Studies on the Prodigiosin Production from Streptomyces coelicolor in Liquid Media by Using Heated Lactobacillus rhamnosus

Article history: Received on: 05/04/2014 Revised on: 02/05/2014 Accepted on: 18/05/2014 Available online: 27/05/2014 Prodigiosin (PG) is not only an antibiotic but it has also been characterized immunosuppressive and anticancer activities. Prodigiosin is produced by Streptomyces coelicolor and Seratia marcescens. In this study, prodigiosin (PG) production of S. coelicolor was optimized using 50μL, 100μL of heated cells at 70C and 100C of Lactobacillus rhamnosus. With 50μL heat-killed cells at 70C L. rhamnosus, prodigiosin (PG) production of S. coelicolor enhanced the maximum prodigiosin concentration (9.79  1.68 mg/L) by six-fold at the early stage in the second day incubation in the comparison with prodigiosin (PG) production of S. coelicolor without heated L. rhamnosus (1.43  0.22 mg/L). There were the affects of the components of L. rhamnosus on PG production. This is the first report that L. rhamnosus affecting on PG production in S. coelicolor.

In the industrial point of view, the optimal conditions in order to increase the production of PG are necessary due to its potential application in future.Starting from the actual situation, .a new strategy was applied to increase the production of PG on Streptomyces coelicolor recently; it is the use of dead cells bacteria as factors that increase the production of PG on S. coelicolor (Luti and Mavituna, 2011;Rigali et al., 2008).Some reports shown that N-acetyl glucosamine, one of main components of peptidoglycan, may trigger antibiotic production in Streptomyces (Tsao et al., 1985).For this reason, Lactic acid bacteria (LAB) -a group of gram-positive bacteria and considered as commercial microorganisms with no pathogenic potential which has a thick peptidoglycan layer containing the activity on N-acetyl glucosamine and membrane products may play a new role as an elicitor in case of PG production in S. coelicolor.In those regard, the aim of this study was therefore to develop the production of PG on S. coelicolor using the heated Lactobacillus rhamnosus LGG.

Microorganisms and growth cultures
Streptomyces coelicolor was obtained from John Innes Centre as a gift.Lactobacillus rhamnosus LGG was isolated and identified in yogurt inside Vietnam.For Streptomyces coelicolor, YEME media were used.For Streptomyces coelicolor inoculation, a stock of 10 8 spores was used.
A loopful of Lactobacillus rhamnosus was inoculated in a test tube containing 5ml MRS broth medium.It was incubated at room temperature for 2 days.Then the bacterial cell suspensions were treated at 50 o C, 70 o C, and 100 o C in 1 h before adding in the Streptomyces coelicolor culture.The morphology of these strains was tested, using gram stain.

Prodigiosin production tests
For study on prodigiosin production in medium, 20 µL of Streptomyces coelicolor prepared stocked were cultured in a shaken flask at 28 o C with shaking at 200rpm in 48 hours.The experiments were carried out in triplicate and the results represented in the arithmetic average.
After 48 hours, 0.2 ml of inoculums was added into new flasks containing 20 mL medium.At this step, the different amounts of heated L. rhamnosus suspension were added, using a volume of 50 µl or 100 µl of L. rhamnosus heated at 70 o C and 100 o C. The flasks were incubated at 28 o C with a shaking at 200 rpm.Every day, one ml of each culture was taken for the analyses.

Isolation of prodigiosin
All the cultures were harvested by centrifuging at 10,000 rpm for 10 minutes.The supernatant (20 mL) and pellet (2 g) were harvested.The prodiogiosin extraction was done with a modification (Helvia et al., 2010).For the supernatant, the pigment was extracted by using chloroform with an equal amount to supernatant.The chloroform fraction was extracted by using methanol with an optimal ratio (1:1).
Further methanol extraction was done for a complete extraction.All methanol layers were collected to evaporate until dryness and the amount pigment obtained on a dry weight basis was calculated.For the pellet, the methanol (10 mL) was used for extraction.Further methanol extraction was done for a complete extraction.All methanol layers were collected to evaporate until dryness and the amount pigment obtained on a dry weight basis was calculated.The calculated amount of pigment obtained in the pellet and supernatant of culture gave the total amount of prodigiosin produced in Streptomyces in different condition.The analyses of results were performed by one-way ANOVA on SPSS 16.0 software.

Temperature effects on Lactobacillus
The survival of Lactobacillus was checked after heated at 50 o C, 70 o C and 100 o C. The heated Lactobacillus samples were spread on MRS agar which were incubated at room temperature from 48 h to 120 h.There were the colonies appearing on the MRS agar in case the Lactobacillus was heated at 50 o C. In order to confirm the Lactobacillus colonies, the gram stain was done.As a result, Lactobacillus could be still alive at 50 o C. Therefore, Lactobacillus heated at 70 o C and 100 o C was used for the optimization of prodigiosin production in Streptomyces coelicolor.

Production of PG on liquid YEME medium
The experiment was carried out on YEME broth medium because the PG production did not occur on GYP broth medium in both the pure culture of S. coelicolor and the culture inoculated with the dead cells of L. rhamnosus.The samples started to be collected after the cultures incubated for 24 hours.Remarkably, The prodigiosin production occurred after second day of incubation (Fig. 1).After sixth day, the prodigiosin production reduced (Fig. 2).The PG yields in 50µL of heat-killed cells at 70 o C of L. rhamnosus were extracted and measured.The results were presented in the table 1.As seeing in figure 1, figure 2 and table 1, the prodigiosin production showed the strongest when using 50 µL of L. rhamnosus cells heated at 70 o C. The tested treatments of heated L. rhamnosus gave the significant difference at the second day for incubation volume of 20 ml when the analyses were performed by one-way ANOVA.The 50µl of heated L. rhamnosus at 70 o C gave the best result and reached the maximum of 9.79 mg/L.The red color in the flask reduced when using 100 µL of L. rhamnosus cells heated at 70 o C.There was a binding of Lactobacillus component with the factors relating to PG synthesis.The increasing the amount of Lactobacillus rhamnosus caused the lower PG.However, the PG production reduced when Streptomyces incubated with the heated L. rhamnosus cells at 100 o C.However, in terms of interpretation at molecular level, the mechanism of this kind of stimulation is not fully understood.Some suggestions were given that the dead cells have lysed compounds that may play role as precursors for the product.That component should be done so far.et al., 1985).Besides, many compounds from biological sources including protein, cell wall fragments, yeast extract or carbohydrates were used to enhance the production of secondary metabolites in microbial, whole-plant, and plant cell cultures (Radman et al., 2003).
As presented in figure 3 and figure 4, the culture started producing PG on the second day of incubation.It reached its maximum of 1.43mg/L on the second day and the maximum wet biomass concentration of 27.65g/l was obtained on the third day.The culture inoculated with 50 µl of heated L. rhamnosus cells at 70 o C rose to the maximum of 9.79 mg/L on the second day, higher than Streptomyces production at six -fold.
In the figure 4, the biomass was maximum at 25.5g/L on the third day of incubation.However, the prodigiosin production highly obtained in the second day.Obviously, the heated L. rhamnosus affected on the early stage of S. coelicolor.After that, prodigiosin was down because the unstability of the heated L. rhamnosus compounds or there were the interaction of S.coelicolor secondary metabolism which reduced the prodigiosin production.Many mechanisms should be propagated so far.

CONCLUSION
The heated cells of L. rhamnosus at 70 o C stimulated the production of prodigiosin on S. coelicolor at the early stage of S. coelicolor.Using the dead cells make more convenient than others compounds that need to be extracted from cells or cell walls due to its simple, less costly.In addition, this study may lead a strategy in order to enhance the production of other antibiotics.However, which components that cause the enhancement of PG production are not known yet.Many studies need to be conducted so far to achieve the knowledge about those components and the mechanism of this interaction.In the industrial point of view, the study played a role as a preliminary step and should be scaled up so far.

Fig. 3 :
Fig. 3: Prodigiosin production measured according to the incubation time of Streptomyces coelicolor.The blue line showed the prodigiosin in Streptomyces coelicolor.The red line showed the prodigiosin produced in Streptomyces coelicolor after incubated with heated L. rhamnosus at 70 o C.

Fig. 4 :
Fig. 4: The biomass and prodigiosin production of Streptomyces coelicolor when incubated with heated L. rhamnosus at 70 o C.

Table . 1
: Production of prodigiosin (mg/L) by S. coelicolor without and with heated L. rhamnosus cells at 70 o C.