INTRODUCTION:

In recent years, petrochemical originated polymers have been used worldwide. However, most of these polymer materials are not biodegradable which will generate significant sources of pollution and harming wildlife when being dispersed in nature [1]. Therefore, new materials within a perspective eco-design or sustainable development are extremely necessary and important. That is the reason why biodegradable polymers can be considered as interesting, safe and alternative sources. Synthesized biodegradable polymers were first mentioned in the 1980s.

Recently, poly (lactic acid) (PLA) polymer which belongs to the family of aliphatic polyesters commonly made from α-hydroxyl acids has been being used and studied increasingly in huge number of applications such as biodegradable plastic, cardiovascular applications or drug delivery system. Practically, various kinds of drugs (hormones, proteins, antibiotics, antitumor, anesthetics, antipsychotics,etc.) have been delivered with these polymeric particles [2]. However, most of the PLA synthesizing methods are mainly based on chemical processes on the basis of metal catalyst-mediated chemical polymerization of lactic acid which may not be compatible with large-scale production owing to the nature of the preparation and high cost of materials employed [3, 4].

Therefore, finding out the natural lactic acid sources was important. Lactobacillus is potential lipase enzyme producer which has been reported as biological catalyst for both ring opening polymerization–similar function as Tin(II) chloride and Tin (II) bis(2-ethylhexanoate) and condensation polymerization [5]. Enzymatic polymerization has been successful in synthesizing many polymer kinds such as poly(lactone), poly(lactide-co-glycolide) (PLGA) and other polyester [6].

As above stated reasons, Lactobacillus rhamnosusPN04 (L. rhamnosusPN04) was used to study on the PLA production [7, 8]. The crude PLA was characterized by thin layer chromatography (TLC), infrared spectroscopy (IR), nuclear magnetic resonance (H-NMR), gel permeation chromatography (GPC) and scanning electron microscope (SEM).

MATERIALS AND METHODS:

Bacterial strain:

L. rhamnosus PN04 isolated from Hottuynia cordata[7, 8], Candida albicans ATCC 14053, Pseudomonas aeruginosa ATCC 27853 and Micrococcus luteus ATCC 10240 were used in the study.

L. rhamnosus preparation for PLA production:

1 mL of L. rhamnosus (10⁹ CFU/mL) was cultured in 100 mL MRS prepared at different pH (4, 7 and 10). After that, the MRS medium containing L. rhamnosus PN04 was inoculated at different temperature (room temperature (RT), 37^oC and 45^oC). In the next three days, all the cultures were used to measure pH to consider the lactic acid secretion. Then, the supernatants of cultures were collected by centrifugation (10000 rpm, 20 minutes, 4^oC).

PLA polymer production:

Because there were many necessary agents in the supernatant such as lactic acid or lipase, the collected supernatants were directly heated at 100^oC. The processes required 6 hours to be dried completely. Then, chloroform was added for dissolving the polymer in residue. In the next day, the liquid phase containing polymer was taken to remove chloroform by evaporation. Then, polymer was harvested. The starting products were determined to be PLA.

Thin layer chromatography (TLC):

In order to detect lactic acid unit in PLA, the TLC plates (G F254, Merck, Germany) were used. The mobile phase was a mixture of n-butanol and acid formic in a ratio of 95:5, respectively [9].

Gel permeation chromatography (GPC):

GPC was used for determining the molecular weight distribution of the polymer. Detector was refractive index detector (RID). Tetrahydrofuran was used as eluent. Column C18 was used in the study. Injection volume (20 µL) and flow rate (1 mL/min) were conditions for the study.

Infrared spectroscopy (IR):

The IR was conducted to analyze the presence of the important functional groups of PLA. The IR spectrum was recorded by IR spectrophotometer (8400S, Shimadzu, Japan).

Nuclear magnetic resonance (H-NMR):

PLA was dissolved in CDCl₃. The H-NMR was then recorded by a NMR spectrophotometer (Brucker AC 250, USA).

Optimization of conditions for changing PLA molecular weight:

Xylene or/and reduced pressure were used to improve the reaction [9]. The conditions for PLA production were kept identically (100^oC, 100rpm and 6 hours).

Antimicrobial activity test:

Agar diffusion test was used to check antimicrobial ability. Pseudomonas aeruginosa ATCC 27853 and Micrococcus luteus ATCC 10240 were cultured in 5mL of LB medium and then were stretched on LB agar plates. Candida albicans ATCC 14053 was cultured in 5mL of potato dextrose broth (PDB) and then was stretched on PDB agar plates. Different amounts of PLA (0.02 g, 0.04 g, 0.06 g)were solubilized in the suitable amount of water (100 µL) and then were put into wells formed in plates. The antimicrobial zones were checked and measured in diameter in the next day. All the experiments were triplicated.

Cytotoxicity test:

The cytotoxicity test was performed by sulforhodamine B assay [10]. It is a simple colorimetric method for determining sensitivity and cytotoxicity of a substance. Sulforhodamine B (SRB), a negatively charged dye which can bind electrostatically with the positively charged parts of proteins will be the main reagent for the assay. The amount of binding dye will reflect the total cellular protein. Positive control of the test was the mixture of cells and camptothecin at the concentration 0.07 µg/mL. The mixture of cells and the solvent that dissolved the polymer (EMEM/10% FBS culture medium) was used as negative control. Cancer cell line used to test were HepG2.

RESULTS AND DISCUSSION:

Determination of optimal culture condition:

The pH values in experimental culture were determined in Table 1. The changes in pH values expressed the organic acid production. The variation of the pH values illustrated the amount of substances which influenced the substrate production for PLA. The media adjusted with pH4 were not suitable for L. rhamnosus to grow while the media adjusted to pH7 and 10 were more suitable for this bacterium. In term of temperature, L. rhamnosus could grow well at RT and 37^oC and 45^oC. However,the culture at 37^oC turned to acid better than the other conditions, pointing that lactic acid was produced more highly to prepare PLA conveniently. Therefore, the temperature (37^oC) was the selective condition for the study.

Table 1: pH values under different culture conditions

Temperature of culturing	pH before culturing	pH after 3 days of culturing
Room pemperature	4	3.82
	7	3.76
	10	5.34
37^OC	4	3.66
	7	3.55
	10	4.32
45^OC	4	3.92
	7	5.3
	10	5.91

Firstly, the solid products after evaporating chloroform were analyzed by GPC for investing the molecular weight (Mw) and polydispersity index (PDI) of the products (Figure 1). As seeing in figure 1, the Mw of PLA prepared from the supernatant originated from the condition (pH=7, 37^oC) was highest, besides that, the PDI value of this sample is also low (1.17) which indicated the homogeneity in size between molecules are high. The reason why this condition could be used to make higher PLA molecular weight was due to some other compounds produced in culture supporting for the biosynthesis.

After GPC analysis, product obtained from culturing condition (pH=7, 37^oC) was chosen for further steps because the condition could produce lactic acid and the other compounds required for high weight PLA. Actually, the chosen one was then hydrolyzed with chloric acid (10%) at 100^oC and performed TLC. The TLC result was shown in Figure 2 confirming the presence of lactic acid in the sample after hydrolysis.

Figure 2 TLC plate observed under 254 nm light. 1: acid lactic (standard), 2: sample (non-hydrolysis), 3: sample (hydrolysis), 4 control (HCl, 10%)

For better understanding about the sample whether the sample was PLA or not, IR and H-NMR studies were conducted. Particularly, PLA has the three important functional groups containing in its structural which were hydroxyl (–OH), carboxyl (–COOH) and ester (COO–). That were the strong evidences for the success in PLA synthesis.

Figure 3 and 4 showed the IR spectrum of interest sample and the monomer (lactic acid), respectively. The intense bands at 2924 cm^-1 and 2870 cm^-1 are from the symmetric and asymmetric valence vibrations of C-H from CH₃, in the sample spectrum. The band shifts related to the C=O group stretch can be observed at 1643 cm^-1 in the monomer spectrum and 1666 cm^-1 in the sample spectrum. The band shifting illustrates a difference in peak intensity which suggest the arrangement of the molecules in the polymer chain. In the sample spectrum, other significant bands are in the region of 1000-1400 cm^-1 which are typical of the ester sequence (–CO–O–). The band around 3500 cm^-1 is related to the stretching of OH group and this raising from the polymer (3441 cm-1) to the monomer (3502 cm^-1 ) due to reaction polyesterification that consumes the OH groups when they react with the acid groups to form the ester bond. The IR spectrum reading is based on the one described by Nikolic et al [11].

Figure 3 IR spectrum of interest solid sample.

Figure 4 IR spectrum of lactic acid.

In the H-NMR analysis, the peaks from 1 ppm to 2 ppm correspond to CH₃ and CH group [12,13], the broader and weaker peak around 6.20 ppm correspond to the COOH terminal group [14]. The peak at 7.4 ppm referred to the solvent (CDCl₃) used in the analysis (Figure 5). Together, the IR and H-NMR studies have confirmed the success PLA production. Thus, from this part, the interest sample could be called as PLA pre-polymer.

Figure 5 H-NMR spectrum of interest sample.

PLA synthesis under xylene and/or reduced pressure conditions

Figure 6Polycondensation of lactic acid under modified conditions. Number-average molecular weight (Mn). Weight-average molecular weight (Mw). polydispersity index (PDI = Mw/Mn)

For the xylene only condition, xylene with the capability of dissolving the PLA was added. With the aid of xylene, it could be seen in figure 6 that the Mw of the polymer was increased slightly from around 2000 g/mole to around 3000 g/mole.

In the reduced pressure condition, although molecular weight induction was not really significant in the comparison to the xylene condition, the PDI value of reduced pressure condition was quite lower than the one of xylene. However, there was a large change of Mw when the two factors were combined, the Mw rose to over 10000 g/mole (Figure 6). Moreover, the PDI value was also low, only 1.15.

Together with these experiments, there was another condition which was the control. In the control, the conventional catalyst (SnCl₂.H2O) was added. With the catalyst, the control showed a better quality of the polymer when the Mw was higher and PDI was lower than other condition. From these results, the Mw of PLA produced from L. rhamnosus can be improved based on the ion transport of this bacterium according to genetic in NCBI. Therefore, the culturing media should be optimized in near future.

Theoretically, the main difference between the spectrums of PLA pre-polymer and PLA polymer is the bands around 3500 cm^-1, which reflects the number of OH group of PLA is relatively low compare to the OH group of the PLA pre-polymer [13]. This fact can be seen clearly from the obtained results indicating the success in raising the Mw of PLA. Also, there were many peaks around 400-700 cm^-1 in the pre-polymer spectrum while being disappeared in the PLA spectrum by IR analysis (Figure 7).

Figure 7 IR spectrum of produced PLA

One of the limitation of direct polycondensation is the presence of byproduct, water which interferes the polymer synthesizing reaction and results in the low molecular weight polymer [15, 16].Therefore, the extra factors have been used for increasing the molecular weight of the polymer: xylene or/and reduced pressure. Both factors have been shown to be very effective in PLA synthesis in combination functioning as increasing molecular weight agents (Mn and Mw raised to 11617 and 13404 g/mole, respectively). In this study, the temperature used for heating the supernatantswas much lower the synthesizedtemperature of PLA, normally, the PLA synthesizing temperature is very high, varies from 150^oC to 190^oC [17]. The reason of setting a high temperature is mainly for removing the water [18], however, with the help of xylene and reduced pressure, the water has been removed effectively. That could be an explanation for the impact of xylene and reduced pressure on increasing the molecular weight of PLA.

Antimicrobial activities:

The advantages of PLA synthesis by bacteria does not stop at removing the dependence of metal catalyst-mediated chemical polymerization and safer for human uses, it also contained the antimicrobial activities thanks to the bacteria secreted substances. The summary of the analyzed data of antimicrobial activities over tested microorganisms shown in the Table 2. The mechanism will be discovered so far. Probably, PLA could carry bacteriocins or antimicrobials produced in bacterial culture to cause the bacterial inhibition.

Table 2: Antimicrobial test results

Microorganism	Dose of 0.02g PLA	Dose of 0.04g PLA	Dose of 0.06g PLA
Pseudomonas aeruginosa	14.33 ± 0.88	27.33 ± 1.45	34.67 ± 0.33
Candida albicans	19.67 ±1 .45	30.33 ± 1.45	39.33 ± 1.20
Micrococcus luteus	28.67 ± 0.67	39.67 ± 0.88	46.33 ± 0.88

*Antibacterial zone in diameter in millimeter

Cytotoxicity activities:

The initial concentration of the stock solution was 1g/mL. It was diluted to the concentration of 100mg/mL in order to perform the test. The test showed cytotoxic activity on HepG2 approximately 90.3 ± 0.99 % at the concentration of 100mg/mL. This activity occurred because L. rhamnosus PN04 culture could produce activity on HepG2 [8]. When PLA was produced in this way, PLA could package with the cytotoxic agents produced by L. rhamnosus PN04. Although PLA had low molecular weight, PLA could show strong activity that will be a promise in pharmaceutical science.

CONCLUSION:

Many strains of L.rhamnosus are being used as probiotics which for improving human health. In this study, L. rhamnosusPN04 was used as the producer of PLA polymer. The PLA has successfully produced from the supernatant of L. rhamnosus PN04 with extra antimicrobial and cytotoxicity activities. Specifically, Lactobacillus has been proven to have the ability of lipase production that is the catalyst for ROP as well as polymeric condensation [5].

The antimicrobial and cytotoxicity activities of PLA was explained thanks to the secreted agents of L. rhamnosus in the supernatant which had been proved to be antibacterial before [19-21]. During the PLA production time, there could be some antimicrobial agents attached to the PLA chains.

Although this study has succeeded in producing the PLA, the obtained molecular weight was still not high. Reality, reaching the low molecular weight is normal since each experiments were done for only 6 hours when in researches, scientists have done the experiment for days [9, 14,18]. If there were more time, there was a high probability that the molecular weight will be increased. Moreover, in the MRS culturing medium, there could be some modifications for increasing the molecular weight as well as increase the quality of PLA.

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Received on 20.02.2018 Modified on 10.04.2018

Research J. Pharm. and Tech 2018; 11(7): 3057-3062.

DOI: 10.5958/0974-360X.2018.00562.0