Antioxidant and antitumor activity of lactic bacteria isolated from natural beverage - Coconut toddy

 

M. Krishna Moorthy¹, Bijaya Kumar Nayak² , Anima Nanda¹*

¹Department of Biomedical Engineering, Sathyabama University, Chennai-600 119, India.

²Department of Botany, K. M. Centre for P.G. Studies (Autonomous), Airport Road, Pondicherry-605 008, India.

 

ABSTRACT:

Probiotics are live microbes, which have the health promoting factors and they are naturally occurring in various kinds of food. During the recent period, the promising interest in naturally fermented food due to its benefits for its richest nutrients and biological property known as probiotic organism, thus we had taken toddy as one of its kind. Toddy is naturally fermented by tapping the male inflorescence of coconut tree. A total of 43 toddy samples were taken, among which 137 organisms were isolated and after the screening for its probiotic nature, one organism was screened out and it was identified by 16S rRNA sequence analysis which confirmed the organism as Lactobacillus fermentum KT183369, and it was further subjected for its application as probiotics. The organism was identified for its antioxidant activity and also for exhibiting the adhesive property into the human beings in order to adhere to the host epithelial and thus was checked using CaCo2 and HT29 cell line assay.

 

 

INTRODUCTION:

Large numbers of potential probiotic organisms are present in the different type of foods and still they are under identification and play a major role in the reservoir for many microbes. Generally fermented foods are consumed in larger amount globally due to its health promoting factors and resistance against diseases causing microorganisms^1,2. India is well known for its various old-fashioned fermented foods and drinks and they are consumed in larger amount among the peoples although the nature of production and the name varies from place to place and its constituent too. Based upon the ingredients, the nature of its efficacy also varies³. Toddy is a very popular and common fermented drink consumed among larger population among the local community of peninsular India², and it is the traditional fermented drink generally produced from Palm and Coconut varieties in India. 

 

 

These alcoholic beverages are prepared from sap by cutting the edge of the inflorescence and a fresh sweetened sap is isolated which forms a major raw material or ingredients for the toddy production. The microbial compositions which are present naturally in the sap undergo a process of fermentation for producing an alcoholic beverage named as toddy^2,4,5,6  Before the process of fermentation the raw sap is generally consisted of larger amount of sugar content present into them, the microbes present in the raw source generally consume all the sugar content for its metabolism and due to its fermentation activity as an alcoholic beverage has been made naturally within 14 – 18 hours based upon the sugar content the time varies. The aims and objectives of this work was to isolate and characterize the organism which acts as probiotic by identifying its application against tumor and exhibiting its efficiency by adhering to host epithelial cells and being a good antioxidant for the benefit towards the host health.

 

MATERIALS AND METHOD:

Toddy collection and bacteria identification:

Toddy sample was collected near Uthukottai, Tiruvallur, Chennai at early morning in a earthen pot and transported to laboratory in a sample container at 3-5°C cold condition within 5 hrs. The sample was serially diluted and plated them using Plate Count Agar and after the incubation period of 24hr at 37°C, various distinguished colonies were isolated based upon the different colony morphology. Each organism was transferred into a sterile Nutrient agar slant and taken for further test.

 

Gene Sequence:

The isolated organism are further classified genetically by using 16s rRNA sequencing with Gene blast technique. The DNA was isolated from the selective organism they are further amplified using PCR and the amplified DNA was purified and sequenced by using the primer (785F 5' GGA TTA GAT ACC CTG GTA 3' and 907R 5' CCG TCA ATT CCT TTR AGT TT 3'[Ma1]). With this the organism was identified as Lactobacillus fermentum KT183369.

 

DPPH Free radical scavenging activity:

MRS broth was prepared and the Lactobacillus fermentum was inoculated, kept for overnight incubation at 37°C, after the incubation period 50µl of Lactobacillus fermentum was taken in a sterile test tube, into which 1 ml of freshly prepared 0.1 mm 2,2-DiPhenyl-2-Picryl hydrazyl hydrate  (DPPH) was added. DPPH was dissolved using ethanol, for control 50 µl of methanol was added into 1ml of DPPH solution and for blank un-inoculated MRS broth was used as blank for this test. Both the test and control sample were made to incubate in dark for 30 min, after the incubation the reduction was measured at absorbance of 250nm with UV spectrophotometer^2,7.

 

Cell Culture of CaCO₂ and HT29:

Both the cell lines (CaCO₂ and HT29) were obtained from National Centre for Cell Science (NCCS), Pune, India. The cells were grown in T25 culture flasks, (Dulbecco’s modified Eagle’s minimal essential medium) DMEM was used along with 10% Fetal Bovine Serum (FBS) used as supplement, L-Glutamine and antibiotic 100 µg/ml of antimycotic drug was also used as a constituent along with the medium. The cells were monitored daily for its morphology and they were maintained at 5% CO₂ incubator at 37°C. The cells should be sterile and vacuole formed during the entire storage period. Cells were maintained until its growth reached to 80% confluence.

 

Adhesion Assay:

Adhesion assay was carried out after 30-60 passages for Caco2 cell lines. Adhesion of the cell suspension, Lactobacillus cultures was prepared at the concentration of 1X10⁵ cells in 4 ml of complete DMEM medium and they were transferred to each well of six-well tissue culture plates. The medium was changed for every alternate day up to 20 days until the confluence has reached 80% growth. After the growth the spent medium was completely removed 24 h before adhesion assay and cells were fed with DMEM medium and this medium should be free from antibiotics and serum.

 

After 24 hr the cell was washed twice with phosphate-buffered saline (pH 7.4) and once again the plates were added with 2ml of DMEM medium and incubated at 37°C for 30 min. Different Lactobacillus cultures at 1 X 10⁹ CFUs was suspended in 1 ml serum free DMEM medium were added to different wells. The plates were incubated at 37°C in 5 per cent CO₂-95 per cent air for 2 h. The monolayers were washed five times with sterile PBS. The adhesion score was measured by enumerating adhered bacteria per 20 different microscopic fields⁷.

 

Percent adhesion:

From the monolayer cells was detached by trypsinization, 1 ml of 0.25 % trypsin-EDTA solution (Himedia, India) was added to each well and the plates were incubated for 15 min at room temperature. Detached cells by trypsinisation were aspirated to a homogenous suspension, then the homogenized cells were serially diluted using saline solution and plated on MRS agar. The plates were incubated for 24-48 h at 37°C and colonies were counted (B1 cfu/ml). Bacterial cells initially added to each well of six-well plates were also counted (B0 cfu/ml). The adhesion percentage was then calculated as, % adhesion= (B₁ / B₀) * 100

 

RESULTS AND DISCUSSION:

Bacterial Identification:

The toddy sample was collected and serially diluted using saline and they were plated on Plate count agar, the different colonies were taken on nutrient agar slant among the various isolates all the organisms were subjected to characterization of probiotic by exposing the organism to human in-vitro condition for its stability as a probiotic organism². Prior to the screening of gram positive organism and it was identified genetically.

 

Genetic Characterization:

The test organism DNA was initially isolated and amplified using PCR then the pure form of DNA approx 1400 bp is taken and they sequenced using the primer (785F 5' GGA TTA GAT ACC CTG GTA 3' and 907R 5' CCG TCA ATT CCT TTR AGT TT 3'[Ma1]) in Big Dye terminator cycle sequencing kit (Applied BioSystems, USA). Sequencing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system service provided at Applied Bio Systems, USA. The isolated organism was identified and submitted to National Council for Bio Informatics (NCBI) for genetic tree construction and it was identified as Lactobacillus fermentum KT183369 (Fig 1).

 

Fig 1. Phylogenetic Tree for Lactobacillus sp.

 

Antioxidant Activity:

The probiotic culture of Lactobacillus fermentum showed strongest radical scavenging activity towards various concentrations ranging from 60 – 100 µL (Fig 2). The OD value was measured at 250nm and the results were tabulated and given in Table 1. The results indicated that the probiotic organism have high level of scavenging activity, thus when the probiotic concentration increased the scavenging activity also decreased generously (Fig 2). Thus the OD value was decreased enormously when the concentration of probiotic organism was in high level.

 

Table 1: Values of Antioxidant – DPPH Method

 

Fig 2: Antioxidant Assay – DPPH method

 

Adhesion Assay:

The Lactobacillus fermentum was investigated for its adhesion potential based on in-vitro study by cell surface hydrophobicity. The binding of the test culture to cell line was investigated by seeing to electron microscope; most of the Lactobacillus fermentum populations tend to get attached with cell line predominately (Fig 3).

 

 

CaCo₂

 

 

HT 29

Fig 3. Adhesive Property of various cell lines

 

Percentage Adhesion:

The percentage observation for adhesion of probiotic organism exhibited a good adhesion property with percent adhesion value of 78% with CaCo2 cell line when compared with HT 29 it exhibited the adhesion percentage of 51.47% based upon the calculation (Table 2). Thus the organism showed a good sort of adhesion of towards the host epithelial cells upon various extremities, which proved that Lactobacillus fermentum can play a vital role as probiotics (Table 2).

Table 2:Observation of percentage of adhesion value with CaCO₂ and HT 29 cell line

 

DISCUSSION:

The more important aspect of a probiotic bacterium includes its ability to adhere to the host epithelial cells into the colonization of gut region for expressing its application to the host’s health. Thus this test organism, Lactobacillus fermentum KT183369 showed a good source of attachment towards the in-vitro cell line of CaCO₂ and HT29 and thus it can be extensively designated as probiotic organism because, probiotics should adhere to mucosal epithelial cells. One of the important criteria for the bacterium is the complex process for its involvement in the attachment. It was found that Lactobacillus fermentum KT183369 has an extensive adhesive property towards the host cell as well as it was also confirmed that it has antioxidant characteristics based on the strongest radical scavenging activity by DPPH assay. Different concentration of probiotic strain was used and the OD was measured at 250nm, the probiotic culture concentration was increased gradually the radical scavenging activity got decreased thus the organism have good antioxidant activity as a major application of probiotic organism when it’s consumed.

 

CONFLICT OF INTEREST: 

The authors declare no conflict of interest.

 

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Accepted on 14.08.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(12): 4317-4320.