Research Article | Open Access

Aditya Chaudhary1, Khushbu Verma1 and Baljeet Singh Saharan2

1Faculty of Agriculture and Veterinary Science, Jayoti Vidyapeeth Women’s University, Jaipur, India.
2Department of Microbiology, CCS Haryana Agricultural University, Hisar, Haryana, India.
J. Pure Appl. Microbiol., 2020, 14 (1): 657-672 | Article Number: 5973
https://doi.org/10.22207/JPAM.14.1.68 | © The Author(s). 2020
Received: 01/12/2019 | Accepted: 20/02/2020 | Published: 13/03/2020
Abstract

A GC-MS based metabolic profiling was carried out to study metabolic differences of lactic acid bacteria isolated from different food sources. Metabolic fingerprinting is a non-targeted procedure where all detectable peaks are considered to establish sample classification. A total of 40 compounds were identified as major metabolites contributing to the difference among five different probiotic lactic acid bacteria. Some of the metabolites identified in this study have been reported as a  defrosting agent, antioxidant, flavour agent, antimicrobial, natural food additive, anti-inflammatory, anti-sleep disorder agent and anti-cancer agents. These results suggest that GC-MS based metabolomic analysis is a useful tool to facilitate future investigations into the characterization of probiotic lactic acid bacteria.

Keywords

Lactic acid bacteria, metabolic profiling, GC-MS, traditional food products and probiotics.

Introduction

Lactobacillus and Pediococcus is a gram-positive facultative anaerobic bacterium found widely in fermented food products and can be investigated for the metabolites present in them by GC-MS based metabolic profiling.

Metabolic fingerprinting is explained as the semi-quantitative investigation of extracellular (exo-metabolome) and intracellular (endo- metabolome) metabolites, respectively (Villas-Boas et al., 2005). These metabolic profiles GC-MS has consistently been the most favoured analytical technique for the analysis of metabolites present in distinct biological samples. Gas chromatography and mass spectrophotometry (GC-MS) present the high chromatographic resolution ascribed to the high sensitivity and specificity of mass spectrophotometry (Villas-Boas et al., 2005). In comparison with other techniques, GC-MS can produce a comparably high reproducibility, high resolution, high-quality sensitivity, and good- throughput analysis, which can be used for analyzing the metabolic products, inclusive of carbohydrates, fatty acids, organic acids, and amino acids (Park et al., 2016). Derivatization is necessary before the investigation by GC-MS as most of the metabolites are non-volatile (Schummer et al., 2009). Nowadays, most adaptable derivatization technique is silylation, which has the ability to derivatize compounds having polar functional groups by mixing TMS (Trimethylsilyl) reagent to develop TMS Compounds (Nordström A, 2004). However, for the study of different biological metabolic fingerprinting, N, O-bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane is generally used as silylation reagent and derivatization method with GC-MS (Alreshidi et al., 2015; Li et al., 2013). For the study of metabolites in the microbial sample, derivatization based GC-MS technique can be applied to identify the intracellular as well as extracellular metabolites of Lactobacillus and Pediococcus species.

Liquid injection GC-MS provides an inexpensive and simple option for the analysis of metabolites produced by probiotic lactic acid bacteria. As the concentration of metabolites varies widely in different probiotic lactic acid bacteria, it is required to develop the analytical plan that would permit concurrent quantification of them in a single run utilizing comparably a small amount of sample along with minimum sample preparation. GC-MS has high sensitivity and hence can be used for the investigation of less common samples that might only be available in minute quantity. In this study, direct-injection GC-MS methodology was used for the profiling of different metabolites, which has the high specificity of mass spectrometry along with the high reproducibility and high resolution of gas chromatography.

The results of this study provide reference data for interpreting the differences in the metabolite profiles of different probiotic lactic acid bacteria isolated from different food sources.

Materials and Methods

Collection of food samples
Food items viz. Dosa batter, jalebi batter, maida dough, sauerkraut and soymilk were used in this experiment. Dosa batter, Jalebi batter, Soymilk were collected from a local market in New Delhi, India. Maida dough and Sauerkraut were prepared at home. The food samples were taken in a sterilized bag and stored at -4° until use.

Strain isolation
1 g or 1 ml of food sample were added into 9 ml of 0.85% (w/v) normal saline. After homogenization, serial dilutions were prepared upto 10-9 with 0.85% (w/v) normal saline and 0.1 ml decimal of appropriate dilutions were plated onto de Man, Rogosa, Sharpe (MRS) agar medium (Himedia, India) (de Man et al., 1960). The agar plates were incubated at 35° for 24 h under anaerobiosis. Morphologically different colonies were picked and re-streaked onto MRS agar plates up to purity. Glycerol stocks of strains were preserved at -20°.

Probiotic Lactic Acid Bacteria
L. plantarum DB-2, L. fermentum J-1, P. acidilactici M-3, L. plantarum SK-3 and P. pentosaceus SM-2 were isolated from Dosa batter, Jalebi batter, Maida dough, Sauerkraut, and Soymilk and were identified in own previous studies with NCBI accession no. MK246169, MK353735, MK461878, MK246167 and MK461882 respectively (Chaudhary and Saharan, 2019) (Table 1). Probiotic attributes such as acid tolerance (Liong and Shah, 2005), bile tolerance (Walker and Gilliland, 1993), antibiotic susceptibility (Thirabunyanon et al., 2009), hemolytic activity (Harrigan, 1998), gelatinase activity (Harrigan, 1990), autoaggregation (Del Re et al., 2000), co-aggregation studies (Del Re et al., 2000), hydrophobicity (Rosenberg et al., 1980), bacteriocin production (Papagianni and Anastasiadou, 2009), lactic acid and hydrogen peroxide production (AOAC, 1995), exopolysaccharide production (Mora et al., 2002) were studied on all the five isolates. Bacterial isolates were identified at the genomic level by using 16 S rRNA gene technique (Hashem et al., 2010; Bollag and Edelstein, 1991).

Table (1):
Genotyping of screened lactic acid bacterial strains.

Name of isolate Source Identity (%) 16S r RNA identification Accession number
DB-2 Dosa Batter 99 Lactobacillus plantarum MK246169
J-1 Jalebi batter 99 Lactobacillus fermentum MK353735
M-3 Maida dough 97 Pediococcus acidilactici MK461878
SK-3 Sauerkraut 100 Lactobacillus plantarum MK246167
SM-2 Soymilk 99 Pediococcus pentosaceus MK461882


Metabolic fingerprinting
Metabolites were extracted by following the method given by Coucheney et al. (2008) with minor modifications.

Sampling for metabolic fingerprinting
Microbial suspensions (20 ml) of isolate (grown in MRS broth for 24 h at 35°C) were disrupted using pulsed, high-frequency sound waves (>20kHz) for five cycles (30 sec. run; one min break) to extract intracellular metabolites. Suspensions procured after sonication were centrifuged for 10 min at 10,000 rpm in order to segregate the extra as well as intracellular metabolites from the cells.

Extraction for metabolic fingerprinting
The supernatant was removed and the metabolites were extracted with a Methanol: Water: Chloroform mixture (2: 0.8: 1): 2.5 ml of cold chloroform and 5 ml of cold methanol (-20°C) and the phases were allowed to separate. Metabolic fingerprints were assessed in the aqueous phase after freeze-drying while in the organic phase, the dried chemical extracts obtained after complete evaporation of the solvent was used.

Generation of metabolic fingerprints and data processing
Derivatization of dried samples
The derivatization method given by Mastrangelo et al. (2015) and Park et al. (2019) was used in this study for the evaluation of metabolic fingerprinting. The freeze-dried samples and dried chemical extracts were methoxymated using methoxyamine in pyridine solution and trimethylsilylated by BSTFA (N,O-Bis(trimethylsilyl)trifluoroacetamide) with 1 % chlorotrimethylsilane (TMCS).

GC-MS analysis
Derivatives from Lactobacillus and Pediococcus strains were analysed using Thermo Trace 1300GC coupled with Thermo TSQ 800 Triple Quadrupole MS (Pogacic et al., 2015). Six-microlitre aliquots were injected in splitless mode. The samples were warmed to 65°C for 15 min and the metabolites were extracted before being adsorbed on the trap at 35°C. The trap was heated at 250°C for 0.1 min, leading to desorption of the metabolites. Metabolites were then separated on TG 5MS column (30m X 0.25mm, 0.25µm thickness) with column makeup of 5% diphenyl; 95% dimethyl polysiloxane. The temperature of the oven was initially 50°C, maintained for 3 min. The temperature was increased at 15°C/min to 220°C. The mass spectrometer was operated in the scan mode within a mass range of m/z 30 to 700. The quadruple mass spectrophotometry parameters were set to the conditions: ion source temperature of 230°C, split flow of 40 ml/min, carrier flow of 1.5 ml/ min, injector temperature and MS transfer line temperature of 250°C. Ionization was done by electronic impact at 70 eV. All samples were analysed in the same GC-MS and injected in a randomized order over the GC-MS run. Blank samples (boiled deionized water) were injected after every sample to verify the instrumental carryover.

Data pre-processing and data analysis
The GC-MS raw data files were converted to netCDF format. Further, the raw data were processed to time- and mass- aligned chromatographic peak areas with the XCalibur 2.2SP1 with Foundation 2.0SP1. Metabolites were identified by comparison of mass spectra and retention times with those of authentic standards from NIST 2.0 Mass Spectral Library (Mastrangelo et al., 2015).

RESULTS AND DISCUSSION

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.

S.No Compound Five isolates Four isolates Three isolates Two isolates One isolate
1 Acetic acid, anhydride with formic acid
2 4-amino-1-butanol
3 Benzaldehyde,2,4,dimethyl
4 Benzoic acid
5 Decane
6 DL-2,3-Butanediol
7 Dodecane
8 Dodecanoic acid
9 Eicosanoic acid
10 Ethanamine, 2-propoxy-
11 Ethanol, 2-(2-propenyloxy)
12 2-Ethoxyethylamine
13 Ethylamine
14 Formamide
15 Geranyl isovalerate
16 Hexadecane
17 Hexadecane,2,6,11,15 tetramethyl
18 n-hexadecanoic acid
19 2-Hydrazino ethanol
20 Hydroperoxide, heptyl
21 Hydroperoxide, 1-methylhexyl
22 Hydroperoxide, pentyl
23 Isopropyl Alcohol
24 Isopropyl myristate
25 L-Lactic acid
26 Methane, nitroso-
27 Methyltartronic acid
28 9 Octadecenamide, (Z)
29 4-Penten-2-ol
30 Phenol,2,4,bis (1,1 dimethylethyl)
31 Propanoic acid, 2-hydroxy-, methylester
32 2-Propanol,1-Hydrazino
33 Propylene glycol
34 Pyrrolo [ 1,2 a ] pyrazine 1,4 dione, hexahydro 3 (2 methylpropyl)
35 R-(-)-1,2 propanediol
36 Squalene
37   Tetracosane
38 Tetradecane
39 Tetradecanoic acid
40 Undecane

 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

b. GC-MS chromatogram of plantarum DB-2 at retention time 10.53 min

c. GC-MS chromatogram of metabolites secreted by fermentum J-1

d. GC-MS chromatogram of fermentum J-1 at retention time 11.37 min

e. GC-MS chromatogram of metabolites secreted by acidilactici M-3

f. GC-MS chromatogram of acidilactici M-3 at retention time 10.70 min

g. GC-MS chromatogram of metabolites secreted by plantarum SK-3

h. GC-MS chromatogram of plantarum SK-3 at retention time 10.49 min

i. GC-MS chromatogram of metabolites secreted by pentosaceus SM-2

j. GC-MS chromatogram of pentosaceus SM-2 at retention time 10.13 min
Fig. 1. GC-MS chromatograms of metabolites secreted by Lactic acid bacteria

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

Retention Time Peak Area Area % Peak Height
9.63 13668.39 0.06 23789.56
9.65 5965.64 0.03 13938.29
9.71 16405.98 0.07 28881.92
10.09 13022.90 0.06 27200.06
10.12 10917.74 0.05 18554.92
10.36 532337.99 2.31 147518.20
10.43 89582.31 0.39 148261.31
10.53 19659374.61 85.29 7235909.56
10.63 1922104.73 8.34 757552.20
10.77 785784.33 3.41 319137.44
Retention Time Metabolites Molecular formula Cas No.
9.63 Methane, nitroso- CH3NO 18501-20-7
Formamide CH3NO 75-12-7
Dodecane CH3(CH2)10CH3 112-40-3
DL-2,3-Butanediol C4H10O2 6982-25-8
9.65 2-Propanol, 1-hydrazino- C3H10N2O 18501-20-7
Isopropyl alcohol C3H8O 67-63-0
Propylene glycol C3H8O2 57-55-6
9.71 L-Lactic acid C3H6O3 79-33-4
Acetic acid, anhydride with formic acid C3H4O3 2258-42-6
R-(-)-1,2-propanediol C3H8O2 4254-14-2
10.09 Methyltartronic acid C4H6O5 595-98-2
Dodecanoic acid C3H10N20 18501-20-7
2-Hydrazinoethanol C2H8N20 109-84-2
10.12 Tetradecane CH2(CH3)10CH2 629-59-4
Benzoic acid C6H5COOH 65-85-0
Undecane C11H24 1120-21-4
10.36 Hexadecane, 2,6,11,15, tetramethyl C20H42 504-44-9
Benzaldehyde, 2,4, dimethyl C9H10O 15764-16-6
Phenol, 2,4, bis (1,1 dimethylethyl) C14H22O 96-76-4
10.43 Decane C10H22 124-18-5
Formamide CH3N0 75-12-7
Tetracosane C24H50 646-31-1
10.53 Hexadecane C16H34 544-76-3
Geranyl isovalerate C15H26O2 109-20-6
Pyrrolo [1,2a] pyrazine 1,4 dione, hexahydro 3 (2 methylpropyl) C11H18N2O2 5654-86-4
10.63 n-Hexadeconic acid C16H32O2 57-10-3
9 Octadecenamide, (Z) C18H35NO 301-02-0
L-Lactic acid C3H6O3 79-33-4
10.77 Methane, nitroso- CH3NO 865-40-7
Ethylamine C2H7N 75-04-7
2-Ethoxyethylamine C4H11NO 110-76-9


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.

Retention Time Peak Area Area % Peak Height
5.04 2763275.83 0.17 4173857.72
11.30 152563796.70 9.55 141697072
11.37 863376582.00 54.03 157335492.57
11.53 497177369.61 31.12 129599071.95
11.61 81982240.17 5.13 26851053.15
Retention Time Metabolites Molecular Formula Cas No.
5.04 Hydroperoxide, pentyl C5H12O2 74-80-6
Ethanol, 2-(2-propenyloxy)- C5H10O2 111-45-5
Hydroperoxide, 1-methylhexyl C7H16O2 762-46-9
Decane C10H22 124-18-5
Undecane C11H24 1120-21-4
11.30 Benzaldehyde, 2,4, dimethyl C9H10O 15764-16-6
Benzoic acid C6H5COOH 65-85-0
2-Propanol, 1-hydrazino- C3H10N2O 18501-20-7
Isopropyl alcohol C3H8O 67-63-0
L-Lactic acid C3H6O3 79-33-4
11.37 Dodecane C12H26 112-40-3
Phenol, 2,4 bis (1,1dimethylethyl) C14H22O 96-76-4
Propanoic acid, 2-hydroxy-,methyl ester, (ñ)- C4H8O3 2155-30-8
Dodecanoic acid C12H24O2 143-07-7
Tetradecane CH2(CH3)10CH2 629-59-4
11.53 Hexadecane C16H34 544-76-3
Geranyl Isovalerate C15H26O2 109-20-6
Pyrrolo [ 1,2 a ] pyrazine 1,4 dione, hexahydro 3 (2 methylpropyl) C11H18N2O2 5654-86-4
n-Hexadeconic acid C16H32O2 57-10-3
2-Hydrazinoethanol C2H8N2O 109-84-2
11.61 Eicosanoic acid C20H40O2 506-30-9
Tetracosane C24H50 646-31-1
Isopropyl myristate C17H34O2 110-27-0
9 Octadecenamide, (Z) C18H35NO 301-02-0
Tetradecanoic acid C14H28O2 544-63-8
Hexadecane, 2,6,11,15 tetramethyl C20H42 504-44-9


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.

Retention Time Peak Area Area % Peak Height
5.02 3774941.17 7.06 3718077.48
10.58 2981495.15 5.57 1994601.20
10.63 1773985.26 3.32 1572423.15
10.70 31245106.39 58.41 9971964.56
10.80 9650577.59 18.04 3715383.92
11.00 4064008.32 7.60 1071788.04
Retention Time Metabolites Molecular Formula Cas No.
5.02 Hydroperoxide, pentyl C5H12O2 74-80-6
Hydroperoxide, heptyl C7H16O2 764-81-8
Ethanol, 2-(2-propenyloxy)- C5H10O2 111-45-5
Benzoic acid C6H5COOH 65-85-0
10.58 Dodecane C12H26 112-40-3
Undecane C11H24 1120-21-4
Isopropyl alcohol C3H8O 67-63-0
Propylene Glycol C3H8O2 57-55-6
L-Lactic acid C3H6O3 79-33-4
10.63 R-(-)-1,2-propanediol C3H8O2 4254-14-2
Tetradecane CH2(CH3)10CH2 629-59-4
Phenol, 2,4 bis (1,1dimethylethyl) C14H22O 96-76-4
Hexadecane, 2,6,11,15 tetramethyl C20H42 504-44-9
Benzaldehyde, 2,4, dimethyl C9H10O 15764-16-6
10.70 2-Propanol, 1-hydrazino- C3H10N2O 18501-20-7
Dodecanoic acid C12H24O2 143-07-7
Methane, nitroso- CH3NO 865-40-7
Tetracosane C24H50 646-31-1
Decane C10H22 124-18-5
10.80 Eicosanoic acid C20H40O2 506-30-9
Hexadecane C16H34 544-76-3
Isopropyl myristate C17H34O2 110-27-0
Geranyl isovalerate C15H26O2 109-20-6
n-Hexadeconic acid C16H32O2 57-10-3
11.00 Ethanamine, 2-propoxy-
Pyrrolo [ 1,2 a ] pyrazine 1,4 dione, hexahydro 3 (2 methylpropyl) C11H18N2O2 5654-86-4
Tetradecanoic acid C14H28O2 544-63-8
9 Octadecenamide, (Z) C18H35NO 301-02-0
Squalene C30H50 111-02-4


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.

Retention Time Peak Area Area % Peak Height
9.75 625128.64 1.03 279968.97
10.33 87526.28 0.14 137242.54
10.42 1067849.36 1.77 1455637.02
10.45 898971.70 1.49 1508377.52
10.49 55699133.56 92.21 13468063.78
10.69 2027689.17 3.36 774610.72
Retention Time Metabolites Molecular Formula Cas No.
9.75 Isopropyl alcohol C3H8O 67-63-0
L-Lactic acid C3H6O3 79-33-4
2-propanol, 1-hydrazino C3H10N2O 18501-20-7
Benzoic acid C6H5COOH 65-85-0
10.33 2-Hydrazinoethanol C2H8N20 109-84-2
Dodecane C12H26 112-40-3
Benzaldehyde, 2,4, dimethyl C9H10O 15764-16-6
Undecane C11H24 1120-21-4
10.42 Propylene glycol C3H8O2 57-55-6
Tetradecane CH2(CH3)10CH2 629-59-4
Dodecanoic acid C12H24O2 143-07-7
Hexadecane, 2,6,11,15 tetramethyl C20H42 504-44-9
10.45 R-(-)-1,2-propanediol C3H8O2 4254-14-2
Hexadecane C16H34 544-76-3
Isopropyl myristate C17H34O2 110-27-0
Decane C10H22 124-18-5
10.49 Methane, nitroso- CH3NO 865-40-7
Phenol, 2,4 bis (1,1dimethylethyl) C14H22O 96-76-4
Geranyl isovalerate C15H26O2 109-20-6
n-Hexadeconic acid C16H32O2 57-10-3
10.69 Ethanamine, 2-propoxy- C5H13NO 42185-03-5
4-Amino-1-butanol C4H11NO 13325-10-5
Pyrrolo [ 1,2 a ] pyrazine 1,4 dione, hexahydro 3 (2 methylpropyl) C11H18N2O2 5654-86-4
9 Octadecenamide, (Z) C18H35NO 301-02-0


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.

Retention Time Peak Area Area % Peak Height
9.72 216757.74 9.37 98425.02
9.79 210048.41 9.08 89220.41
9.82 72569.37 3.14 142459.44
9.86 36228.30 1.57 51748.50
10.13 1586768.57 68.57 739977.40
10.20 191626.29 8.28 115859.62
Retention Time Metabolites Molecular Formula Cas No.
9.72 Methyltartronic acid C4H6O5 595-98-2
L-Lactic acid C3H603 79-33-4
2-propanol, 1-hydrazino C3H10N2O 18501-20-7
Benzoic acid C6H5COOH 65-85-0
9.79 R-(-)-1,2-propanediol C3H8O2 4254-14-2
DL- 2,3 –Butanediol C4H10O2 6982-25-8
Dodecane C12H26 112-40-3
Decane C10H22 124-18-5
Benzaldehyde, 2,4, dimethyl C9H10O 15764-16-6
9.82 Propylene glycol C3H8O2 57-55-6
Tetradecane CH2(CH3)10CH2 629-59-4
Dodecanoic acid C12H24O2 143-07-7
Phenol, 2,4 bis (1,1dimethylethyl) C14H22O 96-76-4
Hexadecane, 2,6,11,15 tetramethyl C20H42 504-44-9
9.86 Isopropyl Alcohol C3H8O 67-63-0
Hexadecane C16H34 544-76-3
Tetracosane C24H50 646-31-1
Tetradecanoic acid C14H28O2 544-63-8
Undecane C11H24 1120-21-4
10.13 Methane, nitroso- CH3NO 865-40-7
Isopropyl myristate C17H34O2 110-27-0
Geranyl isovalerate C15H26O2 109-20-6
n-Hexadeconic acid C16H32O2 57-10-3
Eicosanoic acid C20H40O2 506-30-9
10.20 4-Penten-2-ol C5H10O 625-31-0
Pyrrolo [ 1,2 a ] pyrazine 1,4 dione, hexahydro 3 (2 methylpropyl) C11H18N2O2 5654-86-4
9 Octadecenamide, (Z) C18H35NO 301-02-0
Squalene C30H50 111-02-4

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).

CONCLUSION

Utilization of probiotic bacteria and their metabolites in medicines, dietary supplements, pharmaceutical preparation and food products are enhancing due to their potential health benefits such as anticarcinogenic activities, constipation, reducing lactose intolerance, regulation of immune function, allergenic diseases such as food allergy, etc. These attributes of probiotic lactic acid bacteria have been described by the production of different primary and secondary metabolites that exert their biological effects either directly or by modifying the immune system.

Declarations

ACKNOWLEDGMENTS

Authors are thankful to Hon’ble Chairperson JV’n Vidushi Garg & Hon’ble Founder and Advisor JV’n Dr. Panckaj Garg, Jayoti Vidyapeeth Women’s University, Jaipur (Rajasthan) for their kind cooperation, encouragement and providing the facilities of University Innovation Centre and other laboratories.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.

AUTHORS’ CONTRIBUTION
Ad C conceived and planned the experiments, performed the analysis and wrote the manuscript. K V and B S S supervised the research project.

FUNDING
None.

ETHICS STATEMENT
This research does not contain any studies with human participants or animals performed by any of the authors.

AVAILABILITY OF DATA
The authors declare that [the/all other] data supporting the findings of this study are available within the article.

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