Introduction

The continuous quest for natural compounds for treating human diseases have paved way for the discovery of drugs and high-value pharmacophore leads from the microbes, especially from marine and coastal environment. Explorations of previously unfathomed spaces are required for finding novel therapeutic candidates, and the advances were quite promising. The typical mangrove sediment-associated ecological niches, which harbor novel bacterial strains striving in a stressful habitat, under cold, shady, and high pressure conditions, were found to produce fascinating and structurally complex natural products, which could be isolated and evaluated for the development of potential pharmacophores (Ser et al. 2016). Microbial compounds could successfully be used for treating bacterial infections, inflammation and cancer (National Research Council 1999; Tang et al. 2010; Tabbene et al. 2012). Bioactive compounds from the microorganisms are more accessible in industrial quantities as compared to the plant-derived metabolites.

To date, only a small range of bacteria were investigated for bioactive compounds, yet a huge number of active substances have been isolated that could be used as anticancer, anti-inflammatory, and antimicrobial agents. A significant diversity of natural compounds was derived from the bacteria, which are an unlimited source of potential drugs. The above 120 microbial-derived formulations are present in the clinical use for the treatment of infectious diseases, cancer, and as immune suppressors (National Research Council 1999). They have also been found to possess various therapeutic applications, such as anti-inflammatory formulations (Tang et al. 2010), anti-diabetic adjuvant (Okamura et al. 1993) and antioxidative agents (Tabbene et al. 2012). Many important groups of pharmaceutical compounds were found to be associated with the phylum Firmicutes, such as Bacillus subtilis and B. amyloliquefaciens (Armstrong et al. 2001; Goecke et al. 2010; Thilakan et al. 2016). The investigations of mangrove-associated bacteria for bioactive compounds are higher in Streptomyces species, even though there have been focuses on other less explored strains to develop high-value products (Ser et al. 2016).

The present work aimed to isolate and characterize mangrove sediment-associated bacteria directed with a culture-dependent method to isolate and evaluate their pharmacological properties along with various in vitro models. The bacteria exhibiting significant antioxidant and antimicrobial activities were isolated, identified, and characterized by integrated microbiological, biochemical, and 16S rRNA sequencing. Based on the antibacterial potential against various clinically important pathogens, mangrove sediment-associated B. amyloliquefaciens was used to develop organic extracts, and that were assayed for their antimicrobial activities along with antioxidant potential and anti-inflammatory properties against pro-inflammatory 5-lipoxygenase associated with inflammation. The proton integral of different domains in the organic extracts of the mangrove sediment-associated bacteria were determined to emphasize the significance of electronegative functionalities towards the bioactive properties.

Materials and methods

Sample collection and isolation of mangrove sediment-associated bacteria

A total of ten mangrove sediments were collected from Mangalavanam Mangrove Ecosystem and Bird Sanctuary (Fig. S1), located in Kochi, Kerala at 9° 54′ N 76° 18′ E. The sediment samples were associated with the mangrove species, such as Acanthus ilicifolius and Avicenia officinalis. Isolation was carried out by conventional serial dilution and spread plate techniques in sterile solidified nutrient agar plates (HiMedia, HiMedia Laboratories, LLC, Kelton, PA) (Behera et al. 2014). Briefly, the sediment sample (1 g) was suspended in 9 mL of sterile distilled water and dilutions (10–1–10–7) were made before being plated in the nutrient agar plates supplemented with NaCl (0.5% w/v). The incubation was carried out at 34 °C for 7 days and the well-grown colonies were picked up and subjected to further purification. The bacteria were preserved in nutrient agar slants, stab cultures, and glycerol stocks (50%). They were identified by microbiological, biochemical, and molecular characterization as discussed in the subsequent sections. The potent candidates were selected after preliminary screening and bioactivities were evaluated.

Screening for antibacterial and antioxidant activities

Antibacterial activity of the microbes was analyzed by spot-over-lawn-assay in Mueller–Hinton Agar (HiMedia, Kelton, PA) supplemented with NaCl (1% w/v) (Chakraborty et al. 2014). The isolates were spotted against growth cultures of pathogenic bacteria Vibrio parahemolyticus MTCC 451, Escherichia coli MTCC 443, methicillin-resistant Staphylococcus aureus ATCC 33,592, and Aeromonas caviae MTCC 646 prepared in the nutrient broth at their late log phase. The test pathogenic bacterium methicillin resistant S. aureus ATCC 33592 was obtained from American Type Culture Collection (Manassas, VA, USA), whereas V. parahemolyticus MTCC 451, E. coli MTCC 443, and A. caviae MTCC 646 were maintained in the marine natural product laboratory of Central Marine Fisheries Research Institute, Kochi. They were maintained in the nutrient agar slants supplied with NaCl and in Brain Heart Infusion Broth agar (BHI) as per the manufacturers’ guidelines. The inhibition zone diameter was observed and recorded in every 24 h interval after incubation at 34 °C for 3 days.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a free radical, which was used for screening the antioxidant activity of the studied bacteria. The antioxidant effect is directly proportional to the neutralization of DPPH radical producing a zone of clearance (Velho-Pereira et al. 2015). The bacteria were spotted in the nutrient agar plates supplemented with NaCl along with a sterile Whatman filter paper. DPPH was sprayed into the air-dried filter paper after 48 h of incubation, and observed for the zone of clearance.

Microbiological, biochemical, and molecular characterization of the bioactive isolates

Gram staining was performed to determine the Gram character, cell morphology, size, and arrangement of bacteria. Biochemical characterization of some test strains was performed using HiBacillus identification kit (HiMedia, Kelton, PA). The test kit contained the media for malonate, Vogues Proskauer, citrate, ortho nitrophenyl β-galactoside (ONPG), nitrate reduction, catalase, arginine, sucrose, mannitol, glucose, arabinose, and trehalose utilization tests. Bacteria were inoculated in 5 mL of brain heart infusion broth (BHI) at 35 to 37 °C for 6–8 h until the turbidity was ≥ 1.0 at 620 nm. The kit was opened aseptically and inoculated 50 µL of the test organism before being incubated at 35 to 37 °C for 24–48 h.

Genomic DNAs of the selected bacteria were extracted from 18 h old broth culture supplied with 0.5% NaCl, incubated at 34 °C. DNA was isolated using GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO) and the quality and quantity of the DNA samples were assessed using a nanodrop spectrophotometer (Eppendorf, USA). The 16S region in the DNA was amplified using the forward primer, 8 F (Nandakumar et al. 2007) and the reverse primer, 1492 R (Sigma-Aldrich, MO) (Abada et al. 2019), and some reactions were performed with 27 F (Suganthi et al. 2013) and reverse primer 1544 R (Sigma-Aldrich, MO) (El-Helow 2001). PCR products were sequenced, and Bio-Edit software was used to trim the sequence to remove low quality base pairs and for aligning the sequence, whereas Basic Local Alignment Search Tool (BLASTn) software of the National Centre for Biotechnology Information (NCBI) was used for finding similar sequences in the database. The microbes were identified, and a total of nine sequences were submitted at the NCBI GenBank for acquiring the accession numbers.

The 16S-rRNA-based phylogeny was conceded to confirm the biochemical tests. The sequences were aligned against reference sequences using CLUSTALW program of the BioEdit software. Phylogenetic tree was constructed to demonstrate the evolutionary relationship of the isolated strains against different species in the same genera by neighbor-joining method (Kimura 1980; Saitou and Nei 1987), and an outgroup (NR 024570.1 E. coli strain TU5/41-16S ribosomal RNA) was included. The optimal tree with the sum of branch length of 0.43811664 was shown. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances were used to infer the phylogenetic tree. Evolutionary distances were computed using the Kimura 2-parameter method, and are in the units of the number of base substitutions per site (Nei and Kumar 2000). The analysis involved 14 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 432 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 software (Tamura et al. 2013) The presence of polyketide synthase (pks) gene, which codes for polyketide compounds, was assessed with primers gcf and gcr (Schirmer et al. 2005).

Extraction of bioactive bacterial metabolites

The extracellular bioactive metabolites of the potent strain Bacillus amyloliquefaciens MBMS5 with significant antibacterial and antioxidant activities were extracted with an appropriate organic solvent. In brief, the nutrient agar plates supplemented with NaCl (0.5%) were surface cultured with B. amyloliquefaciens MBMS5 and incubated for 48–72 h at 34 °C for the production of secondary metabolites. The spent agar was exhaustively extracted after removing the surface layer by refluxing on a temperature-controlled water bath with solvent ethyl acetate and further concentrated by a rotary vacuum evaporator (Heidolph, Schwabach, Germany). Ethyl acetate extract (3.9 g) was assayed in vitro for its antioxidant activity and against pro-inflammatory lipoxygenase (5-LOX) associated with anti-inflammatory activity along with disc diffusion assay to assess the antibacterial activity. The scrapped surface culture (intracellular extract) was sonicated at 40% amplitude with a 10 min off and on cycle for 20 min (Cole Parmer, USA).

Antioxidant activity of organic extract and immersion bioautography

Antioxidant activities of the organic extract was assessed by 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS+) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging abilities. DPPH radical scavenging activity was measured by an established method (Brand-Williams et al. 1995). The decolorization was read after 20 min of reaction at an absorbance of 517 nm by a UV–Vis spectrophotometer (Varian Cary 50, Agilent Technologies, Inc., Santa Clara, CA). The ABTS+ assay was performed by a previously described method (Re et al. 1999). The reaction was performed for 6 min, and the absorbance was measured at 734 nm. ABTS+/DPPH scavenging activities were expressed as (A0 − A1)/A0 × 100 (in percentage), wherein A0 and A1 represented the absorbance of the control and the sample, respectively. Scavenging activities of the extracts on ABTS+/DPPH radicals were recorded, and the IC50 values (the concentration at which the sample would inhibit 50% of the radicals/enzyme, µg/mL) was calculated via non-linear regression plot. The α-tocopherol was the standard used in the assessment of antioxidant activity.

Anti-inflammatory activity

The in vitro 5-lipoxygenase (5-LOX) inhibition assay was performed for the assessment of anti-inflammatory activity according to an earlier method (Baylac and Racine 2003). 5-LOX is an enzyme required for the synthesis of pro-inflammatory leukotrienes and lipoxins, whereas the inhibition of the pro-inflammatory enzyme demonstrated the anti-inflammatory activity of the test materials (Gilbert et al. 2011). The reaction was performed for 20 min, and the absorbance was measured at 243 nm by a UV–VIS spectrophotometer. The inhibitory activity of the crude extracts was expressed as (A0 − A1)/A0 × 100 (in percentage), where A0 and A1 represented the absorbance of the control and the sample, respectively. Anti-inflammatory activity of the extracts on 5-LOX were recorded, and the IC50 values (the concentration at which the sample would inhibit 50% of the enzyme, µg/mL) was calculated via the non-linear regression plot. Ibuprofen was the standard used in the assessment of anti-inflammatory activity.

Antimicrobial activity of the crude extract by disc diffusion assay

Agar well diffusion method was used to assess the susceptibility of pathogenic bacteria to the bacterial extracts (Chakraborty et al. 2014). The cultures of V. parahemolyticus MTCC 451, E. coli MTCC 443, methicillin-resistant S. aureus ATCC 33592 and A. caviae MTCC 646 were used as the test pathogens, and the extracts were prepared in 30 µg, 60 µg and 100 µg on the sterile discs. Pathogens were swabbed on Mueller–Hinton agar (MHA), and the extracts prepared in the discs were placed on the medium, whereas chloramphenicol and methanol were used as positive and negative controls, respectively.

Spectroscopic analysis

The proton nuclear magnetic resonance (1H-NMR) analysis of the organic extracts of B. amyloliquefaciens MBMS5 (MK765025) was obtained by Bruker AVANCE III 600 MHz spectrometer (Bruker, Germany). 1H-NMR spectra were distributed into five distinct domains, such as aryl protons (C6H5–H, δH 6.6–8.6), olefinic (–CH=CH–)/protons of the hydride group of alkanoates {–CH2C(=O)OMe, δH 4.6–6.0}, alkane hydrocarbon (primary through tertiary, δH 0.1–2.0), allylic (CH2=CH–Me)/acetyl {–C(=O)Me}/derivatized hydride of alkanoates {–CH2C(=O)O–, δH 2.1–2.5}, methoxyl (–OMe)/functionalized alkanol (–CH2OH, δH 2.6–3.5), and anomeric (due to polysaccharides, δH 3.6–4.5).

Statistical analysis

One-way analysis of variance (ANOVA) was carried out to assess the significant differences between the means of bioactivities. The significant differences were represented as p < 0.05, and the values were presented as the means of triplicates ± standard deviation.

Results

Isolation of mangrove sediment-associated bacteria, antimicrobial, and antioxidant screening

A total of 10 sediment samples associated with the mangrove species A. ilicifolius and A. officinalis were collected and 40 well-grown strains obtained by serial dilution and spread plate techniques were selected for further procedures. The selected strains by spot-over-lawn-assay and preliminary antioxidant screening were further identified by morphological, biochemical, and molecular characterization.

Firmicutes was the predominant class among the bacterial communities with more than 47% of the total isolates screened (Fig. 1). The isolated bacteria were screened against pathogenic bacteria and among the total of 40 isolates, 25 strains, i.e., 62.5% of the isolates were active against at least one of the test pathogens. Antimicrobial spectra of the selected isolates against the pathogenic organisms were shown in the Table 1. A total of five strains exhibited zone of clearance against DPPH representing potential antioxidant properties against the target free radical.

Fig. 1
figure 1

Phylogenetic tree derived for the selected mangrove sediment isolates showing evolutionary relationships of the taxa. Phylogenetic analysis of isolated strains (S2A, S1A, S2B, S2C, S2D, S4A, S10B and S10C) along with the related strains was carried out by neighbor-joining method with the MEGA 7 software. Evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 0.43811664 was shown. Percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) were shown next to the branches. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the Kimura 2-parameter method, and are in the units of the number of base substitutions per site. Rate variation among sites was modeled with a gamma distribution (shape parameter = 1). The analysis involved 14 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 432 positions in the final dataset

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Table 1 Antimicrobial spectrum of the selected isolates by spot-over-lawn-assay
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Morphological, biochemical, and molecular characterization based on the 16S-rRNA phylogeny

The selected isolates, which exhibited potent antimicrobial and antioxidant activities, were further characterized by morphological and biochemical methods along with 16S-rRNA-based phylogeny (Fig. 1). Polymerase chain reaction (PCR) primers used for 16S rRNA amplification and gel profile of the amplified gene of selected mangrove sediment bacteria were presented in Table 2 and Fig. S2. A higher similarity of ≥ 99% was obtained for the isolates of B. amyloliquefaciens MBMS5 (GenBank accession number MK765025), MBMS12 (MN165386); Bacillus cereus MBMS1 (MN204214), MBMS7 (MN204215), MBMS38 (MN240446), MBMS39 (MN165387); Bacillus paramycoides MBMS3 (MN204219), MBMS6 (MN204220); and Bacillus anthracis MBMS8 (MN204226) using BLAST search tool, and the accession numbers were obtained from NCBI-GenBank. The selected bacterium B. amyloliquefaciens MBMS5 with potential antioxidant and antimicrobial activities was selected for further procedures. B. amyloliquefaciens MBMS5 belonged to Gram positive rods (Fig. S3) with the ability to ferment carbohydrates, such as glucose, sucrose, mannitol, arabinose, and trehalose along with the capability to reduce nitrate and exhibited morphology of being white and rhizoidal in margin.

Table 2 Polymerase chain reaction (PCR) primers used for 16S rRNA and pks gene amplification
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Biosynthetic gene clusters like pks was found to be involved in the synthesis of many secondary metabolites, and was found to be responsible for the production of an important biologically active group of compounds in the microorganisms (Thilakan et al. 2016). The occurrences of these gene systems in the microorganisms was found out by targeting specific primers to the pks gene sequence (Ayuso-Sacido and Genilloud 2005). B. amyloliquefaciens MBMS5 (MK765025), which was an antioxidant and antimicrobial producer, was recorded for the presence of pks gene using the primers gcf and gcr (Table 2), wherein the 700 bp fragment obtained was sequenced, and the peptide and frame translations were checked with the EMBOSS Transeq software before being submitted in GenBank under the accession number MN 165388.

Antagonistic activities of the bacterial organic extracts

The crude extracts of B. amyloliquefaciens MBMS5 exhibited potential antibacterial activity against the pathogens A. caviae MTCC 646, V. parahemolyticus MTCC 451 (gastrointestinal infection), E. coli MTCC 443 (gastrointestinal infection) and methicillin-resistant S. aureus ATCC 33592 (skin infection) (Fig. S4) vis-à-vis positive control, chloramphenicol (30 µg), which were represented in Table 3.

Table 3 Antibacterial activities of mangrove sediment-associated B. amyloliquefaciens MBMS5 against pathogenic bacteria for their extracellular organic extracts
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Pharmacological activities of the extracts of B. amyloliquefaciens MBMS5

The extracellular organic extracts exhibited immediate decolorization of spots in free radical (DPPH) by immersion bioautography (Fig. 2), and it was inferred that it could possess higher antioxidant activity than that displayed by the intracellular organic extracts. Organic extracts of B. amyloliquefaciens MBMS5 was assayed in vitro for its antioxidant potential along with the ability to inhibit the pro-inflammatory 5-LOX, which is a significant pharmacological target associated with inflammation. The crude extracts exhibited prospective antioxidant activities as determined by the DPPH and ABTS+ (IC50 3.5 and 3.9 μg/mL, respectively) radical scavenging assays. Notably, the antioxidant activities were significantly greater than those displayed by the commercially available antioxidant α-tocopherol. The inhibitory activity of the organic extract of B. amyloliquefaciens MBMS5 against inducible pro-inflammatory enzyme 5-LOX, which was involved in significant functional roles in the metabolic pathway of inflammation, was significantly higher (IC50 6 μg/mL) than that exhibited by commercially available synthetic anti-inflammatory agent ibuprofen (Table 4).

Fig. 2
figure 2

a Bacterial growth curve of Bacillus amyloliquefaciens MBMS5 at 660 nm. b Zone of clearance exhibited by B. amyloliquefaciens MBMS5 against the free radical DPPH and Bacillus cereus MBMS38 were represented by black and red arrows, respectively. c Spot decolorization by immersion bioautography. The arrows represented the active spots against the free radical DPPH

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Table 4 Pharmacological properties of the extracellular extract of B. amyloliquefaciens MBMS5
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Discussion

Mangrove sediment-associated bacteria were reported to contain biologically active compounds, which could be applied against microorganisms and against oxidants. A significant share of mangrove sediment-associated bacteria with potential bioactive properties form a valuable source of pharmaceutical applications (Bandaranayake 1995). At present, we have isolated 40 bacterial strains affiliated with the mangrove species, A. ilicifolius and A. officinalis. Major share (47%) of the bioactive bacterial isolates was found to belong to the phylum Firmicutes. Mangrove sediment-associated bacteria have gained the attention of many natural product chemists apart from Streptomyces sp. (Sun et al. 2019). It was reported that, among the phylum Firmicutes, Bacillus sp. were reported to possess potential antibacterial and antifungal activities (Penesyan et al. 2010; Chakraborty et al. 2014). One among the most active cultures, B. amyloliquefaciens MBMS5 displayed a broad spectrum of inhibition, being active against the pathogenic bacteria including multidrug-resistant S. aureus. Methicillin-resistant S. aureus (MRSA) was reported to cause nosocomial infections along with increased rates of death and illness (Amalaradjou and Venkitanarayanan 2014).

In the recent times, MRSA had attributed to the multidrug-resistance against variously available antibiotics leading to a serious clinical problem. Even though, they were habitually opportunistic, varied strains were recognized to be destructively pathogenic, and staphylococci was found to inhabit 75–80% adults transiently and about 25% of adults permanently (Archer et al. 2011). The explorations of previously unfathomed spaces for finding novel pharmacophore candidates were quite promising in the era of antibiotic-resistance nosocomial pathogens (Gram et al. 2010). Bacilysocin, a novel antibiotic from Bacillus subtilis strain, showed antifungal activity against Saccharomyces cerevisiae and Candida pseudotropicalis, and antimicrobial activity against S. aureus was reported previously (Tamehiro et al. 2002). Ethyl acetate extracts of the actinobacterial strains belonging to the Streptomyces genus isolated from mangrove sediments of Krishna district in Andhra Pradesh were found to exhibit significant antimicrobial activity against Gram positive bacteria, such as Bacillus subtilis, Bacillus megaterium and S. aureus and Gram negative bacterial isolates, such as Pseudomonas aeruginosa, E. coli, and Xanthomonas campestris (Yasmeen et al. 2016). The compounds, such as tetradecane, 1-tetradecene, octadecane, and cyclotetracosane, which were derived from the extracts of Streptomyces cheonanensis, were found to display potential antimicrobial and antifungal activities (Naragani et al. 2016). According to the previous reports, B. amyloliquefaciens associated with mangrove soil have manifested antibacterial activities against V. parahemolyticus that has been a prominent food pathogen causing gastroenteritis, and various studies on mangrove sediment-associated bacteria proved their antimicrobial potential against different pathogenic organisms (Thongjun et al. 2016; Sun et al. 2019).

Antimicrobial activities of the organic extract of B. amyloliquefaciens MBMS5 was analyzed by disc diffusion assay against pathogens, such as V. parahemolyticus MTCC 451, MRSA ATCC 33592, E. coli MTCC 443 and A. caviae MTCC 646. The extracellular extract displayed significant antimicrobial activity against V. parahemolyticus MTCC 451 with an inhibition zone diameter of 20 mm for 100 µg, as compared to the intracellular extract exhibiting lesser activity (12 mm inhibition zone for 100 µg of the extract), whereas the positive control chloramphenicol produced an inhibition zone of 18–20 mm (at 100 µg). Notably, the extracellular extract exhibited higher activity towards the test pathogen A. caviae MTCC 646 (20 mm for 100 µg of the extract). An inhibition zone of 15–16 mm was apparent against the pathogenic E. coli MTCC 443 by 30 µg of the test extract.

The oxidative attacks by free radicals were found to lead towards crucial diseases, such as cancer, chronic inflammatory diseases and diabetes. Some synthetic anti-oxidant compounds, such as butylated hydroxytoluene (BHT), propyl gallate (PG), tert-butyl hydroquinone (TBHQ) were used to arrest deterioration of food ingredients, even though these compounds could cause potential health hazards, such as carcinogenesis and liver damage (Shahidi 2000). In the recent times, there had been substantial interests in naturally derived antioxidant compounds. Prathiba and Jayaraman (2018) reported significant antioxidant and cytotoxicity against HeLa cell lines by Bacillus VITPS7 isolated from the soil samples of Pichavaram mangrove ecosystem and Marakkanam saltern region in South India.

Naturally derived compounds for treating inflammation are being explored to reduce the side effects produced by many medications. Although non-steroidal anti-inflammatory medications (NSAIDs) have been used to deter the inflammatory pathophysiology, various side effects, such as kidney failure, gastro-intestinal bleeding, stroke, heart attacks, and duodenal ulcers were reported to occur. These deleterious side-effects are liable for many instances of hospitalization and deaths (Sostres et al 2010). Squalene extracted from the Bacillus sp. was proved to possess inhibitory effects on major inflammatory pathways including P38, cyclooxygenases (COX), cytokine tumour necrosis factor-α (TNF-α), inducible nitric oxide synthatase (iNOS), and interleukin (IL)-1β (Thoppil and Bishayee 2011). B. amyloliquefaciens MBMS5 displayed prospective anti-inflammatory property against inducible pro-inflammatory enzyme 5-LOX. It has been reported that organic extract of B. amyloliquefaciens MTCC 12716 displayed potential 5-LOX inhibitory activities (IC90 6.06 μg/mL) (Kizhakkekalam and Chakraborty 2019).

Various oxidation reactions might cause several oxidative stress-induced disorders, such as type-2 diabetes, hypertension and arteriosclerotic vascular disease (Scott 2004). It has been proved that the damage induced by the free radicals could lead to impaired glucose tolerance and type-2 diabetes. Ethyl acetate extract of the Bacillus amyloliquefaciens MBMS5 exhibited potential antioxidant activities (IC50 3.5 and 3.9 µg/mL to quench DPPH and ABTS+ radical species, respectively) coupled with attenuation activity against 5-LOX (IC50 6 µg/mL) (Table 4). These results revealed the promising therapeutic potential of mangrove sediment-associated bacterium B. amyloliquefaciens MBMS5. Notably, the bacteria belonging to Firmicutes represented the predominant phyla, and it comprised greater than 47% of cultivable bacteria against the clinically important pathogens including V. parahemolyticus MTCC 451 and MRSA ATCC 33592.

Proton nuclear magnetic resonance (1H-NMR) analysis of extracellular extracts of B. amyloliquefaciens MBMS5 (Fig. 3) disclosed the possible presence of alkane hydrocarbon (primary through tertiary, δH 0.1–2.0) (proton integral of ∑H 207.57), olefinic (–CH=CH–)/protons of the hydride group of alkanoates with ∑H of 9.65 (δH 4.5–6.5), allylic/acetyl/derivatized hydrides of alkanoates (∑H 20.9), methoxyl/functionalized alkanol (∑H 6.43), anomeric groups (∑H 20.42), which was significantly greater than the intracellular extracts (∑Hs of 73.79, 6.08, 6.73, 1.97, 3.69, respectively) indicating the greater bioactive potentials of the extracellular extract. The mangrove sediment-associated B. amyloliquefaciens MBMS5 isolated in the present study could be a potential therapeutic candidate for the use in the pharmaceutical applications.

Fig. 3
figure 3

1H-NMR spectra of the a extracellular and b intracellular extracts of Bacillus amyloliquefaciens MBMS5

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