INTRODUCTION:

Polysaccharides are biopolymers from the most diverse families. Wide applications were found for many kinds of polysaccharides. In recent years, many attention has been focused on EPS pharmacological actions and bioactivities. The bioactivity of a given polysaccharide is closely related to its structure and physicochemical properties¹.

The producer lactic acid bacteria (LAB) for EPS studied widely for his or her properties and human health potential effects during the last decades. LAB EPS are considered to not only play a crucial role in improving the theology, texture and mouthful of fermented products, but also the beneficial effects on human health, such as anti-tumour, immunomodulating and anti-mutagenicity bioactivity^2-3.

Colon cancer may be a widely expressed cancer thanks to somatic mutations in colon cells occurring during the lifetime of a host. Reviews have been mentioning the role of probiotics and prebiotics in protective activity of colon cancer⁴. Probiotics also play a major role in colon cancer protective and trials are now feasible for “at risk” individuals⁵. Several overlapping mechanisms for probiotic associated anticancer effects have emerged including a reduction in the ability of the enteric flora to convert dietary procarcinogens to carcinogens⁶.

Conventional cancer therapies like surgery, radiotherapy or chemotherapy remain the backbone of cancer therapy to date. However, not every cancerous tissue can be targeted by physical or chemical method. In this context, novel treatment options can be used in cancer therapy. Naturally derived drug-delivery systems like EPS can shuttle therapeutic compounds into the tumor⁷.

In recent years, the applicant of Nano fibular forms as a scaffold to prepare organize metallic NPs has merited substantial attention due to to their important potential applications within the fields of catalysis, electronic nanodevices, optoelectronics, sensors, biomedical and nanocomposites⁸. EPS nanocrystals, which are typically crystalline rod-like particles, can be easily extracted from a variety of renewable sources by controlled acid hydrolysis of EPS. They have some notable properties, such as large aspect ratio, good dissolvability in water, excellent mechanical properties and a high capacity for absorption of guest molecules⁹.EPS nanocrystals have found applications in material science, for instance, the reinforcement of polymers¹⁰.

The present work is an attempt to biosynthesize of extracellular prebiotic then converted to nanocrystals and characterization of these crystals using UV spectroscopy, FTIR and TEM as well as estimation the bioactivity of the EPS and its nanocrystals in vitro.

MATERIALS AND METHODS:

Polysaccharide production:

The culture of Lactobacillus brevis was propagated for EPS production using growth medium MRS broth medium containing (g/l): Peptone, 10.0; Meat extract, 8.0; yeast extract, 4.0; D(+) Glucose, 20.0; Dipotassium hydrogen phosphate, 2.0; Sodium acetate trihydrate, 5.0; Triammonium citrate, 2.0; Magnesium sulfate heptahydrate, 0.2; Magnesium sulfate tetrahydrate, 0.05. pH 6.2¹¹.

The EPS was isolated and purified according to Cerning et al.¹² with some modification. The growth cultures were heated at 100 °C for 5 min to inactivate enzymes potentially capable of polymer degradation and the cells were removed by centrifugation at 8000 rpm for 5 min at 4°C. The EPS was precipitated using 2 vols. of absolute ethanol. After standing overnight at 4°C, the resultant precipitate was collected by centrifugation at 8000 rpm for 20min. The EPS was dissolved in deionized water, dialyzed against deionized water at 4 °C for 24 h and freeze-dried. The freeze-dried powder was dissolved in 10% (w/v) trichloroacetic acid to remove proteins. The supernatant was dialyzed at 4 °C against deionized water for 5 days and freeze-dried. These preparations were referred to as purified EPS and were stored at 4 °C.

Preparation of Nano-Prebiotic EPS:

The Nano-prebiotic EPS (NPEPS) were prepared according to pervious study with some modification¹³. EPS was hydrolyzed by diluted sulfuric acid (64 %, 250 ml) and incubated at room temperature for 24 hours under static conditions. At the end of the incubation period, the color of the suspension becomes dark brown. Then the EPS suspension was diluted with de-ionized water to stop the hydrolysis reaction, allowed to settle overnight until the suspensions were layered and the clear top layer was decanted off. After that, the supernatant washed with de-ionized water until no layered was found by centrifugation at 5000rpm for 10 min. Take the supernatant and repeated the previously step (several time). The final washed was conducted using dialysis page with de-ionized water for several days until the water pH remained constant. Afterwards, the ultrasonication was conducted for 20 min at an output power of 1200W. Finally, the NPEPS suspension sample was subjected to freeze-drying. All processing were done under dark conditions.

Characterization of EPS and NPEPS:

Ultraviolet spectrum (UV):

The UV spectrum analyses of the purified samples were recorded with T80+UV/VIS Spectrometer, PG Instrument Ltd. Range: 190-1000 nm.

Infrared spectrum (IR):

The IR spectrums of the purified samples were determined using a Fourier transform-infrared spectrophotometer (FTIR, Jasco 6100, and Model Japan). The sample was ground with spectroscopic grade potassium bromide powder and then pressed into a 1 mm pellet for FT-IR measurement in the frequency range of 4000–400 cm^-1 (Mid infrared region).

Transmission Electron microscopy (TEM):

This study was undertaken to know the size and shape of NPEPS. The TEM image was carried out using: Electron probe micro-analyzer JEOL – JXA 840A, Model Japan.

Antitumor activity and cytotoxicity of the EPS and NPEPS:

Determination of sample cytotoxicity on colorectal adenocarcinoma ATCC ATB-37 (CACO2), from Homo sapiens colon tissue, epithelial cells and Normal Homo sapiens, lung tissue fibroblast (WI-38) cells MTT protocol was performed¹⁴. The 96 well tissue culture plate was inoculated with 1 X 105 cells/ml (100ul/well) and incubated at 37^oC for 24 hours to develop a complete monolayer sheet. Growth medium was decanted from 96 well microtiter plates after confluent sheet of cells were formed, cell monolayer was washed twice with wash media. Two-fold dilutions of the tested sample were made in RPMI medium with 2% serum (maintenance medium). 0.1ml of each dilution was tested in different wells leaving 3 wells as control, receiving only maintenance medium. The plate was incubated at 37^oC and examined. Cells were checked for any physical signs of toxicity, e.g. partial or complete loss of the monolayer, rounding, shrinkage, or cell granulation. MTT solution was prepared (5mg/ml in PBS) (BIO BASIC CANADA INC). 20ul MTT solutions were added to each well. Place on a shaking table, 150rpm for 5 minutes, to thoroughly mix the MTT into the media. Incubate (37^oC, 5% CO₂) for 1-5 hours to allow the MTT to be metabolized. Dump off the media (Dry plate on paper towels to remove residue if necessary). Resuspend formazan (MTT metabolic product) in 200 ul DMSO. Place on a shaking table, 150 rpm for 5 minutes, to thoroughly mix the formazan into the solvent. Read optical density at 560nm and subtract background at 620nm. Optical density should be directly correlated with cell quantity.

RESULTS AND DISCUSSION:

Characterization of Lactobacillus brevis EPS and NPEPS:

Ultraviolet spectrum (UV):

The UV spectrum analysis of Lactobacillus brevis EPS was showed peak at 258. NPEPS in Nano form observed shifted peak at 270nm which confirms the formation of reduced EPS nanoparticles. This technique has proved to be very useful for the analysis of nanoparticles¹⁵.

Infrared spectrum (IR):

The IR spectrum of the purified EPS (figure 1) exhibits absorbance peaks at: 3274cm^-1 (OH stretching vibration), and 1053cm^-1 (C-O stretching vibration). The broader peak at 1374cm^-1 is due to the stretching vibration of the hydroxyl groups (–OH). The stretching vibration of C-O groups was detected at 1053cm^-1. On the other hands, the weak peak at 519cm^-1 was ascribed to that for the β-configuration of the sugar units in EPS. Each polysaccharide exhibited high absorbance in the region 1200–950 cm^-1, which was within the so-called fingerprint region, where the position and intensity of the bands are specific for polysaccharide, allowing its possible identification, and this region was dominated by ring vibrations overlapped with stretching vibrations of (C–OH) side groups and the (C–O–C) glycosidic band vibration. The FTIR spectrum of NPEPS (figure 2) shown the peak at 3452cm^-1 can be attributed to the stretch vibration of (O–H) stretch group. On the other hand, the peak at 1647cm^-1 is attributed to (C=O) carbonyl and carboxyl stretching group. Furthermore, the peak at 1424 is assigned to (O–H) bend group. Moreover, the absorption bands at 1151 and 1112cm⁻¹ is ascribed to (C–O–C) stretching mode from the glucosidic units¹⁶. We thought that signals presence in chart of Nano form and absence in pure EPS indication to convert to Nano crystalline and changes in structure.

Transmission Electron microscopy of NPEPS:

This experiment was undertaken to know the size and shape of Nano-prebiotic EPS. Under magnification of 50 nm the size of Nano-prebiotic EPS were ranging from 5 to 17 nm. The results show that, the Nano-prebiotic EPS in the reaction mixture has a uniform spherical shape and showing varying sizes as observed in Figure 3.

Fig. 3: TEM images of Nano- prebiotic EPS under different magnification power

Anti-colon cancer and cytotoxicity of EPS and NPEPS:

The inhibitory effect of pure EPS against the growth of CACO2 using MTT method revealed that EPS inhibited the proliferation of the CACO2 cell lines in a dose- dependent manner with IC₅₀338μg/ml. Results indicated that EPS from Lactobacillus brevis have a potent inhibitory effect on CACO2 cell line. The mechanisms of the underlying resistance were not clear up to date. This study also demonstrated the selective sensitivity of EPS against CACO2 cells which exhibited almost complete suppression of cell growth. Consistent with the finding of You et al.¹⁷, obtained polysaccharide revealed a marked inhibition of proliferation of HCT 116 and CACO cells. Liu et al.¹⁸ reported that significant anti-proliferation of HEPG2 cells with UA and viili EPS at concentration of 200μg/ml by MTT assay, and the cell senescences at 12.5μg/ml indicated a modulation manner. All these tests have shown a compatibility of herbal UA and foodborne viili EPS, which indicates that they can be processed together for health or functional food¹⁹. Results showed that cell viability decreases when Nano- prebiotic EPS concentration increases. These results suggest that Nano- prebiotic EPS cause more cytotoxicity with IC50 169.3μg/ml than EPS in natural form with IC50 338μg/ml. EPS and Nano- prebiotic EPS treatment results in a noticeable change in morphology of cells. Therefore, Examination was performed using microscopy. As appeared in Figures 4 and 5, cell shape in the control remained normal; the cells adhered well, with most attaching. Most cells were polygonal, with transparent cytoplasm, better scattering and a few newly cells during the process of adhering. Although cells adhered, they could not spread, and some lost the polygonal and became rounded shape. Viability assays are essential for evaluating the cellular response to toxicants. Nano-prebiotic EPS cytotoxicity is still controversial and the mechanism has not been well identified. Cytotoxicity of the EPS and NPEPS against WI-38 Cell Line were carried out to determine the side effect of EPS and NPEPS on normal cells. Results listed in table 1 indicated that IC50 of EPS and Nano- prebiotic EPS recorded 5036 and 2049ug/ml. But, it should be noted that the IC50 on CACO recorded 338 and 169μg/ml. The results showed that the natural EPS was less cytotoxic than Nano-prebiotic EPS and both were active and safe in recommended concentrations. Results showed that natural synthesis EPS was recommended^20-21-22.

Table 1: Cytotoxicity effect of EPS and NPEPS on CACO2 and WI-38 cell lines

Samples tested	IC50 (μg/ml)
Samples tested	CACO2	WI-38
EPS	338	5036
NPEPS	169	2049

Fig. 4: Morphology changes in CACO2 cells after exposure to EPS from Lactobacillus brevis at concentrations.

Fig. 5: Morphology changes in CACO2 cells after exposure to NPEPS at concentrations.

CONCLUSION:

Exopolysaccharides (EPS) are high molecular weight long chain polymer composed of sugar residues and secreted by microbes into the surrounding environment²³. Many bacteria are able to produce extracellular polymeric substances (EPS) outside of their cell walls^24-25. They play an important role in many industries such as textiles, pharmaceuticals, food, oil recovery and wastewater treatment processes^26-27. Polysaccharides are said to have various applications in the field of medicine, food, agriculture and so on^28-29. The biosynthesis of EPS from probiotic bacteria has been demonstrated. The NPEPS synthesized from EPS are eco friendly and efficient. It showed enhanced antitumor activity against CACO2. Results focused on the possibility of using exopolysaccharide and its Nano form from Lactobacillus brevis for human pharmaceutical use as it enhanced anti-colon cancer.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 12.12.2020 Modified on 25.01.2021

Research J. Pharm.and Tech 2021; 14(11):5998-6002.

DOI: 10.52711/0974-360X.2021.01042