Antibacterial and Antioxidant Activity of Metabolites From Bioconverted Docosahexaenoic Acid Using Gut Bacteria

Docosahexaenoic acid (DHA) is an essential fatty acid necessary for brain development in both infants and adults. However, the role of gut microbiome and their metabolites produced from DHA remain unclear. In present study, the bacterial isolates Lactobacillus spp. Clostridium spp. Escherichia coli; Staphylococcus spp. Enterococcus spp. were used to convert the metabolites from DHA with SM medium supplemented with 200mg of DHA as substrate. The metabolites were extracted after 24 hours of incubation at 37°C and analyzed by GC/MS. The antimicrobial activity of these metabolites conrmed their effectiveness against clinical pathogens such as Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis and Staphylococcus aureus. obtained from the SKS Clinical Laboratory, Salem, Tamil Nadu, India. The Gentamycin containing discs (Nam KhoaBioTek Company, Vietnam) (10µg/mL) were used as positive controls and negative controls were sterilized distilled water containing paper discs. 900µg of the bio-converted DHA extract was weighed and dissolved in 10mL Dimethyl sulfoxide (DMSO) to obtain a concentration of 90µg/mL of the extract; this was the initial concentration of the extract used to determine the antimicrobial activity of the extract. Mueller Hinton agar was prepared by following the medium sterilized at 121ºC for 15 min; the sterilized medium was then poured into sterile petri dishes. The plates were allowed to cool and solidify. The sterilized medium was seeded with 0.1mL of the standard inoculum was spread evenly over the surface of medium with a sterile swab. Wells were bored into the solidied inoculated using a standard broth borer of 6 mm in diameter. 0.1mL of the solution of the extract of concentration 90µg/mL was then introduced into each well on the medium. The inoculated medium was then incubated at 37ºC for 24 h after which each plate was observed for the zone of inhibition of growth which was measured with a transparent ruler and the result recorded in millimeters.

Introduction DHA (docosahexaenoic acid) is an important nutrient needed during critical life stages (such as lactation) and for immunity [1]. It is vital during pregnancy and infancy because it plays a critical role in the brain development [2]. DHA accounts for over 90% of the omega-3 fatty acids in human brain, as well as up to 25% of its overall fat content.It is primarily found in cell membranes, where it improves the uidity of the membranes and gaps between cells. This allows nerve cells to transmit and receive electrical signals more easily [3]. Therefore, adequate levels of DHA appear to make nerve cell communication simpler, faster, and more effective. Low levels in the brain or eyes may cause communication between cells to slow down, resulting in blurry vision or impaired cognitive function.DHA is essential for brain tissue growth and function, especially during infancy and development [4]. For the eyes and brain to grow normally, it accumulates in the central nervous system. The baby's DHAlevels are determined by its ingestion during the third trimester of pregnancy, with the largest accumulation occurring in the brain during the rst few months of life [5]. Antibacterial activity is exhibited by DHA derivatives through a variety of mechanisms, all of which mainly involve bacterial cell membrane perturbation.DHA has anti-in ammatory properties and prevents tumorigenesis, in addition to its antimicrobial and antiviral properties [6,7,8,9]. Following cerebral ischemia, the nerve cell membrane releases DHA, which induces oxidative stress and neurotrophin activation, and is metabolized to NPD1-like DHA, which prevents ischemic nerve cell death. These ndings suggested that DHA combined with NPD1 could prevent ischemic neuronal damage [10].
Later, DHA having a higher unsaturation level than EPA, was demonstrated to have an inhibitory effect on gram negative bacteria that surpasses that of EPA [11]. Long chain fatty acids are well known to be inhibitory on gram positive bacteria even at low concentrations. However, gram negative bacteria are known for their complex lipopolysaccharide layer as compared to the gram positive bacteria. But, PUFA are known to have inhibitory effect on these strains as compared to saturated fatty acids as they are readily incorporated into the outer cell membranes of these organisms, where they signi cantly increase membrane uidity. It is possible that by opening up permeability channels, the concentration gradients necessary between the organism and its environment may be dissipated resulting in fatality of the organism [12].Among these, DHA is one of the most effective fatty acid compounds. In addition to its documented antimicrobial and antiviral properties, DHA possesses anti-in ammatory activity and inhibit tumorigenesis [6,7,8,9]. Although microbial bioconversion of EPA and DHA was reported but the antimicrobial activity of bioconversion products have not been investigated so far [11]. The microbicidal activity of selected LCUFAs and their derivatives has been reported on various enveloped viruses, parasitesand pathogenic bacteria such as Pseudomonas aeruginosa, Bacillus subtilis, Listeria monocytogenes, Helicobacter pylori, Staphylococcus aureus and Neisseria gonorrhea [6,11]. Fish oil decreases the proliferation of tumor cells, whereas arachidonic acid, a long chain n-6 fatty acid, increases their proliferation [13]. Therefore, in this study, we investigated whether the antimicrobial properties of DHA derivatives against clinical isolates, thereby supporting their usein the treatment of chronic infection in patients.

Growth Media
The following ingredients were present in the Sole Carbon Source (SCS) media used to grow normal gut ora (in grams per liter): 5 g (NH 4  The pH wasadjusted to 5.5 using HCL, and the media was sterilized through a 0.22µm lter. Solid medium was prepared byadding 15 g agar per liter of liquid SCS media followed byautoclaving at 121°C [14].

Fecal Sample Collection
The stool samples were taken from four healthy volunteers ranging in age from 20 to 75 years' old who lived in the same neighborhood. Within 15 h of collection, samples were sent to the researchers, packed with para lm, then transported and stored in ice.

Isolation and Characterization of Gut Microbial Flora from Human Fecal Sample
Approximately 125mg of fecal material was dissolved in 5mL of SCS media. To avoid the transfer of residual alternative carbonsources present in the original inoculum, samples were transferred (2.5µL) into fresh SCS media (5mL) and incubated at 37°C for 24-48 h. The isolatesfrom the liquid cultures were obtained by plating cultures on an SCS agar medium plate and incubated at 37°C for 24-48 h [14]. The singlecolonies were picked and re-streaked on SCS plates and further isolates were identi ed by morphological and biochemical characteristics.

Bioconversion of Docosahexaenoic Acid
Bioconversion was described previously [11] and carried out in ve set 50 mL of SM broth with supplement of DHA tablet which contains 200mg of DHA were added to 24 h old cultures of ve different gut bacteria to the ve set of SM broth individually and followed by continued incubation for an 24 h at 37° C and bioconversion was allowed to proceed.
Extraction of Fatty Acids from Bio-converted Broth Bio-converted broth were suspended in 3mL of 4 molL − 1 sodium hydroxide, and incubated at 90°C for 90 min. After cooling, the pH of the sample was adjusted to 2 with Hydrochloric acid. Fatty acids were then extracted by adding 2 mL anhydrous diethyl ether and separated by centrifugation at 5500 × g for 10 min. The upper phase was removed and dehydrated by adding anhydrous sodium sulfate. The dehydrated fatty acids were collected and dried under a stream of nitrogen. Next, 50 µLbistrimethylsilyltri uoroacetamide (BSTFA) was added, and the mixture was incubated at 70°C for 30 min and dried under a stream of nitrogen. The fattyacids were dissolved in 100 µL hexane for GC/MS analysis [15].

GC-MS Analysis
Fatty acid composition analysis was performed on the Shimadzu GCMS QP 2020 that employed a fused silica column, packed with SH-Rxi-%Sil MS (30 m × 0.25 mm ID × 250µmdf) and the components were separated using Helium as carrier gas at a constant ow of 1 ml/min. The injector temperature was set at 280°C during the chromatographic run.1µL of extract sample injected into the instrument the oven temperature was as follows: 40°C (2 min); followed by 280°C at the rate of 10°C min − 1 and 280°C, where it was held for 3 min. The mass detector conditions were: transfer line temperature 280°C; ion source temperature 230°C; and ionization mode electron impact at 70 eV, a scan time 0.2 s and scan interval of 0.1 s. The fragments from 40 to 550 Da. The spectrums of the components were compared with the database of spectrum of known components stored in the GC-MS NIST (2017) library.
Antibacterial activity of bio-converted DHA extract The antimicrobial activity of the compound was determined using agar-well diffusion method. The microorganisms Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis and Staphylococcus aureus were obtained from the SKS Clinical Laboratory, Salem, Tamil Nadu, India. The Gentamycin containing discs (Nam KhoaBioTek Company, Vietnam) (10µg/mL) were used as positive controls and negative controls were sterilized distilled water containing paper discs. 900µg of the bio-converted DHA extract was weighed and dissolved in 10mL Dimethyl sulfoxide (DMSO) to obtain a concentration of 90µg/mL of the extract; this was the initial concentration of the extract used to determine the antimicrobial activity of the extract. Mueller Hinton agar was prepared by following the medium sterilized at 121ºC for 15 min; the sterilized medium was then poured into sterile petri dishes. The plates were allowed to cool and solidify. The sterilized medium was seeded with 0.1mL of the standard inoculum was spread evenly over the surface of medium with a sterile swab. Wells were bored into the solidi ed inoculated using a standard broth borer of 6 mm in diameter. 0.1mL of the solution of the extract of concentration 90µg/mL was then introduced into each well on the medium. The inoculated medium was then incubated at 37ºC for 24 h after which each plate was observed for the zone of inhibition of growth which was measured with a transparent ruler and the result recorded in millimeters.

Isolation and Characterization of Gut Microbial Flora from Human Fecal sample
There were 65 colonies from the human fecal sample. Based on colony morphology, 16 bacteria wereselected for further biochemical characteristics [ Table 1]  Table 2, Table 3, Table 4, Table 5, and Table 6].
Anti-bacterial activity of crude extract from bioconverted DHA From the GC-MS analysis, compounds from bioconverted DHA have varies medicinal properties and most of the compounds have similar property of antimicrobial activity. To ensure that we evaluate the antibacterial activity for their extract. The antibacterial activity of bioconverted DHA extract was presented in [ Table 7]. Among 5 tested microorganisms, the extract featured the strongest antimicrobial effect of Bi dobacterium spp., Eschericia coli, Citrobacter spp. Bioconverted DHA extractagainstB. cereus with the diameter of inhibition zone about 25mm, 26mm, 24mm. Bi dobacterium spp., Lactobacillus spp.,Eschericiacoli,Citrobacterspp.,Enterobacter spp.bioconverted DHA extract showed activity againstStaphylococcus aureus (20mm, 19mm, 18mm, 20mm), Escherichia coli(15mm, 16mm, 14mm, 15mm, 17mm), Pseudomonas aeruginosa (19mm, 18mm, 20mm, 19mm, 20mm), and Salmonella enteritidis (16mm, 15mm, 14mm, 15mm, 13mm). There was no activity found against the DMSO and distilled water. The ndings suggest the potential application of bioconverted DHA extract as the antimicrobial agent, because the diameter of inhibition zone of extract were near to that of positive controls.

Discussion
Lactobacillus spp., Clostridium spp., Escherichia coli, Staphylococcus spp.,andEnterococcus spp. have multiple roles in the human body which stabilize the gastric acid, hepatic bile and digestive enzymes of gastrointestinal tract and it can modulate gut microbiota and microbiota associated metabolic pathways [17].These gut bacteria are predominantly present in both the healthy and diseased person [16]. Therefore, these microbes were used in invivostudy to evaluate the relationship between the gut bacteria and DHA. These gut bacteria converted DHA into 82 metabolites [ Table 8] which have various therapeutic properties like anti-oxidant, anti-bacterial, anti-depressant, anti-tumor, anti-bio lm, anti-melanogenic, anxiolytic effects, anti-obesity, anti-diarrheal, anti-fungal, anti-cancer, anti-in ammatory, alpha-amylase inhibitory, anti-staphylococcal activity, pancreatic lipase inhibitory activity, and immunotherapeutic agent.
Commonly, Lactobacillus spp., Clostridium spp., Escherichia coli, Staphylococcus spp., Enterococcus spp. are present in healthy person[18] but decrease if the person is said to be in a diseased state [19]. This could lead to many disorders like depression and other hormonal changes due to the lack of the therapeutic metabolites. We suggest that to improve the role of these therapeutic metabolites in the diseased person, it should be given to them as a combination of DHA and these microbes as probiotics which can increase the normal ora of intestine.
The most common activity among all metabolites obtained from bioconverted DHA extract is the anti-bacterial activity. Hence, in this studythe crude extract was evaluated against clinical pathogens and it showed effective and strong antibacterial activity against them. In an earlier study, DHA was bioconverted using P. aeruginosa PR3 and the crude extract showed effective antibacterial activity against four gram-positive bacteria, Bacillus subtilis, Listeria monocytogenes, Staphylococcus aureus (ATCC 6538) and S. aureus (KCTC 1916) and seven gram-negative bacteria, Enterobacteraerogenes, Escherichia coli, E. coli O157:H7, Pseudomonas aeruginosa, Salmonella enteritidis and S. typhimurium [11]. Whereas, we analyzed the bioconverted DHA with Lactobacillus spp., Clostridium spp., Escherichia coli, Staphylococcus spp., Enterococcus spp. crude extract which showed aneffective antibacterial activity against the clinical pathogens B. cereus, E. coli, P. aeruginosa, S. typhimurium, S. aureus and S. enteritidiswhen compared to the standard antibiotic Gentamycin.

Conclusion
From this work, itcan be concluded that metabolites from extract of bioconverted DHA with Lactobacillus spp., Clostridium spp., Escherichia coli, Staphylococcus spp., Enterococcus spp. has many medicinal properties such as antibacterial, antioxidant, anxiolytic, phytotoxicity and antiviral activities. This work reported for the rst time the metabolite from this bioconversion to possess an anti-depressant effect. In conclusion, Normal ora of gut can convert the Docosahexaenoic acid into the various therapeutic metabolites.

Declarations
Ethics Approval: Not Applicable.