Appl Biochem Biotechnol (2014) 174:2725–2740 DOI 10.1007/s12010-014-1221-7

Newly Antibacterial and Antiadhesive Lipopeptide Biosurfactant Secreted by a Probiotic Strain, Propionibacterium Freudenreichii Hamidreza Hajfarajollah & Babak Mokhtarani & Kambiz Akbari Noghabi Received: 17 May 2014 / Accepted: 2 September 2014 / Published online: 13 September 2014 # Springer Science+Business Media New York 2014

Abstract A lipopeptide biosurfactant production from a probiotic type strain of Propionibacterium freudenreichii subsp. freudenreichii is being reported here for the first time. This biosurfactant is able to reduce the surface tension of water from 72 to 38 mN/m with an increase of the biosurfactant concentration up to critical micelle concentration value of 1.59 mg/ml. The production of the biosurfactant was found to be much higher in medium containing sunflower oil compared to the glucose-containing medium. The maximum emulsifying activity (E24 =72 %) was attained with used frying sunflower oil, while kerosene and starch had the lowest emulsifying activity. Biosurfactant production seems to be parallel to cell growth. The produced biosurfactant was relatively thermo-stable and no appreciable changes in biosurfactant activity occurred at temperature ranges of 25–85 °C. The analysis of the extracted biosurfactant by thin layer chromatography, infrared spectroscopy, and 1H and 13 CNMR spectroscopy revealed the chemical nature of the biosurfactant as lipopeptide. Produced lipopeptide was evaluated for its antimicrobial and antiadhesive activity and showed significant antimicrobial and antiadhesive action against a wide range of pathogenic bacteria and fungi. A total growth inhibition was observed over Rhodococcus erythropolis, while the best result of antiadhesion was obtained against Pseudomonas aeruginosa. Keyword Propionibacterium freudenreichii . Lipopeptide . Antimicrobial activity . Antiadhesive activity

Introduction Biosurfactants are amphipathic compounds excreted by some microorganisms that exhibit surface activity [1]. They belong to a wide variety of chemical structures, such as glycolipids, Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-1221-7) contains supplementary material, which is available to authorized users. H. Hajfarajollah : B. Mokhtarani (*) Chemistry and Chemical Engineering Research Center of Iran, P.O. Box 14335-186, Tehran, Iran e-mail: [email protected]

K. A. Noghabi National Institute of Genetic Engineering and Biotechnology, P.O. Box, 14155-6343, Tehran, Iran

2726

Appl Biochem Biotechnol (2014) 174:2725–2740

lipopeptides, polysaccharide protein complexes, protein-like substances, lipopolysaccharides, phospholipids, fatty acids, and neutral lipids [2]. However, lipopeptides and glycolipids are the most extensively studied and characterized. Some potential applications of biosurfactants are crude oil recovery [3], oil spill remediation [4], hydrocarbon removal from soils [5], heavy metal removal from contaminated soils [6], and hydrocarbon biodegradation in aquatic environment [7]. Bacteria are the predominant group of surfactant-producing organisms. Pseudomonas sp., Acinetobacter sp., Bacillus sp., and Arthrobacter sp. are the most reported bacteria as biosurfactant producers. However, due to the pathogenic nature of the producing organisms, the application of these compounds is not suitable for use in the food industry [8]. There is limited information on the chemical structure of biosurfactants produced by probiotic microorganisms [9]. Some propionic acid bacteria (PAB) have been granted generally recognized as safe (GRAS) status by the US Food and Drug Administration [10]. Propionibacterium freudenreichii as a non-pathogenic strain is practically used in the food and pharmaceutical industries for vitamin and acid production. In fact, this gram-positive, nonspore forming bacterium, produce vitamin B12 intracellularly and excrete mainly propionic acid and acetic acid extracellularly [11, 12]. However, to the best of our knowledge, there is no single report on the capability of biosurfactant production with Propionibacterium species. Lactic acid bacteria (LAB), another probiotic bacteria, have been characterized for biosurfactant production. Nevertheless, the composition of its biosurfactant has not been extensively studied and only a few have been partially characterized [9]. In this paper, the isolation of a new-type biosurfactant from P. freudenreichii subsp. freudenreichii PTCC 1674 with significant critical micelle concentration (CMC) value has been reported for the first time. This was accomplished by (1) testing the bacterial strain for its ability to produce biosurfactant; (2) measuring surface tension, emulsification activity, and bacterial growth kinetics; (3) preliminary characterization of extracted biosurfactant; (4) evaluating various carbon sources for biosurfactant production; and (5) testing antimicrobial and antiadhesive potential of the biosurfactant.

Materials and Methods Chemicals and Microorganisms All chemicals were purchased from Merck (Germany) unless otherwise stated. P. freudenreichii subsp. freudenreichii PTCC 1674 (P. freudenreichii) was purchased from the Persian Type Culture Collection in lyophilized state and stored at −70 °C in glycerol stocks. For antimicrobial and antiadhesive assays, the following strains were kindly provided by the Faculty of Genetic engineering, National Institute of Genetic Engineering and Biotechnology (Iran): Pseudomonas aeruginosa, Bacillus subtilis, Rhodococcus erythropolis, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Listeria monocytogenes, Alternaria alternantherae, Salmonella typhimurium, and Klebsiella pneumonia. Media and Inoculum Preparation A two-stage inoculum preparation was applied both in glucose mineral salt medium (MSM). The two-stage cultivation almost has positive effects on the production of biosurfactants, and it

Appl Biochem Biotechnol (2014) 174:2725–2740

2727

was observed that the use of inoculum in two stages drastically reduced the lag period of fermentation [13]. The medium for both stages contains (gl−1) glucose 20, peptone 5, yeast extract 10, and KH2PO4 2, (NH4) 2NO3 4. Primary cultivation was performed for 24 h at 30 °C and 125 rpm on a shaker incubator (Kuhner, Germany). Two milliliters of the primary inoculum was used to inoculate into 50 ml of glucose mineral salt medium (seed culture) in a 250-ml flask and incubated at 125 rpm and 30 °C for another 16–18 h. The seed culture was used for the biosurfactant production experiments. The medium used for production experiments consisted of (gl−1) glucose 36, peptone 5, yeast extract 10, KH2PO4 1, K2HPO4 1, (NH4) 2NO3 4, MgCl2.6H2O 1, FeSO4.7H2O 0.1, and CoCl2 0.005. It should be noted that to examine the effect of carbon sources on the biosurfactant production, other carbon sources were used instead of glucose in some experiments. The production experiments were carried out in 30 °C, 130 rpm, and pH between 6.7 and 7. Extraction and Purification of Surface-Active Molecules Biosurfactant production experiments were performed in shake flasks. The biosurfactant was separated from the culture broth using the method of acid precipitation followed by solvent extraction reported elsewhere [14]. After fermentation, bacterial cells were removed from the culture broth by centrifugation (7,000×g, 4 °C, 15 min). The cell-free supernatant was then acidified to pH 2 with 4 N HCl and kept at 4 °C overnight to precipitate the biosurfactant. The crude biosurfactant was then collected by high speed centrifugation of the acidified supernatant (18,000×g, 4 °C, 30 min). For further purification, the crude product was extracted three times with ethyl acetate. After extraction, the solvent was evaporated by vacuum evaporator (Thermo Electron Corporation, Heraeus vacutherm) and the concentrated residue was used as partially purified biosurfactant. Purified biosurfactant was then obtained by preparative layer chromatography (PLC) (silica gel 60, 20×20 cm, Merck). For this purpose, partial purified biosurfactant was dissolved in chloroform and then spotted on the plate. The plate was then developed with the solvent system of hexane/ethyl acetate (11:1). Visualization was carried out under UV transilluminator. In preparative mode, visualized spots were scraped off and were extracted more than six times with 10–15 ml of ethyl acetate. Fractions were separated and then evaporated under vacuum. Oil spreading test revealed that only one of the spots (among three fractions obtained) was biosurfactant. This compound was stored as a pure biosurfactant for further studies. Physical and Surface Activity Properties Five methods including surface tension (ST), interfacial tension (IFT), critical micelle concentration (CMC), oil spreading test (OST), and emulsification activity (E24%) were employed to evaluate the surface-active properties of the produced biosurfactant. ST and IFT were determined with a Data Physics Contact Angle system (OCA20, Germany) equipped with a camera monitor at 25 °C, using the pendent drop method. ST values were measured for at least five times and the averages were reported. Interfacial tension was performed between water and hexadecane. CMC was also determined by preparing different concentrations of biosurfactant and measuring their surface tension. Standard procedure of oil spreading test was performed to find biosurfactant activity [15]. OST is also sometimes used as a reliable way to measure the concentration of biosurfactants [15]. The emulsification activity of produced biosurfactant was measured according to Cooper and Goldenberg [16].

2728

Appl Biochem Biotechnol (2014) 174:2725–2740

Effect of Carbon Sources on Biosurfactant Production In general, three categories of carbon sources, i.e., carbohydrates, hydrocarbons, and vegetable oils, are used for bacterial growth and production of special biomaterials. Therefore, different carbon sources such as glucose, sucrose, maltose, starch, kerosene, glycerol, sunflower oil, and waste frying oil were examined for evaluating surface activity properties of the produced biosurfactant. The results are the average of triplicate experiments. Biosurfactant Production Kinetics Biosurfactant production kinetics was studied in a 5-L fermenter (Infors, Switzerland) with 3 L working volume at 30 °C and 130 rpm. The aeration was controlled at a rate of 1.0 vvm. The medium pH was maintained at about 6.9 by automatic addition of 2 N NaOH or HCl solution to the fermenter according to the signal received from the pH electrode. Cellular growth was expressed in terms of dry cell weight which was calculated from the equation of a calibration curve constructed between optical density and cell dry weight of the P. freudenreichii: CDW (gl−1)=0.34×OD600. Optical density was measured at 600 nm by UV-visible spectrophotometer (PerkinElmer, model Lambda25, USA) during different time intervals up to 144 h. Biosurfactant production over time was also investigated in terms of ST and OST on glucose as a sole carbon source. Chemical Characterization Thin Layer Chromatography Thin layer chromatography (TLC) can be applied to detect the presence of compounds such as lipids, peptides, and carbohydrates. A small quantity of the purified biosurfactant was dissolved in chloroform and 10 μl of this solution was put onto a TLC plate (silica gel 60, Germany) at point of origin near the bottom of the plate. Once dried, the plate was developed in various solvent systems. The solvent system of hexane/ethyl acetate (11:1) resulted in better development. After development, one of the plates was put into a jar saturated with iodine vapors to detect lipids as yellow spots [17]. Another plate was sprayed evenly with the ninhydrin reagent (0.5 g ninhydrin (Sigma-Aldrich) in 100 ml anhydrous acetone) and placed in an oven at 110 °C for 10 min to detect the presence of peptides as red spots [1]. Fourier Transform Infrared Spectroscopy The Fourier transform infrared spectroscopy (FTIR) spectroscopic analysis can be used to elucidate the chemical nature of biosurfactants by identifying the types of chemical bonds or the functional groups present in their chemical structures [2]. The FTIR spectrum was recorded in the 4,000–400 cm−1 region on an FTIR system (PerkinElmer, USA), with the sample dispersed in pellets of KBr. The wave number accuracy and resolution of spectrum were 0.01 cm−1 and 4, respectively. Nuclear Magnetic Resonance Spectroscopy Purified biosurfactant was subjected to further analysis with nuclear magnetic resonance. All 1 H and 13C-nuclear magnetic resonance (NMR) spectra were measured using a Bruker JNMA500 spectrometer (Germany) set at 250 MHz with deuterated chloroform as a solvent.

Appl Biochem Biotechnol (2014) 174:2725–2740

2729

Biochemical Composition of Biosurfactant Carbohydrate content of the biosurfactant was determined by the phenol–sulfuric acid method of Dubois [18] using D-glucose as a standard. Protein content was determined by the method of Bradford [19] using Coomassie brilliant blue with bovine serum albumin as a standard and lipid content was estimated by the procedure of Folch et al. [20]. Propionic Acid Analysis Propionic acid concentration in the broth was analyzed using the HPLC system (Shimadzu 250 mm×4.6 mm; 5 μm particle size) on a reversed-phase C18 column. UV detection was done at 210 nm. The mobile phase was the solution of 1 mmol/L sulfuric acid plus 8 mmol/L Na2SO4 in deionized water (pH 2.8). Flow rate was set to 1 ml/min, oven temperature at 25 °C, and injection volume was 20 μL. Propionic acid standards at concentrations ranging from 1 to 30 gl−1 in MilliQ water were used for calibration. Each sample was injected twice and means were reported. Thermal Properties and Stability Tests The thermal gravimetric analysis (TGA) of the biosurfactant was performed with NETZSCH TG 209F1 Iris system (Germany). About 6–7 mg of the sample was loaded on a platinum pan and its weight loss measured in the ranges of 30–480 °C with the heating rate of 5 °C/min. The experiment was conducted under nitrogen atmosphere. The effects of three environmental parameters (pH, temperature, and salinity) on the surface activity of the biosurfactant produced by P. freudenreichii were determined as described elsewhere [9]. Briefly, 8 mg/ml solution of the biosurfactant was prepared and the ST values were measured at different temperatures, pH, and salinity. Antimicrobial Assay Qualitative Agar Diffusion Test Antimicrobial activity of the produced biosurfactant was initially confirmed using two conventional assays including agar disk diffusion and agar well diffusion. In these experiments, B. cereus and S. aureus were used as indicator microorganisms. Quantitative test with 96 well Plate The antimicrobial activity of the produced biosurfactant against a wide range of microbial strains was determined by the microdilution method in 96-well flat-bottom plastic tissue culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany). The antimicrobial assay procedure was performed according to the method described by Gudina et al. [9]. Triplicate assays were performed at all the biosurfactant concentrations for each strain. The growth inhibition percentages at different biosurfactant concentrations for each microorganism were calculated as: % Growth inhibition ¼ ½1 − Ac =A0   100

ð1Þ

where Ac represents the absorbance of the well with a biosurfactant concentration c and A0 the absorbance of the control well (without biosurfactant).

2730

Appl Biochem Biotechnol (2014) 174:2725–2740

Antiadhesive Assay The antiadhesive activity of the produced biosurfactant against four bacterial strains, S. aureus, P. aeruginosa, B. cereus, and E. coli, was evaluated according to a previously reported adhesion assay [9]. Briefly, the wells of a sterile 96-well flat-bottomed plastic tissue culture plate with a lid were filled with 200 μl of the crude biosurfactant. Several concentrations were tested ranging from 2.5 to 40 g/l. The plate was incubated for 18 h at 4 °C and subsequently washed twice with PBS. Control wells contained buffer (PBS) only. A 200-μl aliquot of a washed bacterial or yeast suspension was added and incubated in the wells for 4 h at 4 °C. Unattached organisms were removed by washing the wells three times with PBS. The adherent microorganisms were fixed with 200 μl of 99 % methanol per well, and after 15 min the plates were emptied and left to dry. Then, the plates were stained for 5 min with 200 μl of 2 % crystal violet used for Gram staining per well. Excess stain was rinsed off by placing the plate under running tap water. Subsequently, the plates were air dried, the dye bound to the adherent microorganisms was resolubilized with 200 μl of 33 % (v/v) glacial acetic acid per well and the optical density readings of each well were taken at 595 nm.

Results Physical Characterization Different carbon sources were used for biosurfactant production to investigate the optimized one and evaluating surface-active properties of the produced biosurfactant. Samples were retrieved for measuring ST, diameter of clear zone (OST), and E24. All tested carbon sources were found to support both growth and biosurfactant production. The results are represented in Table 1. The best activity in terms of surface tension was achieved using vegetable oils (Table 1). The results showed the highest reduction in ST when sunflower oil was used for cultivation of P. freudenreichii. The maximum ST reduction to 29 on the sunflower oil (control, 67 mNm-1) is relatively more than ST reduction on glucose. A possible reason is that during the growth of some microorganisms on vegetable oils, lipase activity of microorganisms probably degraded triacylglycerols to free fatty acids, mono, and diacylglycerols. These compounds have surfactant properties that can reduce the surface tension. To investigate this, the cell free supernatant was extracted with equal volume of n-hexane in order to remove the residual surface-active compounds. After phase separation, the bottom aqueous phase was gathered for ST Table 1 Physical characteristics of produced biosurfactant by P. freudenreichii using different carbon sources

All media cultivated in 30 °C and 130 rpm a 4 % (w/v) was used b Control 66–68 mN/m for all media

Carbon sourcea

Min STb

Max OST (cm)

Max E24%

Glucose

38±0.5

2.8±0.2

57±2

Sucrose

39±0.9

2.1±0.3

49±3

Maltose

42.1±0.8

1.8±0.2

51±1

Kerosene Glycerol

44±0.7 41.8±0.9

1.1±0.1 1.5±0.3

14±1 23±1

Starch

42.6±0.7

0.5±0.1

5±2

Sun flower oil

29.2±0.2

14±0.9

71±3

29±0.3

15±0.8

72±2

Waste frying oil

Appl Biochem Biotechnol (2014) 174:2725–2740

2731

measurement. The ST of the extract was increased from 29 to 38.1 mNm-1 that means some surface-active compounds have been produced due to lipase activity. The resultant extracted biosurfactant was used as a standard for subsequent quantification. The diameters of clear zone in OST and emulsification activities have also been reported in Table 1. Taking into account the diameter of clear zone, it can be concluded that the biosurfactant production on sunflower oil was near five times higher than on glucose as a carbon source. Further experiments confirmed this. The amount of biosurfactant produced in glucose and sunflower oil media were 380 mgl−1 and 1.72 gl−1, respectively (see Biosurfactant production kinetics section). The emulsification activity was strongly influenced by the type of carbon source. Although starch, kerosene, and glycerol supported growth, the biosurfactant production was insignificant. The E24% and OST results on these carbon sources were also poor. The emulsification activity using starch and kerosene was high at the first times of E24 test, but it was very unstable and decreased after 4 or 5 h. The best emulsification activity in glucose media was obtained around 73 h cultivation, where the bacterium is at its late exponential phase (see Biosurfactant production kinetics section). Before and after this time, the emulsification activity was generally low. Biosurfactant Production Kinetics The biosurfactant production kinetics was studied in 5-L fermenter on the glucose mineral salt medium, because it would be easier to investigate the production of biosurfactant from watersoluble-substrate cultures such as glucose than oil contained ones. The biosurfactant production (shown by a decrease in the ST) was somewhat dependent on the growth of bacterium in the fermentation medium using glucose as a carbon source. As seen in Fig. 1, ST dropped till the late exponential phase, reaching its lowest value during the last of exponential phase (control, 67 mNm-1) and increased after that by starting the stationary phase. Figure 1 shows that the amount of biosurfactant is at its high level only during a limited time. The amount of extracted biosurfactant at this time was about 380 mgl−1. Increasing of the surface tension can be a consequence of both biosurfactant consumption by microorganism and acid inhibition. It is evident that the biosynthesis of the biosurfactant was inhibited after 73 h in the conventional process, in which the concentration of propionic acid had reached approximately 14 g L−1. Diameter of clear zone (OST) increased with decreasing surface tension and vice versa. This is a rational behavior since the OST is related to the biosurfactant concentration [15]. Chemical Characterization of Biosurfactant The chemical composition analyses revealed that the biosurfactant produced by P. freudenreichii is most likely a lipopeptide predominantly lipid with relative percent of ~70 % (w/w) and 21.1 % (w/w) protein. A minor fraction of carbohydrate (less than 1 %) was found in some of the collected samples probably resulting from remaining culture media coprecipitated with the biosurfactant during its extraction process. Primary characterization of chemical nature of the biosurfactant was done by thin layer chromatography technique. After spotting and development, the plate was kept into the jar saturated with iodine vapors and yellow spot was appeared. This suggests the presence of polar lipids. Treatment with ninhydrin reagent reveals red spot, indicating the presence of peptides (Fig. 2). These results thereby indicated that the surface-active compound was lipopeptide in nature. For further complementary characterization, the FTIR and NMR spectroscopy were performed.

2732

Appl Biochem Biotechnol (2014) 174:2725–2740

Fig. 1 Biomass, ST, pH, OST, and propionic acid concentration results during fermentation at 30 °C and 130 rpm by P. freudenreichii using glucose as a carbon source

Fig. 2 TLC results. a after treatment with ninhydrin. b after contacting with iodine vap

Appl Biochem Biotechnol (2014) 174:2725–2740

2733

Figure 3 shows the IR spectrum of the produced biosurfactant. The IR spectrum can be used to gain insight into the chemical nature. The IR spectrum of the produced biosurfactant was compared with the IR spectral data of some known biosurfactants. The observed peaks were very similar to the IR spectrum of lipopeptide biosurfactants produced by Bacillus species [21-23]. It could be a sign that the produced biosurfactant may have a similar structure to surfactin. The presence of amine and hydroxyl groups of protein was confirmed by IR spectrum of the biosurfactant. In fact, the peak at around 1,058 cm−1 suggested the presence of amide moieties of protein [24]. Protein-related weak bands, the –C = O amide I (1,645 cm−1), and -NH/-C=O combination of the amide II bands (1,540 cm−1), were also observed. Absorption in the region 1,600–1,700 cm−1 is characteristic for amide I vibrations in proteins. On the other hand, absorption in 1,500–1,650 cm−1 is not normally observed in the FTIR spectra of glycolipid biosurfactants [25]. These indicate the presence of peptide groups and show the lipopeptide structure of the produced biosurfactant. A symmetrical stretched peak near 1,384 cm−1 (carboxyl groups) and peaks in the region 2,850–2,950 cm−1 (−CH stretching mode of CH3 and CH2 groups in alkyl chains) were also detected. The weak bands in the region 1,370–1,470 cm−1 are the result of deformation and bending vibrations of –C-CH2 and -C-CH3 groups in aliphatic chains. Figure 4 shows 1H NMR and 13C NMR spectra of the biosurfactant. 1H NMR analysis of the purified biosurfactant demonstrated NH signals (δ 7.0–7.7) and corresponding CH signals (δ 4–5.3) for the α-amino acids of the peptide moiety along with the fatty acid functional group as a single methyl-related peak (δ 0.85–0.87), −(CH2) n- (δ 1.2–1.46), −C-OH (δ 2.25), and -CO-CH2–CH2- (δ 1.6 ppm). Lipid signals present in biosurfactant consisted of CH2 from δ 21.6 to 35.1, CH3 at δ 13.2, and ester and carboxylic groups signals at δ 178 in the 13C NMR spectrum (Fig. 4b). Critical Micelle Concentration CMC was obtained from the break point of the ST versus the bulk concentration curve of the biosurfactant (Fig. 5). The CMC value of the purified biosurfactant was determined as 1.59 mg/ml, and the biosurfactant was able to reduce the ST of water from 72 to about 38 mNm-1. The produced biosurfactant could also reduce the interfacial tension (IFT) between 82

Transmiance (%)

80

78

76

74

1720

1383

2856

72

1460 1645

3380

1058 1170

1540

2923

70 4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750

Wavenumber (cm-1)

Fig. 3 FTIR spectrum of the produced biosurfactant by P. freudenreichii

500

2734

Appl Biochem Biotechnol (2014) 174:2725–2740

Fig. 4 a) 1H NMR and b) 13C NMR spectra of the purified biosurfactant produced by P. freudenreichii

water and hexadecane from 41 to 5 mNm−1. The ST of some efficient biosurfactants like surfactin and rhamnolipids at their CMCs was reported in the range from 27 to 36 mNm-1 [1]. Thus, the lipopeptide biosurfactant reported in this present investigation can be classified under relatively efficient and effective surfactants. Thermal Properties and Stability Tests Thermal stability of biosurfactants is an important characteristic for its application and commercial utilization especially in the food industry. TG analysis (Fig. 6) shows that biosurfactant degradation was occurred by two well differentiated steps. An initial weight loss (5.93 %) between 40 and 140 °C was attributed to loss of trapped moisture molecules in the structure of the biosurfactant. Moisture release during heating of the biosurfactant suggested

Appl Biochem Biotechnol (2014) 174:2725–2740

2735

80

ST (mN/m)

70

60

50

40

30

0

1

2

3

4

5

6

7

Biosurfactant Concentration (mg/mL)

Fig. 5 Variation of ST versus biosurfactant concentration for determination of CMC

that the biosurfactant was not truly anhydrous. This is followed by a drastic weight loss by 75.81 % at 234 °C, which may be attributed to the decomposition of the unstable component in the biosurfactant. The stable part was decomposed by only 11.98 % over the temperature range from 234 to 486.9 °C. For investigating the surface activity of biosurfactant in various environmental conditions, the ST was measured after keeping the biosurfactant solution at the temperature ranges of 25– 85 °C for 120 h. Figure S1 (a) shows that the produced biosurfactant is thermo-stable. As Fig. S1 (b) shows, the produced lipopeptide is more stable in neutral and alkaline pH. However, the pH ranges of 6–8 were found to be the optimum levels for the biosurfactant activity. In order to examine the surface activity changes of the produced biosurfactant in the presence of salts, various concentrations of NaCl (0–30 gl−1) were used. As Fig. S1 (c) shows, the biosurfactant resists well against salinity to the concentrations up to about 20 gl−1 NaCl. The ST started to increase above this concentration.

Fig. 6 TG graph of the produced biosurfactant by P. freudenreichii

120

100 Sample: 6.720 mg

Weight loss%

80

60

40

20

0 100

200

300

Temperature(C)

400

500

2736

Appl Biochem Biotechnol (2014) 174:2725–2740

Antimicrobial Activity Primary evaluation of antimicrobial capability of produced biosurfactant was performed against two indicator pathogens namely B. cereus and S. aureus. Both agar disk diffusion assay (Fig. S2) and ager well diffusion assay (data not shown) confirmed the antimicrobial action of the lipopeptide. For better understanding of the antimicrobial action quantitatively, growth inhibition was calculated. The isolated lipopeptide biosurfactant in various concentrations showed antimicrobial activity against all the microbial strains tested, albeit to different degrees. Figure 7 represents the growth inhibition capability of the lipopeptide biosurfactant toward bacteria and fungi at concentrations ranging from 50 to 3.2 mg/ml. The lipopeptide produced by P. freudenreichii completely inhibited the growth of R. erythropolis at concentration of 25 mg/ml. Although a complete inhibition was not observed against other microorganisms, high growth inhibition was obtained. Slight inhibition effect was observed against B. cereus at lipopeptide concentration of 25 mg/ml. This showed that the mentioned microorganism was the most resistant one compared to other strains. Antiadhesive Activity Nowadays, the search for novel molecules or biomolecules to combat against drug resistant bacteria becomes imperative. Involvement of biosurfactants in microbial adhesion and desorption has been widely reported, and adsorption of biosurfactants to solid surfaces might constitute an effective strategy to reduce microbial adhesion by pathogenic microorganisms, not only in the biomedical field, but also in other areas, such as the food industry [21]. 120 25 mg/ml 12.5 mg/ml 6.25 mg/ml 3.2 mg/ml

% Microbial Inhibition

100

80

60

40

20

0

E.

li s s s e lis lis sa nia um co ne hera reu ropo b t i cereu ino uri mo ge au su th eu rug cyto rnant phim B. y S. B. n e r p a e e ty no P. K. R. alt S. mo A. L.

Fig. 7 Microbial inhibition percentages obtained from the antimicrobial assays with the crude biosurfactant isolated from P. freudenreichii at different concentrations. Results are averages of triplicate experiments

Appl Biochem Biotechnol (2014) 174:2725–2740

2737

Antiadhesive activity of the produced lipopeptide was evaluated against four bacterial strains: E. coli, S. aureus, P. aeruginosa, and B. cereus. The biosurfactant showed different antiadhesive activities against tested microorganisms, depending upon the concentration of biosurfactant and test organism (Table 2). Highest antiadhesive percentage was obtained for P. aeruginosa (67.1 %) at the concentration of 40 g/l. On the contrary, a lower activity was observed for S. aureus (32.3 %) at the same concentration. The inhibition of microbial adherence is one of the effective means to infection control. Hence, the lipopeptide biosurfactant of this study could be useful for such applications.

Discussion Lactobacilli and Streptococcus are among probiotic bacteria which have been proved to produce biosurfactant [26, 27]; however, characterization of their biosurfactants has not been completely undertaken [9]. Here, a lipopeptide production from a type strain of P. freudenreichii has been reported. This strain is the first-reported biosurfactant producer in the genus Propionibacterium. Bacteria of this genus are recognized as probiotic and nonpathogenic. The results of thin layer chromatography, FTIR, 1H-NMR, 13C-NMR spectroscopy as well as compositional analysis proved the chemical nature of lipopeptide for the produced biosurfactant. The effectiveness of a typical biosurfactant is determined by its ability to lower the surface tension, formation of clear zone in oil spreading test, and the ability to emulsify hydrocarbons. The biosurfactants produced by P. freudenreichii reduced the ST of water to about 38 mNm-1, and IFT between water and hexadecane from 41 to 5 mNm-1. As it was shown by Busscher et al. [28], the minimum reduction in surface tension should be more than 8 mNm-1 to identify any microorganism as a biosurfactant producer. By taking into account this value, P. freudenreichii is a biosurfactant producer strain. The biosynthesized biosurfactant by P. freudenreichii revealed the critical micelle concentration of 1.59 mg/ml. The CMC of the produced biosurfactant is comparable with those produced by other probiotic bacteria. Table 3 shows the CMC and ST at CMC of several biosurfactants produced by some probiotic species. As seen, the majority of biosurfactants produced by probiotic bacteria have CMC values greater than 1 mg/ml. In addition, the minimum ST is almost greater than 35 mNm-1. These observations reveal that the biosurfactants produced by probiotic bacteria (probiotic biosurfactants) have relatively lower surface activity compared to surfactin and rhamnolipids. What makes these biosurfactants noticeable is their potential application in the food and pharmaceutical industries as probiotic biosurfactants. Table 2 Antiadhesive properties of the produced biosurfactant Microorganism PBS

Control

Inhibition of microbial adhesion (%) Biosurfactant (g/L) 2.5

5

10

25

40

S. aureus

0

0

2±0.1

9.1±0.1

16.4±0.3

32.3±0.4

E. coli

0

1±0.4

7.7±0.3

13.1±0.4

25.9±0.5

47.7±0.6

P. aeruginosa

0

4.5±0.4

12.5±0.2

37.2±0.6

45.5±0.4

67.1±0.2

B. cereus

0

2±0.1

2.1±0.1

14.5±0.2

21±0.4

39.1±0.6

2738

Appl Biochem Biotechnol (2014) 174:2725–2740

Table 3 Comparison of the ST and CMC of biosurfactants from some probiotic microorganisms Strain

ST mg/ml

CMC mNm-1

Ref. –

Propionibacterium freudenreichii in this work

38.2

1.6

Lactobacillus fermentum RC-14

39

1

12

Lactobacillus fermentum B54

39

2.5

12

Lactobacillus paracasei Streptococcus thermophilus A

41.8 36

2.5 20

9 26

Lactococcus lactis

40

3.5

41

2

27

Lactobacillus delbrueckii



A key problem in the production of biosurfactants, especially by GRAS microorganisms, has always been their low product concentrations at the end of the fermentation [29]. Little amounts of biosurfactant per liter of culture broth makes downstream separation of the product very costly. B. subtilis RB14 produced only about 170 mgl−1 of a lipopeptide iturin A on the malt residue [30]. Micrococcus sodonesis could only produce 100 mgl−1 of a glycolipid [31]. Phospholipid biosurfactant in micromolar amounts produced by Micrococcus cerificans were reported over pentadecane, hexadecane, tetradecane, and heptadecane [32, 33]. Production has been even lower on simpler substrates. Norcardia butanica yielded 400 mgl−1 of a glycolipid on sucrose substrate, while Brevibacterium species produced a mere 250 mgl−1 [34]. In our case, the production of the biosurfactant was about 380 mgl− and 1.7 gl−1 when glucose and sunflower oil was used as carbon sources, respectively. However, probiotic bacteria constitute a promising source of biosurfactants since these microorganisms are considered GRAS. Furthermore, biosurfactant production can be increased through the optimization of the culture conditions. It is interesting to notice that the change in the carbon source from glucose to vegetable oils (i.e., sunflower oil or waste frying oil) induced the cells to produce more biosurfactant. It can be speculated that the use of vegetable oils as carbon sources instead of glucose induced the cells to use another metabolic pathway, and therefore the amount of biosurfactant produced increased. Another reason is that in the presence of oil, the bacteria are induced to produce more biosurfactant, so they can consume this water-insoluble substrate. Propionic acid bacteria have also proven to be ideal hosts for genetic engineering [35]. The productivity of propionic acid and vitamin B12 has been shown to be increased by genetically modified strains of Propionibacterium [36]. Furthermore, genetic engineering can be an interesting approach for developing new strategies of the increasing the biosurfactant production by Propionibacteria. Propionibacteria produce a considerable amount of acetic acid and propionic acid [37]. For this reason, pH reduction is significant, and as stated in some literature [36], Propionibacteria cannot grow below pH 5.0. Thus, pH control is important. As shown in Fig. 1, pH was controlled between 6.8 and 7 using pH meter mounted on the fermenter. Simultaneous production of acetic acid and propionic acid changes drastically the media conditions and can be responsible for the biosurfactant production inhibition. In fact, it has been found that propionic acid significantly inhibits the cell growth and end product formation [38]. Figure 1 shows with increasing the amount of propionic acid in the culture broth, the biosurfactant production has been inhibited. However, a comprehensive study on the kinetics of inhibition is needed to understand the main effects of acid production on the lipopeptide biosynthesis by P. freudenreichii. The applicability of biosurfactants in many fields depends on their stability at different temperatures, pH values, and salinity. The lipopeptide in this work could tolerate a wide range of salinity. Biosurfactants that tolerate higher salinity are very propitious for bioremediation of

Appl Biochem Biotechnol (2014) 174:2725–2740

2739

petroleum pollution in seawater and hydrocarbon pollution in industrial water. Acidic pH reduced the activity of the biosurfactant. Extreme pH condition could alter the structure and activity of surfactants and reduce their solubility in water [39]. Thermal stability of biosurfactants can also be an important factor for their usage especially in the food industry where heating to achieve sterility is of paramount importance. This is because biosurfactants have several functions in the food industry including agglomeration control of fat globules, improving texture and shelf-life of starch-containing products, stabilizing aerated systems, modifying rheological properties of wheat dough, and improving consistency and texture of fat-based products [40]. The antimicrobial and antiadhesive properties of the biosurfactants have been widely reported [9, 26]. Their antimicrobial and antiadhesive properties make them relevant molecules for use in combating many diseases and infections and as therapeutic agents. However, the biosurfactants with antimicrobial and antiadhesive properties reported till date are produced mostly by the pathogenic strains. The lipopeptide produced by a probiotic and safe strain in this work showed a pronounced antimicrobial and antiadhesive action among tested microorganisms. The antiadhesive activity of the produced biosurfactant against four pathogenic microorganisms i.e., S. aureus (a common cause of infection in community and hospital), E. coli, P. aeruginosa, and B. subtilis are very promising for further studies and applications aiming to reduce/prevent microbial colonization on different materials.

Conclusion In the present study, the crude biosurfactant produced by P. freudenreichii strain was partially characterized for the first time. One reason behind selecting this strain for this work was the lack of study on the capability of biosurfactant production by propionic acid bacteria in literature. Moreover, genus Propionibacteria is known for its benevolent uses to humans in many ways such as production of propionic acid, yogurt, cheese, and vitamin B12. Besides physical and chemical characterization of the produced biosurfactant, the antimicrobial and antiadhesive properties were also studied. In summary, beneficial bacterium P. freudenreichii PTCC 1674 offers promising alternatives for new strategies in the production of probiotic biosurfactants.

References 1. Yin, H., Qiang, J., Jia, Y., Ye, J., Peng, H., Qin, H., et al. (2009). Process Biochemistry, 44, 302–308. 2. Sharafi, H., Abdoli, M., Hajfarajollah, H., Samie, N., Alidoust, L., Abbasi, H., et al. (2014). Applied Biochemistry and Biotechnology, 173(5), 1236-1249. 3. Ghojavand, H., Vahabzadeh, F., & Khodabandeh Shahraki, A. (2012). Journal Petro. Science Engineer, 81, 24–30. 4. Saeki, H., Sasaki, M., Komatsu, K., Miura, A., & Matsuda, H. (2009). Bioresource Technology, 100, 572–577. 5. Mulligan, C. N. (2005). Environmental Pollution, 133, 183–198. 6. Juwarkar, A. A., Nair, A., Dubey, K. V., Singh, S. K., & Devotta, S. (2007). Chemosphere, 68, 1996–2002. 7. Zhang, X., Xu, D., Zhu, C., Lunda, T., & Scherr, K. E. (2012). Chemical Engineering Journal, 209, 138– 146. 8. Sheperd, R., Rockey, J., Sutherland, I., & Roller, S. (1995). Journal of Biotechnology, 40, 207–217. 9. Gudina, E. J., Teixeira, J. A., & Rodrigues, L. R. (2010). Colloid Surf. B: Biointerface, 76, 298–304. 10. Piao, Y., Kawaraichi, N., Asegawa, R., Kiatpan, P., Ono, H., Yamashita, M., et al. (2004). Journal of Bioscience and Bioengineering, 97(5), 310–316. 11. Hajfarajollah, H., Mokhtarani, B., Sharifi, A., Mirzaei, M., & Afaghi, A. (2014). RSC Advances, 4, 13153– 13160.

2740

Appl Biochem Biotechnol (2014) 174:2725–2740

12. Hajfarajollah, H., Mokhtarani, B., Mortaheb, H., & Afaghi, A. (2014). Food Sciene Technology. doi:10.1007/ s13197-014-1383-x. 13. Sen, R., & Swaminathan, T. (1997). Applied Microbiology and Biotechnology, 47, 358–363. 14. Khoshdast, H., Abbasi, H., Sam, A., & Noghabi, K. A. (2012). Biochemical Engineering Journal, 60, 127– 134. 15. Morikawa, M., Hirata, Y., & Imanaka, T. (2000). Biochemistry Biophysical Acta, 488, 211–218. 16. Cooper, D. G., & Goldenberg, B. G. (1987). Applied and Environmental Microbiology, 53(2), 224–229. 17. Das, P., Mukherjee, S., & Sen, R. (2009). Bioresource Technology, 100, 1015–1019. 18. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Analytical Chemistry, 28, 350–356. 19. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254. 20. Folch, J. M., Lees, M., & Stanly, H. S. (1956). Journal of Biological Chemistry, 226, 497–509. 21. Nitschke, M., & Costa, S. G. V. A. O. (2007). Trends Food Sci. Technology, 18, 252–259. 22. Joshi, S., Bharucha, C., & Desai, A. J. (2008). Bioresource Technology, 99, 4603–4608. 23. Arima, K., Kakinuma, A., & Tamura, G. (1968). Biophysics Res. Community, 31(3), 488–494. 24. Jain, R. M., Mody, K., Mishra, A., & Jha, B. (2012). Carbohydrate Polymers, 89, 1110–1116. 25. Ramani, K., Jain, S. C., Mandal, A. B., & Sekaran, G. (2012). Colloid Surf. B: Biointerface, 97, 254–263. 26. Rodrigues, L. R., Teixeira, J. A., Van der Mei, H. C., & Oliveira, R. (2006). Colloid Surf B: Biointerface, 53, 105–112. 27. Thavasi, R., Jayalakshmi, S., & Banat, I. M. (2011). Bioresource Technology, 102, 3366–3372. 28. Busscher, H. J., Neu, T., & Van der Mei, H. C. (1994). Applied Microbiology and Biotechnology, 41, 4–7. 29. Desai, J. D., & Banat, I. M. (1997). Microbiology and Molecular Biology Reviews, 6(1), 47–64. 30. Wahab, K. A., Mohammad Shahedur, R., & Takashi, A. (2009). Journal Environmental Science Supplement., 21, 33–35. 31. Suzuki, T., Tanaka, K., Matsubara, I., & Kinoshita, S. (1969). Agriculture Biological Chemistry, 33(11), 1619–1627. 32. Makula, R., & Finnerty, W. R. (1970). Journal of Bacteriology, 103, 348–355. 33. Makula, M. R., & Finnerty, W. R. (1972). Journal of Bacteriology, 112(7), 398–407. 34. Suzuki, T., Tanaka, H., & Itoh, S. (1974). Agriculture Biological Chemistry, 38(3), 557–563. 35. Zhang, A., & Yang, S. (2009). Biotechnology and Bioengineering, 104, 766–773. 36. Hugenschmidt, S., Schwenninger, S. M., Gnehm, N., & Lacroix, C. (2010). International Dairy Journal, 20, 852–857. 37. Gardner, N., & Champagne, C. P. (2005). Journal of Applied Microbiology, 99, 1236–1245. 38. Miyano, K., Ye, K., & Shimizu, K. (2005). Biochemical Engineering Journal, 6, 207–214. 39. Qiao, N., & Shao, Z. J. (2010). Appl. Microbiology, 108, 1207–1216. 40. Nitschke, M., & Pastore, G. M. (2004). Applied Biochemistry and Biotechnology, 112, 163–172. 41. Saravanakumari, P., & Mani, K. (2010). Bioresource Technology, 101, 8851–8854.

Newly antibacterial and antiadhesive lipopeptide biosurfactant secreted by a probiotic strain, Propionibacterium freudenreichii.

A lipopeptide biosurfactant production from a probiotic type strain of Propionibacterium freudenreichii subsp. freudenreichii is being reported here f...
602KB Sizes 0 Downloads 11 Views