Characterization of multicomponent antioxidants from  Haloferax alexandrinus GUSF-1 (KF796625)

Abstract

The present study was aimed to exploit the haloarchaeon Haloferax alexandrinus GUSF-1 (KF796625) for the presence of biomolecules possessing antioxidant activity. The culture produced a bright orange pigment when grown aerobically in nutrient rich medium with 25% crude solar salt. Biomolecules from cell-free supernatant and from the cells of the culture were individually extracted through the assistance of solvents of different polarities, such as ethanol, methanol and hexane, and monitored for scavenging of stable free radicals. Each of the extracts showed varying capacities to scavenge DPPH^•(20, 31, and 80% DPPH^• RSA; 160.19, 248.29 and 640.76 AAE µg g⁻¹ of cells) at 1 mg mL⁻¹. The extracellular ethanolic extract was polysaccharide in nature, equivalent to 47 µg mL⁻¹ of glucose when assayed with the phenol-sulfuric acid method. The Fourier Transform-Infra Red spectroscopy confirmed the characteristic glycosidic peaks between 2000 and 1000 cm⁻¹. Similarly, the glycerol diether moiety separated from hydroxylated methanolysates through thin-layer chromatography scavenged free radicals (10.47% DPPH^• RSA; 80.03 AAE µg g⁻¹ of cells). Further, the hexanolic extract exhibited spectral characteristics of red carotenoids and resolved into distinct compounds when separated by thin-layer chromatography using different developing systems. All separated compounds were positive for the DPPH^• reaction (13–30% DPPH^• RSA; 100–240 AAE µg g⁻¹). Chemical profiling of the hexanolic extract using the high resolution-liquid chromatography–mass spectroscopy–diode array detector analysis confirmed the presence of different carbon length isoprenoids; C₃₀: tetrahydrosqualene, C₄₀: 3-hydroxyechinenone, astaxanthin, canthaxanthin, lycopene, phytofluene, phytoene and C₅₀: bisanhydrobacterioruberin, monoanhydrobacterioruberin, bacterioruberin and haloxanthin. Thus, we conclude that the synergistic actions of all these components contribute to the antioxidant activity of the culture and that the antioxidant activity of the exopolysaccharide, glycerol dither moiety, tetrahydrosqualene, haloxanthin and 3-hydroxyechinenone is recorded as the first report for Haloferax alexandrinus GUSF-1 (KF796625). Therefore, recommended for use in microbial industrial biotechnology.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-020-02584-9.

Introduction

Free radicals from atoms, molecules, or ions with highly unstable unpaired electrons are reported to be produced by oxidative reactions from biota and the environment (Lu et al. 2010). The balance between free radicals and oxidative stress caused by them in a living system is maintained by antioxidants (Wan et al. 2016; Alvares and Furtado 2018). Nutraceuticals, cosmetics, and food industries seek natural antioxidants, because they harbor phenolic components that scavenge free radicals (Utkina et al. 2004; Alvares and Furtado 2018). The  antioxidant potential of marine halophilic eubacterial bionts isolated from several dominant marine invertebrates (sponges, corals, and bivalves) and haloarchaeal bionts are reported by (Velho Pereira and Furtado 2014; Velho Pereira et al. 2015), but their presence in Haloarchaeal cultures from solar salt is relatively less explored.

Reports of multiple biotechnological potentials in Haloarchaea, which belong to the third domain of life, are available (Singh and Singh 2017). Besides, they are known to degrade organic pollutants (Raghavan and Furtado 2000), mineralize manganese (Naik and Furtado 2019), produce extremozymes (Gaonkar and Furtado 2018; Malik and Furtado 2019), and synthesize nanoparticles (Patil et al. 2014).

Haloferax alexandrinus GUSF-1 (KF796625) retrieved from solar salt of Goa, India was selected because it biosynthesized silver nanoparticles (Patil et al. 2014), produced rhodochrosite (Naik and Furtado 2019) and its ability to scavenge free radicals (Alvares and Furtado 2018), all of significance, in the pharmaceutical and biotechnological industry.

This present study, therefore, is a record of the: (1) extraction of the biomolecules from the cells of Haloferax alexandrinus GUSF-1 (KF796625) assisted by the use of solvents such as ethanol, methanol and hexane; (2) the ability of the extracts to scavenge stable DPPH^• free radicals and (3) the use of TLC, UV–visible, FTIR spectroscopy and HR-LC/MS–DAD for separation, identification and chemical profiling of the biomolecules responsible for the antioxidant activity of Haloferax alexandrinus GUSF-1 (KF796625).

Materials and methods

Chemicals and reagents

All chemicals and reagents used were of analytical grade. 1, 1-diphenyl-2-picrylhydrazyl (DPPH^•) was acquired from Sigma-Aldrich and prepared in methanol (purity 97%)/hexane (purity > 95%) as required. Thin-layer chromatography (TLC) sheets (silica gel GF254 plates) used in this study were from Merck, Darmstadt, Germany.

Growth of Haloferax alexandrinus GUSF-1 (KF796625)

Haloferax alexandrinus GUSF-1 (GenBank accession number KF796625), a haloarchaeon, isolated from natural solar salt by Sequeira (1992), was used in this study. Cells were cultured in 5 L of Tryptone Yeast Extract containing 25% crude solar salt (NTYE) (Raghavan and Furtado 2000) at 42 °C and 150 rpm for 6 days (REMI CIS-24 PLUS, India), resulting in a wet cell mass of 107 g. Separation of cell mass from culture broth was achieved using a high-speed centrifuge at 12,857×g and 4 °C (Biofuge-Heraeus Stratos). The supernatant obtained was passed through a 0.2 μm membrane filter (MF-Millipore^™) to give the cell-free supernatant (CFS).

Preparation of ethanolic extract from CFS, hydroxylated methanolysate and hexanolic extract

Cell-free supernatant was subjected to extraction by the Del Gallo and Haegi (1990) method using 96% cold ethanol. After addition, the mixture was kept standing overnight at -20 °C and centrifuged. The pellet obtained was dispersed in sterile distilled water and checked for the presence of total carbohydrate using the phenol–sulfuric acid method, according to Dubois et al. (1956) with glucose as the standard.

Hydroxylated methanolysate of cells was prepared by the Ross et al. (1981) method. One gram of cells was mixed with methanol:toluene:concentrated H₂SO₄ (3:3:1 v/v), heated at 50 °C in a water bath for 18 h, and extraction of the hydroxylated methanolysate was accomplished by adding hexane.

To the remaining cells, methanol was added in minimum light exposure and kept overnight at 4 °C. The cell debris were then separated by centrifuging to obtain a clear cell-free methanolic extract of cells. Components from this were fractionated with hexane and distilled water according to the method of Asker et al. (2002) to obtain the hexanolic extract. The ethanolic extract, hydroxylated methanolysates and the phase fractionated hexanolic extract were concentrated under N₂ gas, wrapped in aluminium foil to avoid light exposure and stored at −20 °C.

Detection of free radical scavenging activity (RSA) of the ethanolic extract, hydroxylated methanolysate and hexanolic extract

Individual extracts at 1 mg mL⁻¹ were checked for RSA by the method of Alvares and Furtado (2018). Each was mixed with 1 mL of 0.2 mM DPPH^• reagent (prepared in hexane or methanol as required; Supplementary Fig. 1) kept in the dark at room temperature (28 ± 2 °C), for 30 min and the absorbance checked at 517 nm, against hexane/methanol as the reference solution.

The % radical scavenging activity was calculated using Eq. 1;

$$\%DPPH\bullet \text{RSA}=\frac{\text{AB}-\text{AS}}{\text{AB}}\times 100,$$ 1

where AB is the absorbance of 0.2 mM DPPH^• in the respective solvent (hexane or methanol). AS is the absorbance of the reaction mixture (DPPH^• + extract). Ascorbic acid was used as the standard (R² = 0.9835) and results were also expressed as Ascorbic Acid Equivalent (AAE) µg g⁻¹ of cells. The individual reaction mixtures were then scanned between 190 and 800 nm after 30 min of incubation.

Characterization of ethanolic extract, hydroxylated methanolysate and hexanolic extract

UV–Vis spectroscopy

The ethanolic and the fractionated hexanolic extracts were individually scanned between 190 and 800 nm using a UV–visible spectrophotometer (UV-1601, Shimadzu, Kyoto, Japan) and against distilled water/hexane as the reference solution, as required.

TLC analysis

Components in the hydroxylated methanolysate were chromatographed on four different TLC sheets and resolved separately in the developing system consisting of petroleum ether (60–80 °C):diethyl ether (85:15, v/v). The first chromatogram was placed in an iodine chamber to check for colorless compounds (Kushwaha et al. 1974). The second was sprayed with 10% dodecaphosphomolybdic acid and heated at 150 °C for 15 min (Ross et al. 1981). Detection of free radical scavenging activity was carried out by spraying the third chromatogram with 0.2 mM DPPH^• and incubating in the dark. The fourth chromatogram was left untreated.

Similarly, components in the hexanolic extract were chromatographed and resolved using different developing systems: acetone:petroleum ether (60–80 °C) (20:80, v/v), methanol:chloroform (7:93, v/v) (Asker et al. 2002), acetone:hexane (50:50, v/v) (Ronnekleiv and Liaaen-Jensen 1995), and n-heptane:benzene (90:10, v/v) (Tornabene et al. 1969), respectively.

In each case, sets of three different TLC sheets were used. In each set, spotting on a single chromatogram was done at a distance of 1.5 cm apart. After resolving in respective developing system, all the chromatograms were first air-dried at room temperature in the dark. Visible compounds, if any that were separated, were noted. The chromatogram developed in n-heptane:benzene (90:10, v/v) was visualized with iodine vapors. In each case, the detection of free radical scavenging activity was carried out by spraying the second set of chromatograms with 0.2 mM DPPH^•. The third set was left untreated. The retention factor, R_f, of the resolved spots that decolorized the purple DPPH^• was recorded and compared with that reported in literature using identical developing systems.

Select spots from individual chromatograms that were positive for the DPPH^• activity, were scrapped off from the respective resolved untreated chromatogram. Under minimum light, each compound was individually eluted out in the respective solvent, concentrated, and weighed. Each elute was checked for their RSA and UV–Vis spectral characteristics.

Quantification of the purified colored compound was carried out using Eq. 2 (Kushwaha et al. 1974) and as reported by Raghavan and Furtado (2005):

$$\text{Conc}\left(\mu \text{g/g of cellular protein}\right)=\frac{\mathit{AY}}{E^{1\%}},$$ 2

where A is the absorbance at λ_max,  Y= volume in mL (in this case, 1 mL cuvette was used). E^1% = is the extinction coefficient specific for each separated compounds (Britton 1995). C₅₀ carotenoids—2450 at λ₄₉₀ (Kushwaha et al. 1974); Canthaxanthin—2092 at λ₄₇₆ (Warren and Weedon 1958); 3-hydroxyechinenone—2100 at λ₄₇₆ (An et al. 1989).

Fourier Transform Infrared (FTIR) spectroscopy

The ethanolic extract, the compound resolved on silica plate at R_f 0.2 from the hydroxylated methanolysate and hexanolic extract were individually mixed with KBr to form a pellet. Samples were then analyzed in the 4000 and 400 cm⁻¹ spectral region at a resolution of 4.0 cm⁻¹ using the FTIR spectrometer (Shimadzu IR Prestige-21).

High resolution-liquid chromatography–mass spectroscopy–diode array detector (HR-LC/MS–DAD) analysis

Chemical profiling of the constituents in hexanolic extract was achieved using a HR-LC/MS–DAD instrument; Agilent 1290 Infinity UHPLC system, 1260 Infinity Nano HPLC with Chipcube series (Agilent Technologies, USA) and a hypersil gold 3 microns 100 × 2.1 mm C-18 reverse-phase column from Agilent Technologies. The ESI interface consisted of a binary pump, an autosampler and 6550 i funnel QTOF-LC/MS. The two mobile phases used were: (A) 0.1% (v/v) formic acid in water and (B) 70% acetonitrile in 30% (v/v) methanol. The flow rate was adjusted for 0.3 mL/min at 25 °C. The UV–Vis spectrum was obtained between 190 and 600 nm and the chromatograms were processed at 320, 447 and 490 nm, respectively. Separated compounds were identified by comparing the obtained m/z values with the Agilent Mass Hunter Qualitative Analysis Software version (B.05.00), Japanese carotenoid database, Pubchem and earlier reported literature.

Results and discussion

Cells of Haloferax alexandrinus GUSF-1 (KF796625)

Haloferax alexandrinus GUSF-1 (KF796625) produced a bright orange pigment when grown aerobically in 25% NTYE broth and a cell mass of 107 g was recovered from the fully grown culture (Supplementary Fig. 2). The colonies of the culture have been reported to scavenge free radicals by the agar growth method in a previous study (Alvares and Furtado 2018).

Characterization of ethanolic extract

Ethanolic extracts from the cell-free supernatant resulted in a white precipitate soluble in distilled water and exhibited a value of 20 ± 0.4% DPPH^• RSA (160.19 AAE µg g⁻¹ of cells). The UV spectrum of this precipitate showed a single peak at 193 nm (Fig. 1a). The spectrum of the reaction mixture revealed that both peaks of the pure DPPH^• radical seen at 517 and 325 nm were abolished along with the single peak at 193 nm during the decolorization process suggesting its involvement (Fig. 1b). In the FTIR spectrum, absorption peaks were seen between 3900 and 500 cm⁻¹ (Fig. 1c). Characteristic peaks were also seen at 3317 cm⁻¹ for the OH group, at 2912 cm⁻¹ indicating the presence of the C-H group and at 1625 cm⁻¹ for the carbonyl group. A broad stretch at 1041 cm⁻¹ followed by an intense peak at 1124 cm⁻¹ is indicative of the C–OH side groups and the C–O–C glycosidic band vibrations (Zhang et al. 2011). The ethanolic extract tested positive for carbohydrates with 47 mg mL⁻¹ equivalent of glucose, hence, polysaccharide in nature (Supplementary Fig. 3).

Fig. 1.

Characterization of ethanolic extract from Haloferax alexandrinus GUSF-1 (KF796625). a UV spectrum showing a single peak at 193 nm, b DPPH^• decolorization of ethanolic extract monitored by the spectrophotometer; which shows that both the peaks of DPPH^• (325 and 517 nm) are abolished by the ethanolic extract, indicating the free radical scavenging capacity, c FTIR spectrum revealing the characteristic glycosidic peaks between 2000 and 1000 cm⁻¹

Little is reported about exopolysaccharide (EPS) production from haloarchaea with Antón et al. (1988), first reporting the production of EPS by the Haloferax mediterranei (ATCC 33500). Antioxidant activity of EPS has been reported in the Haloterrigena turkmenica by Squillaci et al. (2016), at a value three times lower than our activity, although assayed at the same concentration.

Characterization of hydroxylated methanolysate

The hydroxylated methanolysate extract was pale yellow and of oily consistency and gave a value of 31 ± 0.24% DPPH^• RSA (248.29 AAE µg g⁻¹ of cells). The chromatogram revealed the presence of 6 spots in iodine chamber at R_f 0.2, 0.3, 0.48, 0.59, 0.78 and 0.89, respectively (Fig. 2a). On spraying with 10% dodecaphosphomolybdic acid, a blue spot was visualized at R_f 0.2 (Fig. 2b). Interestingly, the compound at R_f 0.2 decolorized DPPH^• on the chromatogram and on the spot test (Fig. 2c, d) and the RSA of the compound after elution in hexane, gave a value of 10.47 ± 0.21% DPPH^• RSA (80.03 ± 0.32 AAE µg g⁻¹ of cells) (Table 1). These results also confirmed our earlier report (Alvares and Furtado 2018). The UV spectrum exhibited absorption maxima at 256, 263, and 270 nm (Table 1). Notable in the FTIR spectra was the complete absence of ester absorption peaks between 1730 and 1750 cm⁻¹. However, the spectra did show strong absorption bands indicative of long-chain groups (2960 cm⁻¹), OH groups (3300 cm⁻¹), ether C–O–C groups (1120 cm⁻¹) and the isopropyl units between 1365 and 1420 (Fig. 3). This spectrum matched with that reported for the purified glycerol diether moiety from Halobacterium cutirubrum NCMB 763 confirming the identity as glycerol diether moiety (GDEM), a compound that is unique to haloarchaea and which distinguishes it from the eubacterial domain (Ross et al. 1981,1985). Furthermore, the result was also consistent with reports by Braganca and Furtado (2009) and Gaonkar and Furtado (2018). Though GDEM is reported to protect Archaea and haloarchaea against hydrolysis of their membrane phospholipids, their role as scavengers of DPPH^• observed by us in this study is the first record.

Fig. 2.

Characterization of hydroxylated methanolysate from cells of Haloferax alexandrinus GUSF-1 (KF796625): The thin-layer chromatogram was resolved in petroleum ether (40–60 °C): diethyl ether (85:15,v/v) and developed with a iodine vapor, b 10% dodecaphosphomolybdic acid in absolute ethanol and heated at 150 °C, c decolorization of hexanolic DPPH^• solution by compound at R_f 0.2 and d DPPH^• spot assay

Table 1.

Characterization and quantification of glycerol diether moiety extracted from cells of Haloferax alexandrinus GUSF-1 (KF796625) and separated using thin-layer chromatography

Solvent system	Rf of compound with antioxidant activity	DPPH• reaction	Quantity obtained after TLC separation (mg 100 g−1 cells)	% DPPH• RSA; AAE µg g−1 of cells (mean ± SD)	UV–visible absorption spectrum	UV–visible absorption spectral peaks (nm)	Identification
Petroleum ether(60–80 °C): Diethyl ether (85:15, v/v)	0.2	+	6.66	10.47 ± 0.21; 80.03 ± 0.32		256, 263, 270	Glycerol diether moiety (GDEM)

Fig. 3.

FTIR spectrum of glycerol diether moiety extracted from hydroxylated methanolysate from cells of Haloferax alexandrinus GUSF-1 (KF796625)

Characterization of hexanolic extract

The hexanolic extract was bright orange, with absorption peaks both in the UV (190–300 nm) and the visible (300–700 nm) range (Fig. 4a, b). These peaks corroborated with red carotenoids and their derivatives, as reported earlier for the culture (Alvares and Furtado 2018) and also reported for Haloarchaea by Britton et al. (1995). The hexanolic extract decolorized the deep purple-colored stable DPPH^• to straw yellow and showed a highly potent value of 80 ± 0.32% DPPH^• RSA (640.76 AAE µg g⁻¹) (Fig. 4c). The FTIR spectral analysis revealed intense absorption peaks at 3462 cm⁻¹ and stretching vibrations were seen at 2924 cm⁻¹, while absorption bands were observed at 1735, 1637, 1220, and 990 cm⁻¹ (Fig. 5). The constituents of the hexanolic extract resolved by TLC were found to be all positive for the DPPH^• reaction with values in the range of 13–30% DPPH^• RSA; 100–240 AAE µg g⁻¹. The TLC profiles, spectroscopic characteristics and quantification data obtained using the individual elutes' absorption coefficients are recorded in Fig. 6 and Tables 2 and 3.

Fig. 4.

Decolorization of DPPH^• by hexanolic extract from cells of Haloferax alexandrinus GUSF-1 (KF796625). a Bright orange hexanolic extract, b UV–Vis absorption spectrum of hexanolic extract with peaks in the UV and visible region and c the deep purple color of DPPH^• (left) is decolorized by the hexanolic extract rendering it straw yellow (right), an indication of its potent free radical scavenging capacity

Fig. 5.

FTIR spectrum of hexanolic extract from cells of Haloferax alexandrinus GUSF-1 (KF796625)

Fig. 6.

Thin-layer chromatograms of hexanolic extract from cells of Haloferax alexandrinus GUSF-1 (KF796625) developed in a acetone:petroleum ether (20:80; v/v), b methanol:chloroform (7:93; v/v), and c acetone:hexane (50:50; v/v) and viewed as (1) visible spots and (2) after spraying with DPPH^•. The hexanolic extract was also resolved in d n-heptane: benzene (90:10, v/v) and viewed as (1) visible spots, (2) after spraying with DPPH^• and (3) exposed to iodine vapor

Table 2.

Separation of biomolecules from hexanolic extract of Haloferax alexandrinus GUSF-1 (KF796625) achieved using thin-layer chromatography and different developing systems

Solvent system	Rf of separated carotenoids	DPPH• reaction
Acetone–petroleum ether (20: 80, v/v)	0.82	+
	0.72	+
	0.63	+
	0.53	+
	0.51	+
	0.47	+
	0.4	+
	0.36	+
	0.14	+
Methanol–chloroform (7:93, v/v)	0.71	+
	0.43	+
	0.29	+
	0.24	+
Acetone: hexane (50:50, v/v)	0.71	+
	0.58	+
	0.46	+
n-heptane: benzene (90:10, v/v)	0.9	+

Table 3.

Purification and quantification of the antioxidant compounds separated from the hexanolic extract of Haloferax alexandrinus GUSF-1 (KF796625)

Solvent system	Rf of separated compounds with antioxidant activity	Quantity obtained after TLC separation (mg 100 g−1 cells)	% DPPH• RSA; AAE µg g−1 of cells (mean ± SD)	UV–visible absorption spectrum of pure compound	UV–visible absorption spectral peaks (nm)	Quantification of the purified compound using molar extinction coefficient (µg/g of cellular protein) (Kushwaha et al. 1974)	Identification
Acetone–petroleum ether (20:80, v/v)	0.14	8	30 ± 0.04; 240.02 ± 0.5		368, 387, 463, 492, 525	2	Bacterioruberin
	0.36	5.6	17.51 ± 0.1; 140.0 ± 0.21		369, 388, 458	1.009	3-hydroxyechinenone
	0.53	2.93	14.56 ± 0.18; 110.6 ± 0.36		374, 476	2.151	Canthaxanthin
Methanol–chloroform (7:93, v/v)	0.24	8.53	28 ± 0.12; 220.4 ± 0.06		368, 386, 463, 492, 525	2	Bacterioruberin
	0.29	5.06	23.24 ± 0.20; 180.6 ± 0.19		369, 389, 464, 490, 522	0.36	Bisanhydrobacterioruberin
	0.43	4.53	18.49 ± 0.09; 140.8 ± 0.01		368, 387, 464, 489, 522	0.534	Monoanhydrobacterioruberin
Acetone:hexane (50:50, v/v)	0.46	3.73	13.26 ± 0.07; 100.6 ± 0.14		377, 451, 478, 512	0.3151	Haloxanthin
n-Heptane:benzene (90:10, v/v)	0.9	8	26.04 ± 0.12; 200.85 ± 0.1	–	–	–	Tetrahydrosqualene

This separation attained by TLC supported and confirmed the presence of canthaxanthin,  3-hydroxyechinenone, bacterioruberin, bisanhydrobacterioruberin, monoanhydrobacterioruberin, haloxanthin and tetrahydrosqualene. These results are in good agreement and consistent with that reported for Haloferax alexandrinus Strain TMT (Asker et al. 2002), Haloferax volcanii culture (Ronnekleiv et al. 1995) and in Halobacterium cutirubrum as described by Tornabene et al (1969).

Chemical profiling of hexanolic extract

The chemical profile of hexanolic extract by HR-LC/MS–DAD showed 12 peaks, which, in the increasing order of their retention time, eluted a total of 17 different compounds, with different m/z values (Fig. 7a). The diode array detector (DAD) revealed that peaks between R_t 29.943 and 31.68 absorbed at 320, 447, and 490 nm (Fig. 7b–d). These compounds were therefore, confirmed as red carotenoids. A combination of the DAD analyses (UV–Vis), the Agilent Mass Hunter software, the Japanese carotenoid database, and PubChem was used to identify the different compounds according to their m/z values. Compounds thus identified were canthaxanthin, tetrahydrosqualene, 3-hydroxyechinenone, phytoene, astaxanthin, bisanhydrobacterioruberin, monoanhydrobacterioruberin, bacterioruberin, lycopene, haloxanthin, and phytofluene, whose mass spectral data are presented in Table 4.

Fig. 7.

Chemical profile of hexanolic extract from cells of Haloferax alexandrinus GUSF-1 (KF796625). a HR-LC/MS–DAD chromatogram of hexanolic extract revealing a total of 12 peaks labeled according to the increasing retention times (R_t). Identification of the separated compounds was achieved by comparing the m/z values obtained with the Agilent Mass Hunter Qualitative Analysis Software version (B.05.00), Japanese carotenoid database, Pubchem, and reported literature. Diode Array Detector response at b 320, c 447 and d 490 nm

Table 4.

Mass spectral analysis of the hexanolic extract of Haloferax alexandrinus GUSF-1 (KF796625)

Peak no	Retention time a	(m/z)a	za	Iona	Abundancea (conc)	Massa	Molecular formulab	Identificationb
1	1.121	202.17	1	(M + H) +	1.12 × 107	201.17	C11H23NO2	Unidentified
2	4.528	302.19	1	(M + H) +	0.07 × 107	301.18	C16H23N5O	Unidentified
3	6.823	565.3	1	(M + H) +	0.51 × 107	564.3	C40H52O2	Canthaxanthin
4	9.801	430.24	1	(M + H) +	0.31 × 107	429.23	C22H31N5O4	Unidentified
5	20.523	415.20	1	(M + H) +	0.37 × 107	414.20	C30H54	Tetrahydrosqualene
6	23.457	301.13	1	(M + Na) +	0.02 × 107	278.15	C16H22O4	Unidentified
7	25.471	568.45	1	(M + H) +	0.03 × 107	567.45	C40H56O2	3-hydroxyechinenone
		595.38	1	(M + Na) +	0.08 × 107	572.39	C40H60O2	Unidentified
		546.40	1	(M + H) +	0.1 × 104	545.39	C40H64	Phytoene
		597.4	1	(M + H) +	2 × 104	596.6	C40H52O4	Astaxanthin
8	27.226	337.23	1	(M + Na) +	0.04 × 107	314.24	C18H34O4	Unidentified
9	29.943	705.55	1	(M + H) +	0.3 × 104	704.55	C50H72O2	Bisanhydrobacterioruberin
		723.57	1	(M + H) +	0.9 × 104	722.55	C50H74O3	Monoanhydrobacterioruberin
		741.58	1	(M + H) +	0.03 × 107	740.57	C50H76O4	Bacterioruberin
10	30.209	537.4	1	(M + H) +	1 × 103	536	C40H56	Lycopene
11	31.68	739.56	1	(M + H) +	0.07 × 107	738.55	C50H74O4	Haloxanthin
12	35.658	543.2	1	(M + H) +	2.1 × 103	542.2	C40H62	Phytofluene

^aThe hexanolic extract was chromatographed on a C-18 reverse-phase column; Hypersil gold 3 micron 100 × 2.1 mm column. Peaks are labeled based on increasing retention times (R_t) that they are eluted out

^bIdentified using the support system; Agilent Mass Hunter software, the Japanese carotenoid database, and Pubchem

Tetrahydrosqualene (C₃₀ isoprenoid) attributes to stabilize the membrane structure in Halobacterium cutirubrum as reported by Tornabene et al. (1969) and as observed in this study, is recorded for its first-time role as a free radical scavenger (antioxidant). The C₄₀ isoprenoids detected were  3-hydroxyechinenone, astaxanthin, canthaxanthin, lycopene, phytofluene and phytoene. Although all of these are reported in Haloferax alexandrinus strain TMT (Asker et al. 2002), but their biological property and role is not characterized. The antioxidant activity for astaxanthin, canthaxanthin, and lycopene is a known feature, but we demonstrated and recorded, for the first time, the free radical scavenging activity of  3-hydroxyechinenone. Yang et al. (2015) delineated phytofluene and phytoene as precursors in the biosynthetic pathway of C₅₀ carotenoids in Haloarchaea. Phytoene detected in very low concentrations than the other chemical constituents in this study is consistent with that reported by Rodrigo-Baños et al. (2015).

The C₅₀ isoprenoids identified were bacterioruberin along with its precursors; monoanhydrobacterioruberin and bisanhydrobacterioruberin. All of these decolorized DPPH^•, confirming their antioxidant activity. Involvements of these carotenoids in free radical scavenging activity in a whole-cell extract of Haloterrigena turkmenica are known and reports are available (Squillaci et al. 2017). A dodecane C₅₀ carotenoid, haloxanthin with a peroxide group detected in the hexanolic extract, has been identified in Haloferax volcanii (Ronnekleiv et al. 1995), but not in Haloferax alexandrinus GUSF-1 (KF796625) and its role in scavenging free radicals as observed in this study, is a novel report.

Conclusively, the present study is the first report of the antioxidant activity of exopolysaccharide, glycerol diether moiety, tetrahydrosqualene, haloxanthin, and  3-hydroxyechinenone of Haloferax alexandrinus GUSF-1 (KF796625). The synergistic actions of all of these components contribute to the antioxidant activity of the culture. The minimum aseptic conditions required to obtain cell mass possessing such high antioxidant ability gives it an economic advantage over other eubacterial counterparts for large-scale production. Therefore, the haloarchaeon, Haloferax alexandrinus GUSF-1(KF796625) is recommended for use in microbial industrial biotechnology.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Author IJ Furtado designed the experiments, analyzed the observations, and formulated the MS along with JJ Alvares who alone carried out all the experimental benchwork.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.