Appl Biochem Biotechnol DOI 10.1007/s12010-013-0649-5
Evaluation and Functional Characterization of a Biosurfactant Produced by Lactobacillus plantarum CFR 2194 Arenahalli Ningegowda Madhu & Siddalingaiya Gurudutt Prapulla
Received: 30 April 2012 / Accepted: 8 November 2013 # Springer Science+Business Media New York 2013
Abstract The study details the investigations on the ability of Lactobacillus plantarum CFR 2194, an isolate from kanjika, a rice-based ayurvedic fermented product, to produce biosurfactant. Surfactant production, as a function of fermentation time, indicates that the maximum production occurred at 72 h under stationary conditions. Isolation, partial purification, and characterization of the biosurfactant produced have been carried out, and Fourier transform infrared spectroscopy (FTIR) spectra demonstrated that biosurfactants were constituted by protein and polysaccharide fractions, i.e., possessed the structure typical of glycoprotein, which is affected by the medium composition and the phase of growth of the biosurfactant-synthesizing strain. Critical micelle concentration (cmc) of the biosurfactant was found to be 6 g l−1. The emulsification index (EI), emulsification activity (EA), and emulsion stability (ES) values of the biosurfactant have confirmed its emulsification property. Aqueous fractions of the produced biosurfactant exhibited a significant antimicrobial activity against the food-borne pathogenic species: Escherichia coli ATCC 31705, E. coli MTCC 108, Salmonella typhi, Yersinia enterocolitica MTCC 859, and Staphylococcus aureus F 722. More importantly, the biosurfactant from L. plantarum showed antiadhesive property against above food-borne pathogens. The results thus indicate the potential for developing strategies to prevent microbial colonization of food contact surfaces and health-care prosthesis using these biosurfactants. Keywords Biosurfactants . Probiotics . Lactobacillus plantarum . Glycoproteins
Introduction Biosurfactants or bioemulsifiers are a structurally diverse group of surface-active molecules synthesized by microorganisms. Lactic acid bacteria (LAB), in particular Lactobacillus genus,
A. N. Madhu : S. G. Prapulla (*) Fermentation Technology and Bioengineering Department, Council of Scientific and Industrial Research (CSIR), Central Food Technological Research Institute, Mysore 570 020, India e-mail: [email protected]
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are an important group of biosurfactant-synthesizing microorganisms. The use of biosurfactant released by lactobacilli strains is very promising because these microorganisms are natural inhabitants of human microflora and are well known for their probiotic effect [1]. Biosurfactants are environmentally friendly, since they are easily biodegradable [2] and have low toxicity [3], and their unique structures provide new properties that many classical surfactants lack. Originally, biosurfactants attracted attention as hydrocarbon dissolution agents [4], but the interest in these molecules as an alternative to chemical surfactants (carboxylates, sulfonates, and sulfate acid esters), especially in food, pharmaceutical, and oil industry, has been increasing considerably in the recent years [5]. The emulsifying property of microbial surfactant finds many applications in food industry [6]. Attempts have been made to evaluate stable emulsion formation by biosurfactants with edible oils and fats. An extracellular carbohydrate-rich compound from Candida utilis was successfully used as an emulsifying agent in salad dressing formulations [7]. In addition to emulsification, biosurfactants exhibit antiadhesive and antimicrobial properties [1], and this can be used in controlling the adherence of microorganisms to food contact surfaces providing safe and quality products to consumers [8]. Biosurfactant produced from thermophilic Streptococcus pasteurizers retards the colonization of other thermophilic strains of streptococcus responsible for fouling in dairy products [9]. Considering all the interesting properties demonstrated by biosurfactants, they can be projected as multipurpose food additives of future, exhibiting emulsifier, antiadhesive, and antimicrobial activities. They bound to have a significant impact and might open up newer avenues for their application in food industry [5, 7]. The main factor that works against the widespread use of biosurfactants is the economics of their production [10]. An important point that should be considered for the development of cheaper processes is the selection of inexpensive medium, which accounts for 10–30 % of overall costs [11]. Agro-industrial by-products like molasses from sugar industry [12] or wastes like used cooking oil, cassava [13], and distillery waste [14] are promising alternatives. These residues generally contain high levels of carbohydrates or lipids to support growth and surfactant synthesis. The treatment and disposal costs for these residues demand greater attention from industry, which are invariably looking for alternatives to reduce, reuse, and recycle these wastes. The increasing number of patents issued on biosurfactant, regarding their use as additives for food, cosmetics, and pharmaceutical products [15], demonstrates the increasing interest in using these biosurfactants obtained from GRAS microorganisms like lactobacilli. However, biosurfactant is not yet used on a large scale, as many regulations regarding the approval of new food ingredients are required by local governmental agencies. Consumer awareness and changes in food policies will definitely create a breakthrough in the use of biosurfactant as a food additive. The present study is focused on the production of biosurfactant by Lactobacillus plantarum CFR 2194, a kanjika (ayurvedic rice-based fermented product), an isolate which has shown some interesting characteristics like the potency for producing vitamin B12 [16], β-galactosidase activity for lactose intolerance sufferers, and production of short-chain fatty acids like lactate and butyrate [17], which play a pivotal role in the prevention of colon carcinogenesis and also has shown positive indications for the production of silver nanoparticles (unpublished data). Hence, in the present investigation, an attempt has been made to evaluate the potential of the selected potent probiotic isolate, L. plantarum CFR 2194 for biosurfactant production, as an additional property. The study also includes optimization and functional characterization of the isolated biosurfactant.
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Materials and Methods Strains and Culture Conditions L. plantarum CFR 2194, isolated from kanjika, was used for the production of biosurfactant. The LAB strain was stored at −60 °C in de Man–Rogosa–Sharpe (MRS) broth (HiMedia, India), supplemented with 40 % (v/v) glycerol as a cryoprotectant. Prior to use, the culture [1 % (v/v)] was subcultured twice in MRS broth at 37 °C for 12 h. Escherichia coli ATCC 31075, E. coli MTCC 108, Salmonella typhi, Yersinia enterocolitica MTCC 859, and Staphylococcus aureus F 722 were the test pathogens used for studying the antimicrobial and antiadhesive properties of the biosurfactant. All the strains were stored at −60 °C in nutrient broth (HiMedia, India), supplemented with 40 % (v/v) glycerol as a cryoprotectant. Prior to use, the frozen stocks [1 % (v/v)] were transferred twice to nutrient broth and incubated at 37 °C for 12 h. Production of Biosurfactant The fermentative production of biosurfactant from L. plantarum CFR 2194 was carried out in Erlenmeyer flasks containing MRS medium (200 ml). The bacterium was cultured twice in MRS broth for 12 h and used as an inoculum (2 %v/v, 7.51 log cfu ml−1) to inoculate fermentation medium (MRS). The medium was incubated at 37 °C for 4, 18, or 72 h under stationary and shaking conditions (150 rev min−1). Isolation of Biosurfactant Biomass of L. plantarum was harvested by centrifugation (10,000×g, 5 min, 10 °C), washed twice with distilled water, and resuspended in 100 ml of phosphate-buffered saline (PBS) (10 mM KH2PO4/K2HPO4 and 150 mM NaCl with pH adjusted to 7.0). The bacterial suspension was incubated on a magnetic stirrer for 2 h at room temperature with gentle stirring for cell-bound biosurfactant to release. Subsequently, the bacterial biomass was separated by centrifugation (10,000×g, 5 min, 10 °C), and the cell-free supernatant was filtered through Millipore-P20 filters (0.2 μm; Millipore, India). The supernatant was dialyzed against distilled water at 4 °C using dialysis membrane (molecular weight cutoff 6,000–8,000; Sigma, India) and freeze-dried. The aqueous suspension of freeze-dried biosurfactant was acidified with 12 N HCl (pH 2.0); at 4 °C for 16–18 h. The precipitated biosurfactant was collected by centrifugation (10,000 rpm, 4 °C, and 15 min). The precipitate was washed three times in acidic water (pH 2.0 with 12 N HCl) and finally resuspended in distilled water and then freeze-dried and stored at −20 °C. Surface Tension Measurement The biosurfactant production was measured in terms of surface tension using the du Nouy ringtype tensiometer (Kruss, K9) equipped with a 1.9-cm platinum ring [1]. The surface tension measurement was carried out at 20 °C after immersing the platinum ring in the solution for a while, in order to attain the equilibrium. The instrument was calibrated by measuring the surface tension of distilled water. All the measurements were taken in triplicate, and an average value was used to express the surface tension. Sterile PBS with surface tension of 72.2 mN m−1 at 20 °C was used as a control.
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Fourier Transform Infrared Spectroscopy (FTIR) The sample was prepared by dispersing uniformly in a matrix of KBr. Freeze-dried biosurfactant (6 mg) was ground into fine powder in a small mortar with 300 mg of KBr. The finely ground powder was placed in the pellet holder, and the screw on top was tightened gently to form the pellet. The spectroscopy measurements of the prepared samples were performed using the diffusion method with the absorption measurement within the range of wave number from 4,000.0 to 400.0 cm−1, using a FTIR spectrophotometer (Nicolet 5700 model; Thermo Electron Corporation, USA) equipped with a KBr bundle separator and DTGS (deuterated triglycine sulfate) detector. Each sample was scanned 64 times at the resolution 4.0 cm−1 and scanner rate 0.2 cm s−1. The KBr pellet was used as a background reference. Oil Spreading Test The oil spreading test measures the diameter of clear zones caused when a drop of a biosurfactant-containing solution is placed on an oil–water surface [18]. The binomial diameter and biosurfactant concentration allow the determination of the cleaning efficiency of a given biosurfactant. The oil spreading test was carried out with 50 ml of distilled water taken in a Petri dish (25 cm diameter) overlaid with 20 μl of crude oil. Crude biosurfactant dissolved in PBS (10 μl at concentrations ranging from 4 to 15 mg ml−1) was then added to the surface of oil. PBS with no added biosurfactant served as the control. The diameters of clear zones of triplicate assays from the same sample were determined. Determination of Critical Micelle Concentration (cmc) cmc is a measure of the concentration of a solution component which represents a critical value, above which an increasing concentration of that component forces the formation of micelles. It is important for several applications of biosurfactant to establish their cmc, as above this concentration, no further effect is expected in the surface activity. The cmc was determined by plotting the surface tension as a function of the logarithm of biosurfactant concentration and is represented as the point at which the baseline of minimal surface tension intersects the slope where surface tension shows a linear decline. The biosurfactant of different concentrations (2.5–40 g l−1) was tested for surface tension until the surface tension will not reduce anymore. Measurements were done in triplicate. Emulsification Index (EI) Emulsification index was calculated based on the increase in the height of emulsion. A mixture of 1 ml of crude biosurfactant (1 mg ml−1), 4 ml of water, and 6 ml of hydrocarbon/oil (kerosene, xylene, hexadecane, heptane, coconut oil, and sunflower oil) was vigorously shaken for 2 min to obtain maximum emulsification. After 24 h, the height of the emulsion was measured, and emulsification index was calculated using Eq. 1. Water was used as the negative control; Tween 80 (1 %) served as the positive control.
EI24 ¼
Height of emulsion layer 100 Total height
The assay was performed in the same sized graduated glass test tubes [19].
ð1Þ
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Emulsification Activity (EA) and Emulsion Stability (ES) The emulsification activities of the biosurfactants were quantified according to the method developed by Calvo et al. [20] and Cirigliano and Carman [20, 21]. Freeze-dried biosurfactant (1 mg ml−1) prepared in PBS (pH 7.0) was diluted with distilled water to a final volume of 4 ml, and the solution was mixed with 1 ml of a substrate (kerosene, xylene, coconut oil, sunflower oil, hexane, and heptane) in a screw-capped tube, and then, the mixture was vigorously shaken for 2 min in a vortex mixer and was allowed to stand for 10 min before measuring the turbidity at 540 nm. The absorbance was expressed as the emulsification activity. The emulsion stability was expressed on the basis of the emulsification activity [20, 21]. The emulsified solutions were allowed to stand for 10 min at room temperature, and absorbance was measured every 10 min over a period of 60 min. The log of the absorbance was plotted versus time, and the slope (decay constant, Kd) was calculated to express the emulsion stability. Antimicrobial Activity The antimicrobial activity of 1.0, 2.0, or 4.0 % (w/v) aqueous solution of freeze-dried biosurfactant was determined using agar well diffusion method described by Golek et al. [22]. Test organisms, E. coli ATCC 31705, E. coli MTCC 108, S. typhi, Y. enterocolitica MTCC 859, and S. aureus F 722, were grown for 12 h in nutrient broth and pour plated with nutrient agar. The aqueous solutions of biosurfactant preparations (60 μl) were added into the wells on the nutrient agar drilled with a sterile 5-mm-diameter driller. Plates were kept in refrigerator for 2 h to diffuse the spent broth and then incubated at 37 °C for 16 h. Diameters (millimeters) of inhibition zones of bacterial growth around the wells were determined with respect to a control sample, i.e., sterile water. Analyses were carried out in triplicate. Antiadhesive Activity The antiadhesive activity of the crude biosurfactant isolated from L. plantarum CFR 2194 against common food-borne pathogens, mentioned in the previous paragraph, was quantified according to the procedure described by Reid et al. [23]. The sterile 96-well flat-bottomed ELISA plate (HiMedia Pvt Ltd., India) was filled with 200 μl of the crude biosurfactant (3 to 50 mg ml−1). The plate was incubated for 18 h at 4 °C and subsequently washed twice with PBS. Wells containing PBS buffer was taken as the control. An aliquot of 200 μl of washed bacterial cells were added to the wells and incubated for 4 h at 4 °C. Unattached microorganisms were removed by washing the wells three times with PBS. The adherent microorganisms were fixed with 200 μl of methanol (99 % purity) 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. Excess stain was rinsed out 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 absorbance of each well was measured at 595 nm. The antiadhesive activity in terms of microbial inhibition (percent) at different biosurfactant concentrations for each microorganism was calculated as follows: % Microbial inhibitionc ¼ ½1−ðAc =A0 Þ 100 where Ac represents the absorbance of the well at a biosurfactant concentration c, and A0 represents the absorbance of the control well. The antiadhesion assay allows the
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estimation of the biosurfactant concentrations that are effective in decreasing adhesion of the microorganisms studied.
Statistical Analysis Values were expressed as means and standard deviations of triplicate experiments. Statistical analysis was carried out using the Origin 6.1 statistical software (OriginLab Corporation, Northampton, MA, USA).
Results and Discussion Synthesis and Characteristics of Biosurfactant Microorganisms that simultaneously produce more than one substance show additional advantages in terms of contributing to increase the profitability of the process. Nevertheless, in some occasions, metabolites need different conditions of cultivation, thus necessitating the defining of suboptimal compromise conditions to allow reasonable productions of all of them. Additionally, it is necessary to study the appropriate methodology to recover separately each metabolite, particularly when different applications are implicated. L. plantarum CFR 2194, as a vitamin B12 [16] and biosurfactant (present study) producer, is a good example of that. There are several reports describing the ability of different LAB to produce biosurfactants [24, 25], but there is only one study for L. plantarum [26]. Thus, the present study focused on the evaluation of the biosurfactant producing capacity of L. plantarum CFR 2194. Reduction in the surface tension of the PBS cell extract indicated the ability of L. plantarum CFR 2194 to produce biosurfactant. The surface tension values of PBS cell extract are given in Table 1. The reduction in the surface tension of PBS cell extract obtained from cells grown under stationary condition (44.3 mN m−1) was comparatively higher than that of control PBS (72.0 mN m−1). The biosurfactant production was highest at 72 h with a 38.63 % decrease in surface tension under stationary conditions (Fig. 1). The present observation that biosurfactant released by L. plantarum CFR 2194 was maximum under stationary phase of growth is in accordance with the earlier report [27]. Additionally, it was also observed that L. plantarum did not produce extracellular biosurfactants as indicated by a negligible difference in the surface tension values of cell-free supernatant (CFS) obtained after 24, 48, and 72 h of fermentation (65–67 mN m−1)
Table 1 The surface tension of biosurfactant from L. plantarum CFR 2194 grown under stationary and shaking conditions Fermentation time (h)
Surface tension of PBS cell extract, mN m−1 Stationary
Shaking
Control PBS
72.2±0.00
–
24
64.46±0.25
67.2±0.15
48
53.56±0.35
56.6±0.26
72
44.30±0.20
50.3±0.15
Results are expressed as mean ± standard deviations of values from triplicate experiments
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Fig. 1 Effect of time on surface tension of the surfactant produced by L. plantarum CFR 2194 against control PBS. Results are expressed as mean ± standard deviations of values from triplicate experiments
in comparison with that of MRS control (70.8 mN m−1). Hence, it could be inferred that the biosurfactant produced is cell bound. Microbial surfactants are characterized by a strong ability to lower surface tension. For instance, surfactin from Bacillus subtilis causes a minimal surface tension of 27 mN m−1 and has a cmc of 11 mg l−1, while the synthetic surfactant sodium dodecyl sulfate has a minimal surface tension of only 37 mN m−1 and a cmc of 2.0–2.9 g l−1 [28]. Strains of lactobacilli synthesize surfactants which are efficient in comparison with synthetic ones. Lactobacillus acidophilus RC14 releases biosurfactant with a cmc of 1.0 g l−1 and a corresponding surface tension of 39 mN m−1 [28]. The results obtained in the current study showed that the mean value of a coefficient of surface tension of a crude biosurfactant solution in a PBS buffer, after their extraction from biomass of L. plantarum CFR 2194, reached 44.30±0.20 mN m−1. The cmc for the crude biosurfactant is shown in Fig. 2. The sample was found to be the most surface active, with cmc of 6 g l−1.
Fig. 2 Effect of concentration of the surfactant produced by L. plantarum CFR 2194 on surface tension. Results are expressed as mean ± standard deviations of values from triplicate experiments
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Structure Analyses of Biosurfactants by Means of FTIR The FTIR spectra (Fig. 3) indicated that the biosurfactant is proteinaceous in nature. The absorbance maxima recorded at wavelengths of 3,408.9, 1,661.2, and 1,556.0 cm−1 are typical of stretching →N–H bonds and CO–N and N=O bonds, indicating the presence of proteins in the sample analyzed. The absorption peak at 2,948 cm−1 corresponds to the presence of bonds occurring in aliphatic chains (–CH3, –CH2–). The occurrence of a spectrum over wavelength range of 1,200–1,000 cm−1 indicated the presence of the polysaccharide fraction of biosurfactant. Strong absorption at a wavelength of 1,087.2 cm−1 is typical of oscillations of C–O–C bonds. The FTIR spectra demonstrated that the chemical composition of biosurfactant synthesized by L. plantarum CFR 2194 is nonhomogeneous and is composed of protein and polysaccharide fraction. Literature data indicated that only a few microorganisms synthesize the surfaceactive compounds as chemically pure ones [29]. For instance, Pseudomonas aeruginosa synthesizes rhamnolipids with various chemical structures, whereas Torulopsis bombicola yeast synthesizes a mixture of at least six to nine different sophorolipids [30, 31]. Oil Displacement Test (ODT) The oil displacement test is an indirect measurement of surface activity of a surfactant sample tested against oil; a larger diameter represents a higher surface activity of the testing solution [18]. Surface activities of the crude biosurfactant were investigated in comparison with that of Tween 80 and PBS as positive and negative controls, respectively. The diameter of the clear zone formed was measured as the activity of biosurfactant (Table 2). As can be seen from the results, diameter of the clear zone increased with an increase in the concentration of the biosurfactant. Oil layer was dispersed maximum at a biosurfactant concentration of 15 mg/ml equivalent to that of the positive control Tween 80 (0.1 % w/v) indicating high surface-active properties at higher concentrations. Although the mechanism of oil displacement by biosurfactant has not been fully understood at molecular level, it is considered as a sensitive and simple method for the measurement of surface active nature of the biosurfactant.
Fig. 3 FTIR spectrum of biosurfactant produced by L. plantarum CFR 2194
Appl Biochem Biotechnol Table 2 Comparison of oil spreading efficiency of standard surface-active agents and crude biosurfactant Agents
Diameter of the clearing zone (mm)
PBS control
0
Tween 80 (0.1 %)
3.4±0.12
Crude biosurfactant (4 mg ml−1)
1.2±0.09
Crude biosurfactant (10 mg ml−1) Crude biosurfactant (15 mg ml−1)
1.9±0.28 2.3±0.15
PBS was used as control with a diameter of 0.0 mm as no clearing zone occurs. Results are expressed as mean ± standard deviations of values from triplicate experiments
Emulsification Index, Emulsification Activity, and Emulsion Stability The emulsification index of the produced biosurfactant (1 mg ml−1) was tested against different substrates like kerosene, xylene, coconut oil, sunflower oil, hexane, and heptane. Tween 80 was used as a standard. Among the hydrocarbons, heptane gave the highest EI value of 38.2, followed by xylene (16.22), kerosene (15.2), and hexane (13.6). EI was significantly higher against certain vegetable oils like coconut oil (37.9) and sunflower oil (19.43), which can have wider implication for food formulations (Fig. 4). The emulsification activity of the biosurfactant from L. plantarum CFR 2194 was measured with a variety of water-immiscible substrates (Table 3). The stabilization ability (Table 3) of the biosurfactant from isolate described as the decay content, Kd values (the slope of the emulsion decay plot) are presented in Table 3. Additionally, emulsion decay plots were constructed for a variety of emulsifying substrates in the presence of the biosurfactants, after which the respective Kd values were calculated (Table 3). From the results, it is observed that coconut oil and sunflower oil were efficiently emulsified. In addition, kerosene and xylene were also emulsified to a considerable extent. Interestingly, EA with n-alkanes increased with
Fig. 4 Emulsification index of biosurfactant for different substrates. Results are expressed as mean ± standard deviations of values from triplicate experiments
Appl Biochem Biotechnol Table 3 Emulsification activity and emulsification stability of biosurfactant from L. plantarum CFR 2194 Emulsification activity (OD540 nm)a
Decay constant (Kd, 10−3)b
Kerosene
0.77
−3.26
Xylene
0.83
−3.29
Coconut oil
1.81
−0.51
Sunflower oil Hexadecane
1.15 1.04
−5.23 −4.29
Heptane
0.34
−7.53
Substrate
Results are expressed as mean ± standard deviations of values from triplicate experiments a
The emulsification assay was performed in the presence of the biosurfactant as described in the text. After an initial 10-min holding period, absorbance readings were taken every 10 min for 60 min b The log of the absorbance was plotted versus time, and the slope (decay constant, Kd) was calculated
the increase in the number of carbon atoms. Emulsions formed with vegetable oils were more stable than emulsions formed with hydrocarbons (Table 3). In the similar study, Moldes et al. [32] and Bello et al. [33] reported that biosurfactants obtained after growing Lactobacillus pentosus on sugars from agricultural residues, shown to stabilize octane/water and gasoline/water emulsions after 24 h of incubation. Antimicrobial Activity Biosurfactant obtained from the lactic acid bacteria have been indicated to exhibit antibiotic properties, which predisposes this group of chemical compounds for potential application in the production of antibiotics [34, 35]. From this perspective, experiments were conducted to determine the antimicrobial activity of preparations of biosurfactant synthesized by L. plantarum CFR 2194 against some of the food-borne pathogens. There are few reports on the antimicrobial activity of biosurfactants isolated from lactobacilli; only biosurfactants obtained from Streptococcus thermophilus A and Lactobacillus lactis 53 have been reported to exhibit a significant antimicrobial activity against several bacterial and yeast strains isolated from explanted voice prostheses [34, 36]. In the present
Table 4 Antimicrobial activities of the biosurfactant from L. plantarum CFR 2194 Microorganism
Zone of inhibition 4 mg ml−1
25 mg ml−1
Escherichia coli ATCC 31075
−
+
Escherichia coli MTCC 108
++
+++
Salmonella typhi Yersinia enterocolitica MTCC 859
− −
− ++
Staphylococcus aureus F 722
+
++
“+” sign indicates inhibition of microbial growth, whereas “−” sign indicates inability to inhibit the growth of the tested microorganisms; + indicates zone of inhibition around 5 mm around the application of biosurfactant, “++” zone of inhibition around 5–10 mm around the application of biosurfactant, and “+++” zone of inhibition more than 15 mm around the application of biosurfactant
Appl Biochem Biotechnol Table 5 Antiadhesive properties of biosurfactant from L. plantarum CFR 2194 Microorganism
Biosurfactant (mg ml−1) 4.0
Escherichia coli ATCC 31075
8.04±0.2
25.0 54.43±0.1
Escherichia coli MTCC 108
10.65±0.1
56.78±0.3
Salmonella typhi
13.92±0.1
61.60±0.2
Yersinia enterocolitica MTCC 859
15.23±0.3
64.72±0.4
Staphylococcus aureus F 722
25.90±0.1
67.18±0.2
Negative controls were set at 0 % to indicate the absence of biosurfactant. Positive percentages indicate the reductions in microbial adhesion when compared to the control. Results are expressed as means ± standard deviation of results from triplicate experiments
study, the antimicrobial activity of the preparations of biosurfactant increased with an increase in the concentration (Table 4). As can be seen from the results, the highest antimicrobial activity was observed at 4 and 25 mg ml−1 aqueous solutions of biosurfactant preparations against E. coli ETEC and S. aureus, respectively. Complete inhibition for E. coli ATCC and Y. enterocolitica was observed at a concentration of 25 mg ml−1 of biosurfactant preparation; thus, results indicate the antimicrobial potential of the biosurfactant synthesized by L. plantarum CFR 2194. However, no inhibition was observed with respect to the growth of S. typhi at both concentrations of the biosurfactant. Antiadhesive Activity In addition to the antimicrobial properties, the isolated biosurfactant exhibited a considerable antiadhesive activity against most of the microorganisms tested. Involvement of biosurfactants in microbial adhesion and desorption has been widely described, and adsorption of biosurfactants isolated from lactobacilli to solid surfaces might constitute an effective strategy to reduce microbial adhesion and combating colonization by pathogenic microorganisms, not only in the biomedical field but also in other areas, such as the food industry [1, 5, 37, 38]. The antiadhesive activity of the biosurfactant was evaluated against a few pathogens as listed in Table 5. The biosurfactant showed antiadhesive activity against all the tested microorganisms, but the antiadhesive effect depends on the concentration and the microorganism tested. The antiadhesive activity was higher against S. aureus (67.18±0.2 % inhibition) at a biosurfactant concentration of 25 mg ml−1. For Y. enterocolitica, the highest antiadhesive percentages were 64.72±0.4 %, whereas the lowest antiadhesive percentages were for E. coli ATCC (54.43±0.1 %). Regarding the other pathogenic bacteria, high antiadhesive percentages were obtained for E. coli ETEC (56.78±0.3 %) and S. typhi (61.60±0.2 %).
Conclusion The study carried out demonstrates that the strain L. plantarum CFR 2194 was able to synthesize the cell-bound biosurfactant. The spectral (FTIR) analysis of biosurfactant synthesized revealed the presence of protein and polysaccharide fractions. Considerable emulsifying activity of the biosurfactant revealed that they could be used as emulsion-forming agents for hydrocarbons and oils, giving stable emulsions. The study has also effectively demonstrated
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the antimicrobial and antiadhesive properties of the biosurfactant isolated from L. plantarum CFR 2194 against food-borne pathogens. The results obtained suggest the possible use of this biosurfactant as an alternative antimicrobial agent in the medical field for applications against microorganisms responsible for diseases and infections in the urinary, vaginal, and gastrointestinal tracts as well as in the skin, making it a suitable alternative to conventional antibiotics. Acknowledgments A.N. Madhu is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for the award of Senior Research Fellowship. Esther Magdalene Sharon, a project student, was kindly acknowledged for help in carrying out the experiments. Dr. Purnima Kaul Tiku, a scientist, of the Protein Chemistry and Nutrition, CFTRI, is greatly acknowledged for the help in using the tensiometer. The authors thank the Director, CFTRI, for supporting the work.
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Singh, P., & Cameotra, S. S. (2004). Trends in Biotechnology, 22(3), 142–146. Mohan, P. K., Nakhla, G., & Yanful, E. K. (2006). Water Research, 40, 533–540. Flasz, A., Rocha, C. A., Mosquera, B., & Sajo, C. (1998). Medical Science Research, 26(3), 181–185. Mulligan, C. N. (2005). Environmental Pollution, 133(2), 183–198. Nitschke, M., & Costa, S. G. V. A. O. (2007). Trends in Food Science and Technology, 18, 252–259. Banat, I. M., Makkar, R. S., & Cameotra, S. S. (2000). Applied Microbiology and Biotechnology, 53, 495– 508. Shepherd, R., Rockey, J., Sutherland, I. W., & Roller, S. (1995). Journal of Biotechnology, 40, 207–217. Hood, S. K., & Zottola, E. A. (1995). Food Control, 6(1), 9–18. Busscher, H. J., van der Kuij-Booij, M., & van der Mei, H. C. (1996). Journal of Industrial Microbiology and Biotechnology, 16(1), 15–21. Makkar, R. S., & Cameotra, S. S. (2002). Applied Microbiology and Biotechnology, 58, 428–434. Cameotra, S. S., & Makkar, R. S. (1998). Applied Microbiology and Biotechnology, 50, 520–529. Makkar, R. S., & Cameotra, S. S. (1997). Journal of the American Oil Chemists Society, 74, 887–889. Nitschke, M., & Pastore, G. M. (2003). Applied Biochemistry and Biotechnology, 105–108, 295–301. Dubey, K., & Juwarkar, A. (2001). World Journal of Microbiology and Biotechnology, 17, 61–69. Shete, A. M., Wadhawa, G., Banat, I. M., & Chopade, B. A. (2006). Journal of Science of Industrial Research, 65(2), 91–115. Madhu, A. N., Giribhattanavar, P., Narayan, M. S., & Prapulla, S. G. (2010). Biotechnology Letters, 32, 503– 506. Madhu, A. N., & Prapulla, S. G. (2012). World Journal of Microbiology and Biotechnology, 28, 901–908. Rodrigues, L. R., Teixeira, J. A., van der Mei, H. C., & Oliveira, R. (2006). Colloids and Surfaces B: Biointerfaces, 49, 78–85. Cooper, D. G., & Goldenberg, B. G. (1987). Applied and Environmental Microbiology, 55(2), 224–227. Calvo, C., Martinez, C. F., Mota, A., Bejar, V., & Quesada, E. (1998). Journal of Industrial Microbiology and Biotechnology, 20, 205–209. Cirigliano, M. C., & Carman, G. M. (1985). Applied Environmental Microbiology, 50, 846–850. Golek, P., Bednarski, W., Brzozowski, B., & Dziuba, B. (2009). Annals of Microbiology, 59(1), 119–126. Reid, G., Heinemann, C., Velraeds, M. M. C., van der Mei, H. C., & Busscher, H. J. (1999). Methods in Enzymology, 310, 426–432. Portilla, O. M., Moldes, A. B., Torrado, A. M., & Domínguez, J. M. (2008). Journal of Agriculture Food Chemistry, 56, 8074–8080. Portilla, O. M., Rivas, B., Torrado, A., Moldes, A. B., & Domínguez, J. M. (2008). Journal of Science of Food and Agriculture, 88, 2298–2308. Anukam, K. C., & Reid, G. (2007). Research Journal of Microbiology, 2(1), 81–87. Bartosz, B., Wlodzimierz, B., & Bartlomiej, D. (2009). Journal of Science of Food and Agriculture, 89, 2467–2476. Velraeds, M. M. C., van der Mei, H. C., Reid, G., & Busscher, H. J. (1996). Colloids and Surfaces B: Biointerfaces, 8, 51–61.
Appl Biochem Biotechnol 29. Busscher, H. J., van Hoogmoed, C. G., Geertsema-Doornbusch, G. I., van der Kuijl-Booij, M., & van der Mei, H. C. (1997). Applied Environmental Microbiology, 63, 3810–3817. 30. Lang, S., & Wullbrandt, D. (1999). Applied Microbiology and Biotechnology, 51, 22–32. 31. Schippers, C., Gebner, K., Muller, T., & Scheper, T. (2000). Journal of Biotechnology, 83, 189–198. 32. Moldes, A. B., Paradelo, R., Vecino, X., Cruz, J. M., Gudina, E., Rodrigues, L., Teixeira, J. A., Dominguez, J. M., & Barral, M. T. (2013). BioMed Research International. doi:10.1155/2013/961842. Article ID 961842, 6 pages. 33. Bello, X. V., Devesa-Rey, R., Cruz, J. M., & Moldes, A. B. (2012). Journal of Agriculture and Food Chemistry, 60, 1258–1265. 34. Rodrigues, L. R., Van der Mei, H. C., Teixeira, J. A., & Oliveira, R. (2004). Applied Environmental Microbiology, 70, 4408–4410. 35. Reid, G., Gan, B. S., Ens, W., Weinberger, S., & Howard, J. C. (2002). Applied Environmental Microbiology, 68, 977–980. 36. Rodrigues, L. R., Teixeira, J. A., Van der Mei, H. C., & Oliveira, R. (2006). Colloids and Surfaces B: Biointerfaces, 53, 105–112. 37. Rodrigues, L. R., Banat, I. M., van der Mei, H. C., Teixeira, J. A., & Oliveira, R. (2006). Journal of Applied Microbiology, 100(3), 470–480. 38. Falagas, M. E., & Makris, G. C. (2009). Journal of Hospital Infection, 71, 301–306.
Evaluation and Functional Characterization of a Biosurfactant Produced by Lactobacillus plantarum CFR 2194 Arenahalli Ningegowda Madhu & Siddalingaiya Gurudutt Prapulla
Received: 30 April 2012 / Accepted: 8 November 2013 # Springer Science+Business Media New York 2013
Abstract The study details the investigations on the ability of Lactobacillus plantarum CFR 2194, an isolate from kanjika, a rice-based ayurvedic fermented product, to produce biosurfactant. Surfactant production, as a function of fermentation time, indicates that the maximum production occurred at 72 h under stationary conditions. Isolation, partial purification, and characterization of the biosurfactant produced have been carried out, and Fourier transform infrared spectroscopy (FTIR) spectra demonstrated that biosurfactants were constituted by protein and polysaccharide fractions, i.e., possessed the structure typical of glycoprotein, which is affected by the medium composition and the phase of growth of the biosurfactant-synthesizing strain. Critical micelle concentration (cmc) of the biosurfactant was found to be 6 g l−1. The emulsification index (EI), emulsification activity (EA), and emulsion stability (ES) values of the biosurfactant have confirmed its emulsification property. Aqueous fractions of the produced biosurfactant exhibited a significant antimicrobial activity against the food-borne pathogenic species: Escherichia coli ATCC 31705, E. coli MTCC 108, Salmonella typhi, Yersinia enterocolitica MTCC 859, and Staphylococcus aureus F 722. More importantly, the biosurfactant from L. plantarum showed antiadhesive property against above food-borne pathogens. The results thus indicate the potential for developing strategies to prevent microbial colonization of food contact surfaces and health-care prosthesis using these biosurfactants. Keywords Biosurfactants . Probiotics . Lactobacillus plantarum . Glycoproteins
Introduction Biosurfactants or bioemulsifiers are a structurally diverse group of surface-active molecules synthesized by microorganisms. Lactic acid bacteria (LAB), in particular Lactobacillus genus,
A. N. Madhu : S. G. Prapulla (*) Fermentation Technology and Bioengineering Department, Council of Scientific and Industrial Research (CSIR), Central Food Technological Research Institute, Mysore 570 020, India e-mail: [email protected]
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are an important group of biosurfactant-synthesizing microorganisms. The use of biosurfactant released by lactobacilli strains is very promising because these microorganisms are natural inhabitants of human microflora and are well known for their probiotic effect [1]. Biosurfactants are environmentally friendly, since they are easily biodegradable [2] and have low toxicity [3], and their unique structures provide new properties that many classical surfactants lack. Originally, biosurfactants attracted attention as hydrocarbon dissolution agents [4], but the interest in these molecules as an alternative to chemical surfactants (carboxylates, sulfonates, and sulfate acid esters), especially in food, pharmaceutical, and oil industry, has been increasing considerably in the recent years [5]. The emulsifying property of microbial surfactant finds many applications in food industry [6]. Attempts have been made to evaluate stable emulsion formation by biosurfactants with edible oils and fats. An extracellular carbohydrate-rich compound from Candida utilis was successfully used as an emulsifying agent in salad dressing formulations [7]. In addition to emulsification, biosurfactants exhibit antiadhesive and antimicrobial properties [1], and this can be used in controlling the adherence of microorganisms to food contact surfaces providing safe and quality products to consumers [8]. Biosurfactant produced from thermophilic Streptococcus pasteurizers retards the colonization of other thermophilic strains of streptococcus responsible for fouling in dairy products [9]. Considering all the interesting properties demonstrated by biosurfactants, they can be projected as multipurpose food additives of future, exhibiting emulsifier, antiadhesive, and antimicrobial activities. They bound to have a significant impact and might open up newer avenues for their application in food industry [5, 7]. The main factor that works against the widespread use of biosurfactants is the economics of their production [10]. An important point that should be considered for the development of cheaper processes is the selection of inexpensive medium, which accounts for 10–30 % of overall costs [11]. Agro-industrial by-products like molasses from sugar industry [12] or wastes like used cooking oil, cassava [13], and distillery waste [14] are promising alternatives. These residues generally contain high levels of carbohydrates or lipids to support growth and surfactant synthesis. The treatment and disposal costs for these residues demand greater attention from industry, which are invariably looking for alternatives to reduce, reuse, and recycle these wastes. The increasing number of patents issued on biosurfactant, regarding their use as additives for food, cosmetics, and pharmaceutical products [15], demonstrates the increasing interest in using these biosurfactants obtained from GRAS microorganisms like lactobacilli. However, biosurfactant is not yet used on a large scale, as many regulations regarding the approval of new food ingredients are required by local governmental agencies. Consumer awareness and changes in food policies will definitely create a breakthrough in the use of biosurfactant as a food additive. The present study is focused on the production of biosurfactant by Lactobacillus plantarum CFR 2194, a kanjika (ayurvedic rice-based fermented product), an isolate which has shown some interesting characteristics like the potency for producing vitamin B12 [16], β-galactosidase activity for lactose intolerance sufferers, and production of short-chain fatty acids like lactate and butyrate [17], which play a pivotal role in the prevention of colon carcinogenesis and also has shown positive indications for the production of silver nanoparticles (unpublished data). Hence, in the present investigation, an attempt has been made to evaluate the potential of the selected potent probiotic isolate, L. plantarum CFR 2194 for biosurfactant production, as an additional property. The study also includes optimization and functional characterization of the isolated biosurfactant.
Appl Biochem Biotechnol
Materials and Methods Strains and Culture Conditions L. plantarum CFR 2194, isolated from kanjika, was used for the production of biosurfactant. The LAB strain was stored at −60 °C in de Man–Rogosa–Sharpe (MRS) broth (HiMedia, India), supplemented with 40 % (v/v) glycerol as a cryoprotectant. Prior to use, the culture [1 % (v/v)] was subcultured twice in MRS broth at 37 °C for 12 h. Escherichia coli ATCC 31075, E. coli MTCC 108, Salmonella typhi, Yersinia enterocolitica MTCC 859, and Staphylococcus aureus F 722 were the test pathogens used for studying the antimicrobial and antiadhesive properties of the biosurfactant. All the strains were stored at −60 °C in nutrient broth (HiMedia, India), supplemented with 40 % (v/v) glycerol as a cryoprotectant. Prior to use, the frozen stocks [1 % (v/v)] were transferred twice to nutrient broth and incubated at 37 °C for 12 h. Production of Biosurfactant The fermentative production of biosurfactant from L. plantarum CFR 2194 was carried out in Erlenmeyer flasks containing MRS medium (200 ml). The bacterium was cultured twice in MRS broth for 12 h and used as an inoculum (2 %v/v, 7.51 log cfu ml−1) to inoculate fermentation medium (MRS). The medium was incubated at 37 °C for 4, 18, or 72 h under stationary and shaking conditions (150 rev min−1). Isolation of Biosurfactant Biomass of L. plantarum was harvested by centrifugation (10,000×g, 5 min, 10 °C), washed twice with distilled water, and resuspended in 100 ml of phosphate-buffered saline (PBS) (10 mM KH2PO4/K2HPO4 and 150 mM NaCl with pH adjusted to 7.0). The bacterial suspension was incubated on a magnetic stirrer for 2 h at room temperature with gentle stirring for cell-bound biosurfactant to release. Subsequently, the bacterial biomass was separated by centrifugation (10,000×g, 5 min, 10 °C), and the cell-free supernatant was filtered through Millipore-P20 filters (0.2 μm; Millipore, India). The supernatant was dialyzed against distilled water at 4 °C using dialysis membrane (molecular weight cutoff 6,000–8,000; Sigma, India) and freeze-dried. The aqueous suspension of freeze-dried biosurfactant was acidified with 12 N HCl (pH 2.0); at 4 °C for 16–18 h. The precipitated biosurfactant was collected by centrifugation (10,000 rpm, 4 °C, and 15 min). The precipitate was washed three times in acidic water (pH 2.0 with 12 N HCl) and finally resuspended in distilled water and then freeze-dried and stored at −20 °C. Surface Tension Measurement The biosurfactant production was measured in terms of surface tension using the du Nouy ringtype tensiometer (Kruss, K9) equipped with a 1.9-cm platinum ring [1]. The surface tension measurement was carried out at 20 °C after immersing the platinum ring in the solution for a while, in order to attain the equilibrium. The instrument was calibrated by measuring the surface tension of distilled water. All the measurements were taken in triplicate, and an average value was used to express the surface tension. Sterile PBS with surface tension of 72.2 mN m−1 at 20 °C was used as a control.
Appl Biochem Biotechnol
Fourier Transform Infrared Spectroscopy (FTIR) The sample was prepared by dispersing uniformly in a matrix of KBr. Freeze-dried biosurfactant (6 mg) was ground into fine powder in a small mortar with 300 mg of KBr. The finely ground powder was placed in the pellet holder, and the screw on top was tightened gently to form the pellet. The spectroscopy measurements of the prepared samples were performed using the diffusion method with the absorption measurement within the range of wave number from 4,000.0 to 400.0 cm−1, using a FTIR spectrophotometer (Nicolet 5700 model; Thermo Electron Corporation, USA) equipped with a KBr bundle separator and DTGS (deuterated triglycine sulfate) detector. Each sample was scanned 64 times at the resolution 4.0 cm−1 and scanner rate 0.2 cm s−1. The KBr pellet was used as a background reference. Oil Spreading Test The oil spreading test measures the diameter of clear zones caused when a drop of a biosurfactant-containing solution is placed on an oil–water surface [18]. The binomial diameter and biosurfactant concentration allow the determination of the cleaning efficiency of a given biosurfactant. The oil spreading test was carried out with 50 ml of distilled water taken in a Petri dish (25 cm diameter) overlaid with 20 μl of crude oil. Crude biosurfactant dissolved in PBS (10 μl at concentrations ranging from 4 to 15 mg ml−1) was then added to the surface of oil. PBS with no added biosurfactant served as the control. The diameters of clear zones of triplicate assays from the same sample were determined. Determination of Critical Micelle Concentration (cmc) cmc is a measure of the concentration of a solution component which represents a critical value, above which an increasing concentration of that component forces the formation of micelles. It is important for several applications of biosurfactant to establish their cmc, as above this concentration, no further effect is expected in the surface activity. The cmc was determined by plotting the surface tension as a function of the logarithm of biosurfactant concentration and is represented as the point at which the baseline of minimal surface tension intersects the slope where surface tension shows a linear decline. The biosurfactant of different concentrations (2.5–40 g l−1) was tested for surface tension until the surface tension will not reduce anymore. Measurements were done in triplicate. Emulsification Index (EI) Emulsification index was calculated based on the increase in the height of emulsion. A mixture of 1 ml of crude biosurfactant (1 mg ml−1), 4 ml of water, and 6 ml of hydrocarbon/oil (kerosene, xylene, hexadecane, heptane, coconut oil, and sunflower oil) was vigorously shaken for 2 min to obtain maximum emulsification. After 24 h, the height of the emulsion was measured, and emulsification index was calculated using Eq. 1. Water was used as the negative control; Tween 80 (1 %) served as the positive control.
EI24 ¼
Height of emulsion layer 100 Total height
The assay was performed in the same sized graduated glass test tubes [19].
ð1Þ
Appl Biochem Biotechnol
Emulsification Activity (EA) and Emulsion Stability (ES) The emulsification activities of the biosurfactants were quantified according to the method developed by Calvo et al. [20] and Cirigliano and Carman [20, 21]. Freeze-dried biosurfactant (1 mg ml−1) prepared in PBS (pH 7.0) was diluted with distilled water to a final volume of 4 ml, and the solution was mixed with 1 ml of a substrate (kerosene, xylene, coconut oil, sunflower oil, hexane, and heptane) in a screw-capped tube, and then, the mixture was vigorously shaken for 2 min in a vortex mixer and was allowed to stand for 10 min before measuring the turbidity at 540 nm. The absorbance was expressed as the emulsification activity. The emulsion stability was expressed on the basis of the emulsification activity [20, 21]. The emulsified solutions were allowed to stand for 10 min at room temperature, and absorbance was measured every 10 min over a period of 60 min. The log of the absorbance was plotted versus time, and the slope (decay constant, Kd) was calculated to express the emulsion stability. Antimicrobial Activity The antimicrobial activity of 1.0, 2.0, or 4.0 % (w/v) aqueous solution of freeze-dried biosurfactant was determined using agar well diffusion method described by Golek et al. [22]. Test organisms, E. coli ATCC 31705, E. coli MTCC 108, S. typhi, Y. enterocolitica MTCC 859, and S. aureus F 722, were grown for 12 h in nutrient broth and pour plated with nutrient agar. The aqueous solutions of biosurfactant preparations (60 μl) were added into the wells on the nutrient agar drilled with a sterile 5-mm-diameter driller. Plates were kept in refrigerator for 2 h to diffuse the spent broth and then incubated at 37 °C for 16 h. Diameters (millimeters) of inhibition zones of bacterial growth around the wells were determined with respect to a control sample, i.e., sterile water. Analyses were carried out in triplicate. Antiadhesive Activity The antiadhesive activity of the crude biosurfactant isolated from L. plantarum CFR 2194 against common food-borne pathogens, mentioned in the previous paragraph, was quantified according to the procedure described by Reid et al. [23]. The sterile 96-well flat-bottomed ELISA plate (HiMedia Pvt Ltd., India) was filled with 200 μl of the crude biosurfactant (3 to 50 mg ml−1). The plate was incubated for 18 h at 4 °C and subsequently washed twice with PBS. Wells containing PBS buffer was taken as the control. An aliquot of 200 μl of washed bacterial cells were added to the wells and incubated for 4 h at 4 °C. Unattached microorganisms were removed by washing the wells three times with PBS. The adherent microorganisms were fixed with 200 μl of methanol (99 % purity) 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. Excess stain was rinsed out 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 absorbance of each well was measured at 595 nm. The antiadhesive activity in terms of microbial inhibition (percent) at different biosurfactant concentrations for each microorganism was calculated as follows: % Microbial inhibitionc ¼ ½1−ðAc =A0 Þ 100 where Ac represents the absorbance of the well at a biosurfactant concentration c, and A0 represents the absorbance of the control well. The antiadhesion assay allows the
Appl Biochem Biotechnol
estimation of the biosurfactant concentrations that are effective in decreasing adhesion of the microorganisms studied.
Statistical Analysis Values were expressed as means and standard deviations of triplicate experiments. Statistical analysis was carried out using the Origin 6.1 statistical software (OriginLab Corporation, Northampton, MA, USA).
Results and Discussion Synthesis and Characteristics of Biosurfactant Microorganisms that simultaneously produce more than one substance show additional advantages in terms of contributing to increase the profitability of the process. Nevertheless, in some occasions, metabolites need different conditions of cultivation, thus necessitating the defining of suboptimal compromise conditions to allow reasonable productions of all of them. Additionally, it is necessary to study the appropriate methodology to recover separately each metabolite, particularly when different applications are implicated. L. plantarum CFR 2194, as a vitamin B12 [16] and biosurfactant (present study) producer, is a good example of that. There are several reports describing the ability of different LAB to produce biosurfactants [24, 25], but there is only one study for L. plantarum [26]. Thus, the present study focused on the evaluation of the biosurfactant producing capacity of L. plantarum CFR 2194. Reduction in the surface tension of the PBS cell extract indicated the ability of L. plantarum CFR 2194 to produce biosurfactant. The surface tension values of PBS cell extract are given in Table 1. The reduction in the surface tension of PBS cell extract obtained from cells grown under stationary condition (44.3 mN m−1) was comparatively higher than that of control PBS (72.0 mN m−1). The biosurfactant production was highest at 72 h with a 38.63 % decrease in surface tension under stationary conditions (Fig. 1). The present observation that biosurfactant released by L. plantarum CFR 2194 was maximum under stationary phase of growth is in accordance with the earlier report [27]. Additionally, it was also observed that L. plantarum did not produce extracellular biosurfactants as indicated by a negligible difference in the surface tension values of cell-free supernatant (CFS) obtained after 24, 48, and 72 h of fermentation (65–67 mN m−1)
Table 1 The surface tension of biosurfactant from L. plantarum CFR 2194 grown under stationary and shaking conditions Fermentation time (h)
Surface tension of PBS cell extract, mN m−1 Stationary
Shaking
Control PBS
72.2±0.00
–
24
64.46±0.25
67.2±0.15
48
53.56±0.35
56.6±0.26
72
44.30±0.20
50.3±0.15
Results are expressed as mean ± standard deviations of values from triplicate experiments
Appl Biochem Biotechnol
Fig. 1 Effect of time on surface tension of the surfactant produced by L. plantarum CFR 2194 against control PBS. Results are expressed as mean ± standard deviations of values from triplicate experiments
in comparison with that of MRS control (70.8 mN m−1). Hence, it could be inferred that the biosurfactant produced is cell bound. Microbial surfactants are characterized by a strong ability to lower surface tension. For instance, surfactin from Bacillus subtilis causes a minimal surface tension of 27 mN m−1 and has a cmc of 11 mg l−1, while the synthetic surfactant sodium dodecyl sulfate has a minimal surface tension of only 37 mN m−1 and a cmc of 2.0–2.9 g l−1 [28]. Strains of lactobacilli synthesize surfactants which are efficient in comparison with synthetic ones. Lactobacillus acidophilus RC14 releases biosurfactant with a cmc of 1.0 g l−1 and a corresponding surface tension of 39 mN m−1 [28]. The results obtained in the current study showed that the mean value of a coefficient of surface tension of a crude biosurfactant solution in a PBS buffer, after their extraction from biomass of L. plantarum CFR 2194, reached 44.30±0.20 mN m−1. The cmc for the crude biosurfactant is shown in Fig. 2. The sample was found to be the most surface active, with cmc of 6 g l−1.
Fig. 2 Effect of concentration of the surfactant produced by L. plantarum CFR 2194 on surface tension. Results are expressed as mean ± standard deviations of values from triplicate experiments
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Structure Analyses of Biosurfactants by Means of FTIR The FTIR spectra (Fig. 3) indicated that the biosurfactant is proteinaceous in nature. The absorbance maxima recorded at wavelengths of 3,408.9, 1,661.2, and 1,556.0 cm−1 are typical of stretching →N–H bonds and CO–N and N=O bonds, indicating the presence of proteins in the sample analyzed. The absorption peak at 2,948 cm−1 corresponds to the presence of bonds occurring in aliphatic chains (–CH3, –CH2–). The occurrence of a spectrum over wavelength range of 1,200–1,000 cm−1 indicated the presence of the polysaccharide fraction of biosurfactant. Strong absorption at a wavelength of 1,087.2 cm−1 is typical of oscillations of C–O–C bonds. The FTIR spectra demonstrated that the chemical composition of biosurfactant synthesized by L. plantarum CFR 2194 is nonhomogeneous and is composed of protein and polysaccharide fraction. Literature data indicated that only a few microorganisms synthesize the surfaceactive compounds as chemically pure ones [29]. For instance, Pseudomonas aeruginosa synthesizes rhamnolipids with various chemical structures, whereas Torulopsis bombicola yeast synthesizes a mixture of at least six to nine different sophorolipids [30, 31]. Oil Displacement Test (ODT) The oil displacement test is an indirect measurement of surface activity of a surfactant sample tested against oil; a larger diameter represents a higher surface activity of the testing solution [18]. Surface activities of the crude biosurfactant were investigated in comparison with that of Tween 80 and PBS as positive and negative controls, respectively. The diameter of the clear zone formed was measured as the activity of biosurfactant (Table 2). As can be seen from the results, diameter of the clear zone increased with an increase in the concentration of the biosurfactant. Oil layer was dispersed maximum at a biosurfactant concentration of 15 mg/ml equivalent to that of the positive control Tween 80 (0.1 % w/v) indicating high surface-active properties at higher concentrations. Although the mechanism of oil displacement by biosurfactant has not been fully understood at molecular level, it is considered as a sensitive and simple method for the measurement of surface active nature of the biosurfactant.
Fig. 3 FTIR spectrum of biosurfactant produced by L. plantarum CFR 2194
Appl Biochem Biotechnol Table 2 Comparison of oil spreading efficiency of standard surface-active agents and crude biosurfactant Agents
Diameter of the clearing zone (mm)
PBS control
0
Tween 80 (0.1 %)
3.4±0.12
Crude biosurfactant (4 mg ml−1)
1.2±0.09
Crude biosurfactant (10 mg ml−1) Crude biosurfactant (15 mg ml−1)
1.9±0.28 2.3±0.15
PBS was used as control with a diameter of 0.0 mm as no clearing zone occurs. Results are expressed as mean ± standard deviations of values from triplicate experiments
Emulsification Index, Emulsification Activity, and Emulsion Stability The emulsification index of the produced biosurfactant (1 mg ml−1) was tested against different substrates like kerosene, xylene, coconut oil, sunflower oil, hexane, and heptane. Tween 80 was used as a standard. Among the hydrocarbons, heptane gave the highest EI value of 38.2, followed by xylene (16.22), kerosene (15.2), and hexane (13.6). EI was significantly higher against certain vegetable oils like coconut oil (37.9) and sunflower oil (19.43), which can have wider implication for food formulations (Fig. 4). The emulsification activity of the biosurfactant from L. plantarum CFR 2194 was measured with a variety of water-immiscible substrates (Table 3). The stabilization ability (Table 3) of the biosurfactant from isolate described as the decay content, Kd values (the slope of the emulsion decay plot) are presented in Table 3. Additionally, emulsion decay plots were constructed for a variety of emulsifying substrates in the presence of the biosurfactants, after which the respective Kd values were calculated (Table 3). From the results, it is observed that coconut oil and sunflower oil were efficiently emulsified. In addition, kerosene and xylene were also emulsified to a considerable extent. Interestingly, EA with n-alkanes increased with
Fig. 4 Emulsification index of biosurfactant for different substrates. Results are expressed as mean ± standard deviations of values from triplicate experiments
Appl Biochem Biotechnol Table 3 Emulsification activity and emulsification stability of biosurfactant from L. plantarum CFR 2194 Emulsification activity (OD540 nm)a
Decay constant (Kd, 10−3)b
Kerosene
0.77
−3.26
Xylene
0.83
−3.29
Coconut oil
1.81
−0.51
Sunflower oil Hexadecane
1.15 1.04
−5.23 −4.29
Heptane
0.34
−7.53
Substrate
Results are expressed as mean ± standard deviations of values from triplicate experiments a
The emulsification assay was performed in the presence of the biosurfactant as described in the text. After an initial 10-min holding period, absorbance readings were taken every 10 min for 60 min b The log of the absorbance was plotted versus time, and the slope (decay constant, Kd) was calculated
the increase in the number of carbon atoms. Emulsions formed with vegetable oils were more stable than emulsions formed with hydrocarbons (Table 3). In the similar study, Moldes et al. [32] and Bello et al. [33] reported that biosurfactants obtained after growing Lactobacillus pentosus on sugars from agricultural residues, shown to stabilize octane/water and gasoline/water emulsions after 24 h of incubation. Antimicrobial Activity Biosurfactant obtained from the lactic acid bacteria have been indicated to exhibit antibiotic properties, which predisposes this group of chemical compounds for potential application in the production of antibiotics [34, 35]. From this perspective, experiments were conducted to determine the antimicrobial activity of preparations of biosurfactant synthesized by L. plantarum CFR 2194 against some of the food-borne pathogens. There are few reports on the antimicrobial activity of biosurfactants isolated from lactobacilli; only biosurfactants obtained from Streptococcus thermophilus A and Lactobacillus lactis 53 have been reported to exhibit a significant antimicrobial activity against several bacterial and yeast strains isolated from explanted voice prostheses [34, 36]. In the present
Table 4 Antimicrobial activities of the biosurfactant from L. plantarum CFR 2194 Microorganism
Zone of inhibition 4 mg ml−1
25 mg ml−1
Escherichia coli ATCC 31075
−
+
Escherichia coli MTCC 108
++
+++
Salmonella typhi Yersinia enterocolitica MTCC 859
− −
− ++
Staphylococcus aureus F 722
+
++
“+” sign indicates inhibition of microbial growth, whereas “−” sign indicates inability to inhibit the growth of the tested microorganisms; + indicates zone of inhibition around 5 mm around the application of biosurfactant, “++” zone of inhibition around 5–10 mm around the application of biosurfactant, and “+++” zone of inhibition more than 15 mm around the application of biosurfactant
Appl Biochem Biotechnol Table 5 Antiadhesive properties of biosurfactant from L. plantarum CFR 2194 Microorganism
Biosurfactant (mg ml−1) 4.0
Escherichia coli ATCC 31075
8.04±0.2
25.0 54.43±0.1
Escherichia coli MTCC 108
10.65±0.1
56.78±0.3
Salmonella typhi
13.92±0.1
61.60±0.2
Yersinia enterocolitica MTCC 859
15.23±0.3
64.72±0.4
Staphylococcus aureus F 722
25.90±0.1
67.18±0.2
Negative controls were set at 0 % to indicate the absence of biosurfactant. Positive percentages indicate the reductions in microbial adhesion when compared to the control. Results are expressed as means ± standard deviation of results from triplicate experiments
study, the antimicrobial activity of the preparations of biosurfactant increased with an increase in the concentration (Table 4). As can be seen from the results, the highest antimicrobial activity was observed at 4 and 25 mg ml−1 aqueous solutions of biosurfactant preparations against E. coli ETEC and S. aureus, respectively. Complete inhibition for E. coli ATCC and Y. enterocolitica was observed at a concentration of 25 mg ml−1 of biosurfactant preparation; thus, results indicate the antimicrobial potential of the biosurfactant synthesized by L. plantarum CFR 2194. However, no inhibition was observed with respect to the growth of S. typhi at both concentrations of the biosurfactant. Antiadhesive Activity In addition to the antimicrobial properties, the isolated biosurfactant exhibited a considerable antiadhesive activity against most of the microorganisms tested. Involvement of biosurfactants in microbial adhesion and desorption has been widely described, and adsorption of biosurfactants isolated from lactobacilli to solid surfaces might constitute an effective strategy to reduce microbial adhesion and combating colonization by pathogenic microorganisms, not only in the biomedical field but also in other areas, such as the food industry [1, 5, 37, 38]. The antiadhesive activity of the biosurfactant was evaluated against a few pathogens as listed in Table 5. The biosurfactant showed antiadhesive activity against all the tested microorganisms, but the antiadhesive effect depends on the concentration and the microorganism tested. The antiadhesive activity was higher against S. aureus (67.18±0.2 % inhibition) at a biosurfactant concentration of 25 mg ml−1. For Y. enterocolitica, the highest antiadhesive percentages were 64.72±0.4 %, whereas the lowest antiadhesive percentages were for E. coli ATCC (54.43±0.1 %). Regarding the other pathogenic bacteria, high antiadhesive percentages were obtained for E. coli ETEC (56.78±0.3 %) and S. typhi (61.60±0.2 %).
Conclusion The study carried out demonstrates that the strain L. plantarum CFR 2194 was able to synthesize the cell-bound biosurfactant. The spectral (FTIR) analysis of biosurfactant synthesized revealed the presence of protein and polysaccharide fractions. Considerable emulsifying activity of the biosurfactant revealed that they could be used as emulsion-forming agents for hydrocarbons and oils, giving stable emulsions. The study has also effectively demonstrated
Appl Biochem Biotechnol
the antimicrobial and antiadhesive properties of the biosurfactant isolated from L. plantarum CFR 2194 against food-borne pathogens. The results obtained suggest the possible use of this biosurfactant as an alternative antimicrobial agent in the medical field for applications against microorganisms responsible for diseases and infections in the urinary, vaginal, and gastrointestinal tracts as well as in the skin, making it a suitable alternative to conventional antibiotics. Acknowledgments A.N. Madhu is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for the award of Senior Research Fellowship. Esther Magdalene Sharon, a project student, was kindly acknowledged for help in carrying out the experiments. Dr. Purnima Kaul Tiku, a scientist, of the Protein Chemistry and Nutrition, CFTRI, is greatly acknowledged for the help in using the tensiometer. The authors thank the Director, CFTRI, for supporting the work.
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