1. Background
Lactic acid bacteria (LAB) are a group of gram-positive, acid-tolerant, generally non-sporullating rod or cocci microorganisms that produce lactic acid as the primary end product of carbohydrate fermentation (1). They are industrially important microorganisms and are used for several applications such as fermentation of dairy, meat, vegetables, production of enzymes, macromolecules, and metabolites (2). LAB is also known for the production of antimicrobial substances such as H2O2, CO2, acetaldehyde, diacetyl, D-isomers of amino acids, reuterin, and bacteriocins (3). Among these, bacteriocins have promising applications in food industry as natural preservatives (4). Bacteriocins are ribosomally-synthesized extracellular peptides or proteins, which have bactericidal or static activity against other closely related species, food-borne pathogens, and spoilage bacteria. The food preservation can be achieved by applying bacteriocin or bacteriocin producing starter culture (4, 5).
Optimum growth and bacteriocin production depends on various factors such as pH, temperature, NaCl concentrations, and carbohydrate source (4, 6-10). These parameters are generally specific to producer strains (4). The growth of microorganisms depends on the rate of chemical reactions and protein structure, which are greatly influenced by temperature. Similarly, pH plays an important role in the growth of microorganisms. Variations in cytoplasmic pH inhibit the enzyme activity, and changes in external pH might cause the ionization of nutrient molecules that result in the reduction of their availability and less growth. The expression of biosynthetic genes for the production of bacteriocin may also be regulated by pH (11). Leaes et al. (12) reported that temperature and initial pH affected the expression of genes essential for the production of antimicrobial peptides. Although most of the bacteria do not require Na+ for the growth, low NaCl concentrations can enhance the growth of LAB. The carbon source is essential for the synthesis of cell components and other metabolites (13).
Reports are available on optimization of growth conditions for many LAB, but soil isolate Pediococcus pentosaceus and Weissella confusa are least documented. The production of bacteriocins in high quantity is essentially required by food industries due to their applications. Therefore, optimization of culture conditions to produce bacteriocin is the logical extension of the authors’ previous study (1). Previously, P. pentosaceus LB44 and W. confusa LM85 were isolated from soil samples collected from dairy and mulberry rhizosphere, respectively. These isolates were identified and characterized to produce bacteriocin-like inhibitory substances, which inhibited the growth of food-borne and clinical pathogens (1).
2. Objectives
The current study aimed at optimizing the culture conditions for higher growth and production of bacteriocins by soil isolates, P. pentosaceus LB44, and W. confusa LM85.
3. Methods
3.1. Bacterial Strains and Growth Conditions
P. pentosaceus LB44 and W. confusa LM85 were routinely grown and maintained on TGYE agar medium (g/L tryptone 5, glucose 1, yeast extract 3, agar 1.5 %, pH 7.0) at 37°C (14). MRS broth medium (g/L peptone 10, beef extract 10, yeast extract 5, dextrose 20, polysorbate-80 1, ammonium citrate 2, sodium acetate 5, magnesium sulphate 0.5, manganese sulphate 0.2, di-potassium phosphate 2, pH 6.8) (15) was used to produce bacteriocin. Indicator strain Micrococcus luteus MTCC 106 was grown in the nutrient broth (g/L peptic digest of animal tissue 5, NaCl 5, beef extract 1.5, yeast extract 1.5, pH 7.4). All the media components used in the current study were purchased from HiMedia, India and Sisco Research Laboratories, India.
3.2. Preparation of Cell-Free Supernatant and Determination of Antimicrobial Activity
Bacteriocins are secreted in cell-free supernatant (CFS) during the growth of bacteria. Therefore, CFS was used to determine the antimicrobial activity. The LB44 and LM85 isolates were grown in MRS medium at 37°C in a BOD incubator (Laby, India) for 18 hours. The cultures were centrifuged at 10,000 rpm for 15 minutes (Sigma, Germany). After centrifugation, CFS was collected and filter-sterilized with 0.2 µm syringe filter (Axiva, India). Agar well diffusion assay (AWDA) was performed as described by Kaur and Tiwari (1). Briefly, soft nutrient agar (0.8%) medium (5 mL) seeded with ~106 cells of freshly grown culture of indicator strain M. luteus was overlaid on nutrient base agar plate. Wells of 6 mm diameter were cut out with sterile cork borer and CFS (100 µL) was loaded. The plates were incubated at 37°C in a BOD incubator overnight. After incubation, the growth inhibition zone was observed and diameter was measured in millimeter (mm).
3.3. Optimization Conditions for Growth and Bacteriocin Production
3.3.1. pH
MRS medium was set to pH 3 to 10 with 1N NaOH and HCl. Overnight grown culture of isolate LB44 and LM85 were inoculated to an initial OD600 0.02. The sets were incubated at 37°C for 18 hours. The growth was measured using a spectrophotometer (Techcomp, Hong Kong) at 600 nm and antimicrobial activity of CFS was determined using AWDA as described above.
3.3.2. Temperature
Isolate LB44 and LM85 were inoculated in MRS medium to initial OD600 0.02 and incubated at 30, 37, 42, and 50°C in a BOD incubator for 18 hours. The growth was measured using a spectrophotometer at 600 nm and antimicrobial activity of CFS was determined using AWDA as described above.
3.3.3. NaCl Concentrations
MRS medium was prepared containing different concentrations of NaCl in different sets from 2% to 10%. The LB44 and LM85 isolates were inoculated to an initial OD600 0.02 in each set and incubated at 37°C for 18 hours. MRS medium without NaCl was used as control. The growth and antimicrobial activity were detected as described above.
3.3.4. Different Carbon Sources
The effect of different carbon sources on growth and bacteriocin production was studied as described by Vijay Simha et al. (8). MRS medium containing different concentrations of glucose and lactose (10, 20, 30 and 40 g/L) were prepared in different sets and filter-sterilized. These sets were inoculated with LB44 and LM85 at an initial OD600 0.02 followed by incubation at 37°C for 18 hours. Growth and antimicrobial activity were determined as described above. MRS medium without glucose or lactose was used as control.
3.4. Statistical Analysis
The experiments were performed in triplicate and values were expressed as mean of the 3 independent experiments with standard deviation (mean ± SD). The graphs were plotted using SigmaPlot 11.0.
4. Results
4.1. Effect of Different pH
P. pentosaceus LB44 grew up to OD600 1.2 to 1.5 and demonstrated antimicrobial activity up to ~ 18 mm at pH 5.0 to 8.0. The optimum growth of these isolates was at pH 6.8 to 8.0 (OD600 1.5). They grew to very less extent (OD600 0.1 to 0.3) with no antimicrobial activity under highly acidic (pH 3.0 and 4.0) and alkaline (pH 9.0 and 10.0) conditions. Therefore, extreme conditions were lethal for the growth and activity. A similar pattern of growth of W. confusa LM85 was also observed, but it could grow under highly alkaline conditions and also demonstrated significant activity. At pH 9.0, W. confusa LM85 grew up to OD600 1.4 and showed 17 mm growth inhibition zone. Interestingly, the isolate LM85 grew at pH 10.0 up to OD600 0.7 and demonstrated 12 mm zone of growth inhibition. At pH 5.0, it grew up to OD600 0.8 and showed 15 mm zone of growth inhibition (Figure 1A and 1B).
Figure 1.
Growth and Antimicrobial Activity of Isolates LB44 (A) and LM85 (B) at Different pH
4.2. Effect of Different Temperatures
Pediococcus pentosaceus LB44 grew at 30, 37, and 42°C, but its optimum growth was recorded at 37°C (OD600 1.2). The growth at 30 and 42°C were OD600 1.0 and 0.7, respectively. It grew only 50% at 42°C, compared with 37°C, but LB85 isolate grew with full extent at 42°C. The LB44 Isolate did not grow at all at 50°C, whereas LM85 grew almost half with OD600 0.6 at 50°C. Therefore, LB44 was sensitive to higher temperatures, whereas LM85 grew at higher temperatures up to 50°C. Antimicrobial activity demonstrated by these isolates was consistent to growth as shown in Figure 2A and 2B.
Figure 2.
Growth and Antimicrobial Activity of Isolates LB44 (A) and LM85 (B) at Different Temperatures
4.3. Effect of Different Concentrations of NaCl
Both of the isolates demonstrated optimum growth (OD600 ~ 1.3) and antimicrobial activity (the growth inhibition zone ~ 21 mm) in the medium without NaCl. The growth continuously reduced as the concentration of NaCl increased. The antimicrobial activities of these isolates also decreased by increasing the concentration of NaCl, and reduced to nil at 6% NaCl and above. These isolates could tolerate, grow, and produce bacteriocin only with up to 4% NaCl. The responses of both isolates were almost similar against different tested NaCl concentrations (Figure 3A and 3B).
Figure 3.
Growth and Antimicrobial Activity of Isolates LB44 (A) and LM85 (B) at Different Concentrations of NaCl
4.4. Effect of Different Carbon Sources
Carbon source is important for growth and affects bacteriocin production, which is a highly energetic process. Therefore, different concentrations of the 2 most commonly used carbon sources, glucose and lactose, were supplied in the culture medium, and growth and bacteriocin production were monitored. P. pentosaceus LB44 grew almost equally up to OD600 ~ 1.6 at 10 to 40 g/L of glucose. The production rates of bacteriocin were similar (20 mm growth inhibition zone) at 20% to 40% glucose, but reduced at 10% glucose by 17 mm. Weissella confusa LM85 showed similar growth and bacteriocin production in the presence of different concentrations of glucose. Both isolates demonstrated much less growth (OD600 ~ 0.3 - 0.4), and antimicrobial activity was nil when grown in a medium without glucose (Figure 4A and 4B).
Figure 4.
Growth and Antimicrobial Activity of Isolates LB44 (A) and LM85 (B) at Different Concentrations of Glucose
In contrast, the growth of P. pentosaceus LB44 was lower at 10 g/L (OD600 0.4) and 20 g/L (OD600 1.4) lactose, but increased with increasing the concentration of lactose. It grew optimally up to OD600 1.6 at 30 to 40 g/L lactose. There was no bacteriocin production up to 20 g/L lactose used in the culture medium and very low antimicrobial activity was recorded at 30 g/L (inhibition zone ~ 9 mm). The optimum activity (growth inhibition zone ~16 mm) was recorded at 40 g/L lactose. Almost similar patterns of growth and bacteriocin production were also demonstrated by W. confusa LM85 (Figure 5A and 5B). In comparison to glucose, growth and bacteriocin production by both isolates were lower at similar concentrations of lactose. Both isolates demonstrated bacteriocin production at 10% to 20% glucose, but it was nil in the presence of lactose at these concentrations. These results suggested that both isolates utilized glucose more efficiently than lactose.
Figure 5.
Growth and Antimicrobial Activity of Isolates LB44 (A) and LM85 (B) at Different Concentrations of Lactose
5. Discussion
The production of bacteriocin is an energetic process, which show growth associated phenomenon. Different conditions including carbon source are important for the production of bacteriocin. It is reported that antimicrobial activity follows similar patterns of growth curve of most LAB strains (14, 16, 17). Therefore, growth was optimized under different conditions such as pH, temperature, different concentrations of NaCl and carbon source. The LB44 Isolate grew and demonstrated activity at acidic and near neutral pH, whereas LM85 demonstrated growth and activity at acidic, near neutral, and highly alkaline pH. Lactobacillus casei and Lactococuss lactis subsp. lactis showed optimum growth and bacteriocin production at pH 6.5 to 7.0 (4). Pediococcus pentosaceus MTCC 5151 showed optimum activity at pH 5.5 (18). pentosaceus NRC AM1 and P. pentosaceus NRC AM4 grew well at pH 4.0 to 8.0 (19).
P. pentosaceus LB44 and W. confusa LM85 optimally grew and demonstrated activity at 30 to 37°C. The growth and activity of LB44 was almost half, compared with LM85 at 42°C. Pediococcus pentosaceusLB44 did not grow or show activity at 50°C, but W. confusa LM85 grew and demonstrated activity up to half compared to 37°C. The optimum temperature for the growth of P. pentosaceus strains was 28 to 35°C (20). P. pentosaceus NRC AM1 and P. pentosaceus NRC AM4 grew well at 10 to 45°C (19). The optimum temperatures for the production of pediocin by P. pentosaceus ST18 and P. pentosaceus ACCEL were 30 and 37°C, respectively (21, 22). The maximum bacteriocin production by P. acidilactici 13 and P. pentosaceous NCDC 273 was reported at 37°C (8, 13). Optimum bacteriocin production by L. casei LA-1 was reported at 26 to 37°C (4). Weissella cibaria grew at 20 to 37°C, but there was no growth at 45°C (OD600 0.169). The optimum growth and activity of W. cibaria 110 was reported at 30°C (23). Pal et al. (17) reported bacteriocin production by W. paramesenteroides DFR-8 at 25 to 40°C, but optimum activity was at 35°C. The optimum production of bacteriocin by Lactococcus lactis subsp. lactis A164 and Lactobacillus rhamnosus GP1 were reported at pH 6.0 and 30°C (16, 24). The soil isolate W. confusa LM85 grew at alkaline pH 9 and 10, and higher temperature up to 50°C, which was a unique feature and not reported till date, to the best of the authors’ knowledge. The growth at broader range of pH and temperature provides extra advantage for the application of isolates and their bacteriocins in different foods.
Growth of both isolates decreased with increase in NaCl concentrations. Similar results were also reported for P. pentosaceus NRC AM4 where growth decreased with increase in NaCl concentrations (19). Pediococcus acidilactici 13 grew up to 10% NaCl, but grew optimally in the absence of NaCl (13). The growth of LAB is sometimes better in the presence of low salt concentration, usually 1% to 2% and is inhibited above 3% NaCl, while few LAB are more resistant to NaCl (13). Delgado et al. (6) stated that NaCl was required to maintain osmotic pressure in the cells, but not required for the production of bacteriocin.
Both isolates grew optimally and demonstrated activity in 10% to 20% glucose. The growth and activity were less in the presence of lactose at similar concentrations. Similar observation was reported by Pal et al. (17) where optimum bacteriocin production by W. paramesenteroides DFR-8 was higher in the presence of glucose as compared to those of other carbon sources including lactose. Delay in growth and antimicrobial activity by P. pentosaceous NCDC 273 in the presence of lactose was also reported by Vijay Simha et al. (8). These findings suggested that the growth and bacteriocin production were higher in the presence of glucose as compared to that of lactose. This could be due to higher utilization of glucose in comparison with that of lactose during the growth of LB44 and LM85 isolates. The optimized condition for growth and bacteriocin production by these isolates are helpful to recover higher yield for their possible applications.
5.1. Conclusions
The culture conditions were optimized for higher growth and production of bacteriocin by soil isolates P. pentosaceus LB44 and W. confusa LM85. The optimum growth conditions for bacteriocin production by isolate LB44 was MRS medium supplemented with glucose at pH 6.8 and incubation temperature 37°C. The LM85 isolate grew in highly alkaline conditions and higher temperatures, but LB44 failed to grow and produce bacteriocin under such conditions. The growth and activity of both isolates were higher in the medium without NaCl. Glucose was more effective than lactose for growth and bacteriocin production. Therefore, optimized culture conditions are useful for higher yield of bacteriocins and their industrial applications.
