Metabolic fingerprinting
The exo- and endo-metabolites of probiotic isolates in culture supernatant provides a window for explaining the comprehensive nature of metabolites. The metabolite profiling provides information about the nutritional as well as of toxic effects, if any of metabolites on the interactive effects of the dietary components on the health of the gut of the host.

In total, 40 metabolites were detected from the culture supernatants of these five isolates. Out of 40 compounds obtained in the solvent phase, only 17 compounds viz. Isopropyl alcohol; L-lactic acid; 2-Propanol, 1-hydrazino; Decane; Benzaldehyde, 2,4, dimethyl; Dodecane; Tetradecane; Phenol, 2,4 bis (1,1 dimethylethyl); Hexadecane, 2,6,11,15 tetramethyl; Hexadecane; Geranyl isovalerate; Pyrrolo [1,2a] pyrazine 1,4 dione, hexahydro 3 (2 methylpropyl); n-Hexadeconic acid; 9 Octadecenamide, (Z); Benzoic acid; Undecane; Dodecanoic acid were common to all the tested isolates as described in Table 2. Out of 17 compounds, Isopropyl alcohol; Dodecane, Hexadecane, Tetradecane and Pyrrolo [1,2a] pyrazine 1,4 dione, hexahydro 3 (2 methylpropyl); Benzaldehyde, 2,4, dimethyl and Geranyl isovalerate; Phenol, 2,4 bis (1,1 dimethylethyl); Hexadecane, 2,6,11,15 tetramethyl; n-Hexadeconic acid; 9 Octadecenamide, (Z), have been reported as defrosting agent, antioxidant, flavour agent, antimicrobial, natural food additive, anti-inflammatory, anti-sleep disorder agent, respectively (Bharat et al., 2013).

Table (2):
Metabolic profile of biocomponents identified in solvent extract of probiotic lactic acid bacteria isolated by GC-MS.

■ represents Lactobacillus plantarum DB-2
■ represents Lactobacillus fermentum J-1
■ represents Pediococcus acidilactici M-3
■ represents Lactobacillus plantarum SK-3
■ represents Pediococcus pentosaceus SM-2

All five bacterial strains secreted different metabolites. The metabolites, their retention time, peak area, area % and peak height are represented in Table 3 and the GC-MS chromatograms are shown in Figure 1.

a. GC-MS chromatogram of metabolites secreted by plantarum DB-2

Total scans for L. plantarum DB-2 was 5709. Total run time was 19.42 min (5.00 – 24.42 min) (Figure 1a). The maximum peak area of 19659374.61 at the peak height of 7235909.56 was observed with 85.29% area covered at retention time 10.53 min (Table 3a, Figure 1b).

Table (3):
Metabolic profile of biocomponents identified in solvent extract of probiotic Lactic acid bacteria isolates by GC-MS.
a.) Lactobacillus plantarum DB-2

L. fermentum J-1 has total of 5713 scans. Total run time was 19.43 min (5.00 – 24.43 min) (Figure 1c). The maximum peak area of 863376582.00 at the peak height of 157335492.57 was observed with 54.03% area covered at retention time 11.37 min (Table 3b, Figure 1d).

b). Lactobacillus fermentum J-1.

P. acidilactici M-3 has total of 5715 scans. Total run time was 19.44 min (5.00 – 24.44 min) (Figure 1e). The maximum peak area of 31245106.39 at the peak height of 9971964.56 was observed with 58.41% area covered at retention time 10.70 min (Table 3c, Figure 1f).

c). Pediococcus acidilactici M-3.

L. plantarum SK-3 has total of 5719 scans. Total run time was 19.45 min (5.00 – 24.45 min) (Figure 1g). The maximum peak area of 55699133.56 at the peak height of 13468063.78 was observed with 92.21% area covered at retention time 10.49 min (Table 3d, Figure 1h).

d). Lactobacillus plantarum SK-3.

L. pentosaceus SM-2 has total of 5860 scans. Total run time was 19.93 min (4.50 – 24.43 min) (Figure 1i). The maximum peak area of 1586768.57 at the peak height of 739977.40 was observed with 68.57% area covered at retention time 10.13 min (Table 3e, Figure 1j).

e). Pediococcus pentosaceus SM-2.

Five metabolic compounds were distributed in four of the selected isolates viz. Isopropyl myristate; Methane, nitroso-; Propylene glycol; R-(-)-1,2 propanediol and Tetracosane. Compounds- Methane, nitroso-; Propylene glycol; R-(-)-1, 2 propanediol were present in
L. plantarum DB-2, P. acidilactici M-3, L. plantarum SK-3 and P. pentosaceus SM-2. A metabolite – Isopropyl myristate was secreted by L. fermentum J-1, P. acidilactici M-3, L. plantarum SK-3 and P. pentosaceus SM-2. Compound – Tetracosane was reported in L. plantarum DB-2, L. fermentum J-1, P. acidilactici M-3 and P. pentosaceus SM-2. Out of five metabolic compounds, Isopropyl myristate has been reported with medicinal activity against skin disorders. Out of 40, 3 metabolic compounds i.e. Eicosanoic acid, 2-Hydrazino ethanol and Tetradecanoic acid have been reported in three of the selected isolates. A metabolite – Eicosanoic acid was secreted by L. fermentum J-1, P. acidilactici M-3, and P. pentosaceus SM-2. Compound – 2-Hydrazino ethanol was found in supernatant of L. plantarum DB-2, P. acidilactici M-3 and P. pentosaceus SM-2. A metabolic compound – Tetradecanoic acid was present in L. fermentum J-1, P. acidilactici M-3 and P. pentosaceus SM-2 (Table 2).

Six out of 40 metabolic compounds viz. DL-2, 3-Butanediol; Ethanamine, 2-propoxy-; Ethanol, 2-(2-propenyloxy); Hydroperoxide, pentyl; Methyltartronic acid and Squalene were present in two of the selected isolates. Metabolites – DL-2, 3-Butanediol and Methyltartronic acid were secreted by L. plantarum DB-2 and L. plantarum SK-3. Compounds – Ethanamine, 2-propoxy-; Ethanol, 2-(2-propenyloxy) and Hydroperoxide, pentyl were found in the supernatant of P. acidilactici M-3 and L. plantarum SK-3. Metabolite – Squalene was synthesized in the culture supernatant of P. acidilactici M-3 and P. pentosaceus SM-2 (Table 2). Squalene is a triterpine compound, originally found in shark liver oil and has a high therapeutic potential. Squalene is a therapeutic agent having anti-cancerous, anti-tumour, chemo-preventive, antioxidant, and sunscreen properties along with anti-microbial activity (Ezhilan and Neelamegam, 2012). In industry, it is scarcely produced by plant sources along with the shark liver oil. Microbial production of Squalene is an attractive alternative to label the issue with an objective to increase its productivity and purity by using biotechnological interventions. Although, a detailed study is required for the commercialization of the production of Squalene from native probiotic bacteria.

9 metabolites out of 40 were produced by only one selected isolate viz. Acetic acid, anhydride with formic acid; 4-amino-1-butanol; 2-Ethoxyethylamine; Ethylamine; Formamide; Hydroperoxide, heptyl; Hydroperoxide, 1-methylhexyl; 4-Penten-2-ol and Propanoic acid, 2-hydroxy-, methylester. Compounds – Acetic acid, anhydride with formic acid; 2-Ethoxyethylamine; Ethylamine; Formamide were produced by L. plantarum DB-2. Metabolite – 4-amino-1-butanol was found to be synthesized by L. plantarum SK-3. Metabolite – Hydroperoxide, heptyl was present in P. acidilactici M-3. Compounds – Hydroperoxide, 1-methylhexyl and Propanoic acid, 2-hydroxy-, methylester were secreted by L. fermentum J1. Metabolite – 4-Penten-2-ol was produced by P. pentosaceus SM-2
(Table 2).

Probiotics has a vital role in well-being and health beyond basic nutrition. Probiotics have potential health benefits such as antimicrobial activity, antimutagenic, anticarcinogenic activities, constipation, regulation of immune function, improving gastrointestinal health, reducing lactose intolerance, and allergenic diseases such as food allergy, etc. Due to these health benefits, probiotic bacteria and their metabolites are gaining importance in pharmaceutical preparation, food products, medicines and dietary supplements (Da Cruz et al., 2009; Settanni and Moschetti, 2010; Fernandez et al., 2013). Production of different primary and secondary metabolites by the probiotic bacteria, employs their biological effects directly or by modifying the immune system. The metabolites of lactic acid bacteria provide therapeutic benefits in preventing and curing various diseases and also responsible for the development of flavour, aroma and texture in food products (Chen et al., 2014). The metabolites produced by lactic acid bacteria in this study have been remarked with various functional as well as therapeutic properties viz. food additives, flavouring agents, anti-inflammatory, anti-cancerous, potential against skin disorders, cholesterol-lowering and anti-diarrheal properties and endorse their huge potential in food and pharmaceutical industries.

Many researchers have observed similar metabolic products in many lactic acid bacteria influencing human metabolism when incorporated into the system as probiotics or the food fermented by the respective bacteria. Cagno et al. (2009) observed tomato juice fermented with L. plantarum POM1 and POM35 for the effect of metabolites produced by the bacterial starters (by GC-MS technique) on the sensory and health-promoting properties of tomato juice and found a large number of volatile compounds in fermented tomato juice as compared to the control. Lee et al. (2009) isolated the metabolites from lactic acid bacteria in grape wines through GC-based metabolic profiling and ¹H-NMR and identified various organic acids such as lactic acid, tartaric acid and volatile compounds such as benzoic acid, dodecanoic acid, dodecamethyl cyclohexasiloxane, isopropyl myristate, tetradecanoic acid, 1-hexadecanol, hexadeconoic acid and octadecanoic acid responsible for wine odour and flavour. Vanaja et al. (2011) observed the metabolites secreted by L. plantarum LPcfr and found compounds viz. hexadecane, hexadeconic acid, dodecanal having antifungal and antioxidant properties and fatty acids such as stearic and palmitic acids having antifungal properties. Sheela et al. (2012) found antimicrobial compounds (fatty acids such as palmitic acid and stearic acid and Phenol, 2, 4-bis (1, dimethylethyl)) after GC-MS in symbiotic cow milk beverage fermented with L. kefiranofaciens, Candida kefir and Saccharomyces boulardii. The fermented cow milk symbiotic beverage had the greater anti-diarrhoeal effect as compared to other milk beverages, thereby indicating the role of antibacterial metabolites produced by the probiotic bacteria. Padmavathi et al. (2014) studied the anti-biofilm efficacy and anti-quorum sensing of the metabolites produced by marine Vibrio alginolyticus G16 against Serratia marcescens and found the active compound as phenol, 2, 4-bis (1,1-dimethylethyl) after purification and mass spectrometric analysis.

Sheela et al. (2015) studied the biocontrol efficacy of symbiotic strawberry juice and found most of the metabolites were of antibacterial, anti-diarrhoeal (17-pentatriacontene) and of antioxidative nature. Effect of probiotics and prebiotics was evaluated on the metabolites isolated from human microbiota and found these metabolites helping in the regulation of colonic cell proliferation, inactivation of toxic compounds and enhancement of health-promoting properties (Vitali et al., 2012). Hong-Xin et al. (2015) used GC-MS analysis to identify aroma compounds viz. decane, dodecanoic acid, hexadecanoic acid, hexanoic acid 4-hexadecyl ester and phenol 2,4 bis (1,1-dimethylethyl) during ripening period (180 days) of cheese fermented with L. casei LC2W and found these metabolites help in accelerating the ripening process.

The reliability of the technique in investigating the effect of metabolic compounds on colonic metabolic signature has been recently studied (Maccaferri et al., 2010, Vitali et al., 2010, Garner et al., 2007).