher concentration of S. oficinalis L. saponin needed to obtain a saturated solution of the surfactant. This conclusion is also consistent with the higher CMC values recorded for S. officinalis L. than for S. mukorossi. An important parameter used to compare the surface activity of surfactants is their critical micelle concentration. This represents the concentration of a surfactant, above which the surfactant molecules form micelles. The lower is the CMC, the less surfactant is needed to saturate interfaces. The critical micelle concentration of the extract (1.00 g L−1 ) can also be assessed using the surface tension measurements. A comparison of CMC values for different plant extracts containing saponins is presented in Table S2. The CMC of the isolated extract is ten times higher than the value found for Sapindus mukorossi, but similar to that found for Quillaja saponaria bark extract [46]. What is more, even though the investigated extract was not purified, its CMC is lower than the values of the same parameter for extracts from Camellia oleifera [47] and Ziziphus joazeiro [48], equal to 1.814 and 1.110 g L−1 respectively. The influence of pH on the surface tension of the extract solution was also evaluated; the results are given in Table 1. For a solution with strong acid properties (pH 2.7) the surface tension

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Fig. 3. Particle size distribution of the Saponaria officinalis L. extractobtained from NTA (2D and 3D plot) and a typical peak obtained from DLS.

at a concentration of 0.75 CMC (0.75 g L−1 ) is equal to 38 mN m−1 , whereas at a concentration of 0.25 CMC (0.25 g L−1 ) the value is 36 mN m−1 . As the pH was increased the surface tension decreased, reaching a minimum at pH 6.0. Further increasing pH caused an increase in surface tension. For a more concentrated solution (0.75 CMC) the difference between the highest and lowest surface tensions is 8.5 mN m−1 , while for a more dilute solution (0.25 CMC) the difference is 6 mN m−1 . The experiment demonstrates the significant dependence of surface activity on the acidity of the extract solution. The influence of pH on the surface tension of natural surfactant solutions has also been investigated by other researchers. Wojciechowski et al. [49] analysed the impact of the pH of Quillaja saponaria solution on its surface tension. They found that the plant surfactant solution, at concentrations above 0.1 g L−1 , had the lowest surface tension at pH 2.7. However, increasing of the pH resulted in an increase in surface tension. Ribeiro et al. [48] investigated a saponin extract from Agave sisalana and Ziziphus joazeiro, and recorded a slight increase in surface tension at pH 9 compared with pH 5 for an extract solution at a concentration close to the CMC.

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Fig. 4. The influence of Saponaria officinalis L. extract on the relative cell metabolic activity measured as optical density changes.

sis is clearly bimodal indicating the existence of two fractions of suspended particles. The most frequent diameter (dmod ) of the first fraction (indicated in Fig. 3 near the first peak) is 59 nm. The second fraction has the dmod value of 112 nm. The peak maximum of the second particle fraction was at 112 nm. Nevertheless, the total mean diameter determined from the whole particle size distribution obtained by NTA (dMEAN ) was 92 nm, with standard deviation (SD) 31 nm. Some particles larger than 200 nm were also found in the sample. Note that before the analysis the samples were filtered and the minimum TL (track length of particle diffusion paths) was adjusted to eliminate artefacts from larger particles’ light reflexes. Fig. 3 (right side) presents a 3D plot of particle size versus intensity, obtained using NTA, which indicates clearly the two main fractions of the particles. Tippel et al. [52] analysed a Quillaja saponaria saponin solution at close to CMC and found the average particle size to be 7.5 nm, which is over ten times smaller. On the other hand, the isolated extract exhibits smaller particles than, for example a biosurfactant produced by Klebsiella sp. strain RJ-03, a solution of which contained particles with sizes ranging from 930 nm to 56 969 nm with an average of 16 042 nm [53].

3.3. Emulsification properties 3.5. Toxicity of the extract The emulsification properties of the extract were also investigated (Fig. S3). An increasing quantity of emulsifiers S. officinalis L. saponins in the system caused a rise in absorption, reflecting a decrease in the size of hexadecane droplets dispersed in the water phase, and an increase in the emulsion stability. Urum and Pekdemir [50] also recorded a slight increase in the emulsification properties of surfactant solution when analysing Quillaja saponaria extract. However, above a concentration of 5 g L−1 , no changes were observed. The results presented here are consistent with the results obtained for dodecyl polyglucoside (APG-12) by Wang et al. [51], who found a positive correlation between the surfactant concentration and the emulsification properties. 3.4. Particle size distribution of the extract Fig. 3 presents the results of the determination of hydrodynamic diameter (d). The maximum of the DLS curve (grey) corresponds to the average particle size dDLS = 103 nm. Unlike in typical DLS measurements, with the nanoparticle tracking analysis (NTA) technique it is easy to observe particle fractions. The black curve and bars correspond to the particle size distribution of the S. officinalis L. extract particles. The size distribution obtained from NTA analy-

The amphiphilic nature of the surfactants makes them highly biologically active. Hence, if a surfactant is being considered for large-scale use, its toxicity should be analysed in depth [54]. To evaluate the potential inhibitory impact of the tested extract on soil bacteria, the MTT toxicity assay was conducted. This test measures the metabolic activity of the bacteria cells in a sample. The metabolic activity of the control samples was taken to be 1.0, and all of the results presented are referred to this value (Fig. 4). For the strain Pseudomonas putida DA1 an inhibitory effect of the S. officinalis L. extract was observed at a concentration above 2.0 CMC (2.0 g L−1 ). However, even for higher concentrations the cell metabolic activity is not lower than 75%. The strain Achromobacter sp. SA1 appeared to be more sensitive to the tested surfactant. For concentrations higher than 1.0 CMC a gradual decrease in metabolic activity is noted, and a 50% decrease in metabolic activity was observed at a concentration of 4.0 CMC (4.0 g L−1 ). Other authors have also investigated the toxicity of saponins. For example H. acida ethanolic extract was found to have a minimal inhibitory effect on several bacterial strains, Escherichia coli, Proteus mirabilis or Pseudomonas aeruginosa, at a level of 1 g L−1 , and on Staphylococcus aureus at 0.5 g L−1 [55]. Ethanolic extract of C. adenogonium leaves

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Table 2 Hydrophobicity and membrane permeability of Pseudomonas putida DA1 and Achromobacter sp. SA1 cells depending on the concentration of Saponaria officinalis L. extract in the sample. Property

Membrane permeability (␮M min−1 )

Hydrophobicity (%)

Microbial strain Concentration of Saponaria officinalis L. extract (CMC, g L−1 )

0.00 0.10 0.25 0.50 1.00 1.50 2.00 0.00 0.50 1.00 3.00

Pseudomonas putida DA1

Achromobacter sp. SA1

0.12 ± 0.01 0.14 ± 0.01 0.14 ± 0.01 0.17 ± 0.01 0.18 ± 0.01 0.19 ± 0.01 0.18 ± 0.01 25.0 ± 1.3 8.8 ± 0.4 1.6 ± 0.1 9.6 ± 0.5

0.39 ± 0.02 0.40 ± 0.02 0.39 ± 0.02 0.40 ± 0.02 0.45 ± 0.02 0.49 ± 0.02 0.48 ± 0.02 4.2 ± 0.2 0.5 ± 0.0 1.7 ± 0.1 2.4 ± 0.1

showed an inhibitory effect on P. aeruginosa and E. coli at a concentration of 1.25 g L−1 [56]. The saponins from S. officinalis L. have higher growth-inhibition concentration than the values for rhamnolipids. Their short-term inhibitory effects on P. fluorescens and P. aeruginosa have been measured at 0.138 g L−1 and 0.164 g L−1 respectively [57]. When the inhibitory concentrations of the S. officinalis L. extract are compared with those for synthetic surfactants, the values for synthetic surfactants are found to be over 30 times lower. The half-maximal effective concentrations of linear alkylbenzenesulphonic acid and quaternary ammonium benzalkonium chloride influencing P. putida growth were found to be 33.4 mg L−1 and 6.9 mg L−1 respectively. 3.6. The influence of Saponaria officinalis L. on bacterial cell surface properties The microbial adhesion to hydrocarbons assay was used to evaluate the influence of Saponaria officinalis L. extract on cell surface hydrophobicity. This method indicates the relative number of bacteria cells characterised by higher adhesion to the organic phase than to the water phase. If all bacterial cells were transferred to the organic phase, their hydrophobicity is taken to be 100%. In spite of the fact that the bacteria in each tested system were hydrophilic, the addition of extract containing saponins caused a further decrease in their hydrophobicity. Comparing the two tested strains, the cells of P. putida DA1 were more hydrophobic than those of Achromobacter sp. SA1 (Table 2). The influence of Saponaria officinalis L. extract on the cell surface hydrophobicity was more significant for the strain P. putida DA1. The hydrophobicity of the sample without the addition of surfactant was 25%. The highest impact of the extract was observed for a sample containing extract at a concentration of 1 CMC (the hydrophobicity decreased to 1.6%). These results are comparable to the results obtained by Sałek et al. [58] for Achromobacter sp. 4 (2010) strain, whose cells were also hydrophilic when cultivated with surfactants. Cell surface hydrophobicity is an important property of bacterial cells. It determines the adhesion of bacteria to various surfaces, which is important in medicine, bioremediation and the food industry [59]. Baumgarten et al. [60] reported that the Pseudomonas putida strain DOT-T1E released outer membrane vesicles in the presence of, among others, long-chain alcohols, which caused a significant increase in the cell surface hydrophobicity. In our studies the presence of saponins in the extract caused a noticeable decrease in the hydrophobicity of P. putida DA1 cells. This indicates that the properties and responses of bacteria to different environmental conditions vary within the genus.

Table 3 The diesel oil biodegradation by chosen bacterial strains in the presence and without Saponaria officinalis extract. Diesel oil biodegradation (%)

without extract 0.5 CMC 1.0 CMC 3.0 CMC

Pseudomonas putida DA1

Achromobacter sp. SA1

43 ± 2 39 ± 3 63 ± 3 48 ± 2

55 ± 3 43 ± 2 47 ± 2 49 ± 3

On the other hand, the bacterial membrane acts as a barrier to the assimilation of hydrophobic organic compounds by microorganisms. The uptake of organic substances by microorganisms is relevant in the biodegradation of those compounds. Transmembrane transport is considered to be the rate-limiting step of the biodegradation process [41]. In order to examine the influence of Saponaria officinalis L. extract containing saponins on the transmembrane transport process, the inner membrane permeability of the two tested bacterial strains Pseudomonas putida DA1 and Achromobacter sp. SA1 was examined in the presence of different concentrations of surfactant (Table 2). The control samples were prepared without the addition of Saponaria officinalis L. extract. The ability of saponins to penetrate the microbial membrane was evaluated through the activity of cytoplasmic ˇ-galactosidase. As shown in Table 2, compared with the control samples, the addition of surfactants slightly increased the permeability of the bacterial membranes of both tested strains. The membrane permeability of Achromobacter sp. SA1 increased, when the concentration of extract containing saponins was greater than or equal to the critical micelle concentration (0.39 ␮M min−1 in the control sample, and 0.45 and 0.49 ␮M min−1 in samples with the addition of extract in concentrations of 1 and 1.5 CMC). The presence of Saponaria officinalis L. extract at concentrations greater than or equal to 0.5 CMC caused a slight increase in ˇ-galactosidase release by the strain Pseudomonas putida DA1 (0.12 ␮M min−1 for the control sample and 0.17 ␮M min−1 for the sample containing extract at concentration 0.5 CMC). Similar results for membrane permeability changes were obtained in the analysis of three different surfactants, Tween 80, TritonX-100 and Brij 30, by Liu et al. [61]. They measured the influence of these surfactants on the permeability of Sphingomonas sp. GY2 B membrane. They found that at low concentration these surfactants do not have a significant effect on the bacterial membrane. A high impact was recorded only for high concentrations (8 CMC) of Triton X-100 and Brij 30. Besides surfactants, there are many other factors that affect membrane permeability. 3.7. Biodegradation test The addition of saponins may bring several benefits in the biodegradation of hydrocarbons. The addition of a natural surfactant may influence the desorption of pollutants from soil or sediment particles and increase their bioavailability to bacteria which are able to degrade the hazardous compounds [23,41,46]. With respect to the Pseudomonas putida DA1 strain, the biodegradation proceeded most effectively at an extract concentration of 1.0 CMC (63%), while without the extract the level of biodegradation was 43% (Table 3). By contrast, the process is least effective at a concentration of 0.5 CMC (39%). In general, when a surfactant is tested at a concentration above the CMC it increases the rate of biodegradation of diesel by Pseudomonas putida DA1, which means that the addition of saponins may have a positive effect on this process. For the strain Achromobacter sp. SA1 the biodegradation process was most effective (55%) in the absence of the extract. The addition of saponins caused a decrease in biodegradation. The extract at a concentration of 0.5 CMC led to the least effective hydrocar-

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bon biodegradability (43%). However, with increasing surfactant concentration, there was a slight increase in the rate of biodegradation. For concentrations of 1.0 CMC and 3.0 CMC it reached 47% and 49%, respectively. The influence of S. officinalis L. saponins on diesel oil biodegradation by the two tested strains is ambiguous. The negative effect of Quillaja saponaria saponins on polycyclic aromatic hydrocarbon (PAH) biodegradation was reported by Soeder et al. [62]. However, Kobayashi et al. [26] used Quillaja saponins to enhance biodegradation of PAH and obtained an increase in the effectiveness of the process by 2.1-times. This research and the results of the present study indicate that the influence of saponins on biodegradation depends strongly on the type of degrading bacterial strain. 4. Conclusions The above presented and discussed results have shown that the extract from Saponaria officinalis L. contains gypsogenin, hederagenin, hydroxyhederagenin and quillaic acid aglycone structures. The extract has good emulsifying properties. The critical micelle concentration of the extract studied in water solution is 1.00 g L−1 . In view of its properties it may serve as an alternative to synthetic surfactants. In addition, it shows no toxic impact on the tested environmental bacterial strains, and therefore it may be used in bioremediation. However, the influence of saponins from Saponaria officinalis L. on biodegradation is dependent on the genus of bacterial strains. A positive effect of saponins at a concentration of 1 CMC on diesel oil biodegradation was observed only for bacteria from the genus Pseudomonas. Acknowledgements This study was supported by The National Science Centre Poland under decision no. DEC-2012/07/B/NZ9/00950. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.11. 035. References [1] M. Thakur, G. Jerz, D. Tuwalska, R. Gilabert-Oriol, S. Wybraniec, P. Winterhalter, H. Fuchs, A. Weng, J. Chromatogr. B: Analyt Technol. Biomed. Life Sci. 955 (2014) 1. [2] S. Böttger, M.F. Melzig, Phytochem. Lett. 4 (2011) 59. [3] K. Koike, Z. Jia, T. Nikaido, J. Nat. Prod. 62 (1999) 1655. [4] Z. Jia, K. Koike, T. Nikaido, J. Nat. Prod. 61 (1998) 1368. [5] S.G. Sparg, M.E. Light, J. van Staden, J. Ethnopharmacol. 94 (2004) 219. [6] Z. Jia, K. Koike, T. Nikaido, J. Nat. Prod. 62 (1999) 449. [7] M. Korkmaz, H. Özc¸elik, Afr. J. Biotechnol. 10 (2011) 9533. [8] Y. Lu, D. Van, L. Deibert, G. Bishop, J. Balsevich, Phytochemistry 113 (2014) 108. [9] T.A. Kuznetsova, L.A. Ivanushko, I.D. Makarenkova, E.I. Cherevach, L.A. Ten’kovskaya, Bull. Exp. Biol. Med. 156 (2014) 366. ´ [10] B. Sadowska, A. Budzynska, M. Wieckowska-Szakiel, M. Paszkiewicz, A. ˙ J. Med. Stochmal, B. Moniuszko-Szajwaj, M. Kowalczyk, B. Rózalska, Microbiol. 63 (2014) 1076. [11] A. Weng, K. Jenett-Siems, P. Schmieder, D. Bachran, C. Bachran, C. Görick, M. Thakur, H. Fuchs, M.F. Melzig, J. Chromatogr, B. Analyt, Technol. Biomed. Life Sci. 878 (2010) 713. [12] S. Di Bucchianico, G. Venora, S. Lucretti, T. Limongi, L. Palladino, A. Poma, Micros. Res. Techniq. 71 (2008) 730.

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