Characterization of pigment produced by high carotenoid yielding bacteria Paracoccus marcusii RSPO1 and evaluation of its anti-diabetic, anti-microbial and antioxidant properties

Abstract High pigment producing bacteria was isolated and identified as Paracoccus marcusii RSPO1 using biochemical and 16s rRNA identification. Bacterial pigment production was optimised using parameters like inoculum size, nitrogen source, pH, temperature, and agitation speed. Carotenoids production was 7,240 ± 41 µg L−1 after optimization. The silica column purified pigment was characterized using UV-visible spectroscopy, TLC, FTIR, LC-ESI-MS and NMR, which revealed its composition as astaxanthin, zeaxanthin, ζ-carotene and β-zeacarotene. The inhibition assays against α-amylase and α-glucosidase showed IC50 values as 226 µg ml−1 and 0.7548 µg ml−1 respectively. The MIC of 1000 µg ml−1 of carotenoid was found to be effective against Escherichia coli and Enterobacter aerogenes when tested for antibacterial activity. Moreover, antioxidant activity of carotenoid sample was also determined where antioxidant potential of extracted carotenoid for DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) inhibition were 65 ± 0.06% and 42 ± 07% respectively at 20 µg ml−1 concentration. Graphical Abstract


Introduction
Carotenoids are fat-soluble micro constituents that have beneficial effects to human health.They belong to the family of tetraterpenes with over 1100, coloured compounds that are present in plants, fruits and microorganisms (Yabuzaki 2017).These carotenoids are capable of giving protection against cancer (Saini et al. 2020), cardiovascular diseases (Di Pietro et al. 2020) and macular degeneration (Abdel-Aal et al. 2013).They are divided into two groups on the basis of presence of oxygen (Arvayo-Enríquez et al. 2013) (Figure S1).The conjugated double-bond chain in carotenoids, determines colours, and also behaves as chromophores that absorbs light and plays important role in protecting cells from the UV radiation damaging effects, as well as exerts antioxidant activity (Wang et al. 2014).Briefly, when cell is irradiated, a carotenoid molecule undergoes electron energy exchange which quenches the irradiated energy and results the carotenoid molecule to transform into triplet state which reacts with triplet oxygen and prevents oxidation reactions.The molecule is even capable to interact with energy-activated singlet oxygen resonantly on returning to its single state level, thus protecting the cellular medium from this radical oxygen species (Reis-Mansur et al. 2019).Apart from antioxidant properties certain carotenoids such as astaxanthin and fucoxanthin are known to have anti-bacterial activity against organisms like Helicobacter pylori, Escherichia coli, Streptococcus mutans, Listeria monocytogenes, Staphylococcus aureus and Pseudomonas aeruginosa, and at the same time it has no adverse effects on normal gut flora such as Lactobacillus spp.(Karpiński et al. 2021).Other medical application of carotenoids includes their anti-diabetic potential.Carotenoids, such as, β-carotene, astaxanthin, zeaxanthin, bixin, lutein, lycopene have been reported to exhibit significant antidiabetic potential.They also exert prophylactic role in diabetic complications (Sahu and Dewanjee 2021).Thus, due to this wide range of applications carotenoids grew huge from $766 million market in 2007, to $1004 million in 2018, with an annual CAGR of 2.3% (Li et al. 2011;Rivera Vélez 2016).And according to a report of 2021, the market for natural carotenoid is expected to reach $1.9 billion by 2028, at a CAGR of 7.4%, (https://www.meticulousresearch.com/product/natural-carotenoid s-market-5232, Accessed 29th April, 2023).Therefore, attempts to explore bacterial carotenoids has been made by various researchers, and Afra et al. (2017) have used one-factor-at-a-time strategy to optimise Arthrobacter's pigment synthesis and identified pigments using various analytical techniques.Further, this pigment's biological activities were assessed, and its EC 50 antioxidant activity was shown to be 4.5 mg ml −1 .On an oesophageal cancer cell line, KYSE30, it had modest anticancer activity, but no inhibition was seen in normal HDF (Human Dermal Fibroblast) cells.This pigment exhibited no bactericidal properties.Another study by Tapia et al. (2021) has shown antiproliferative effects of carotenoids from Dienococcus sp bacteria over three cancer cell lines (20.6% Neuro-2a, 26.3% Saos-2 and 13.2% MCF-7).Similarly, antibacterial, antioxidant and cytotoxic activities of bacterial carotenoids extracted from Rhosopseudomoans strain was also evaluated by Koyyati et al. (2019) and has found some promising results.Therefore, to carry out similar exploration of bacterial carotenoids, the objective of this work was to isolate the high carotenoid producing bacterium and improving its carotenoid production by optimization of various parameters.Further, various analyses were carried out for the identification of carotenoid produced by the selected strain.This carotenoid pigment was evaluated for its biological activities to explore the therapeutic potential of bacterial carotenoids so as to compete with conventional sources of carotenoids.

Results and discussion
In this study, primarily 20 pigment producing bacterial species were isolated from different sources; their colony characteristics and Gram's nature were noted and are shown in Table S1.

Primary screening
Primary screening of 20 isolates using 2%v/v glycerol supplemented nutrient broth gave 6 isolates with high growth out of which 3 highest growing isolates at 72h were selected for secondary screening as shown in Figure S2.

Secondary screening
Isolates C1, PO and P2 were subjected to secondary screening using variable carbon sources at 2% concentration.The growth media after incubation for 24, 48 and 72 h were subjected for evaluation of growth and pigment production.Pigments were extracted in methanol.Each isolate showed better pigment production in lactose and starch supplemented nutrient broths as shown in Figure S3(a-d).The isolate PO however showed highest pigment production, quantitatively 2 folds higher in 2%w/v starch supplemented nutrient broth (0.52 ± 0.037 absorbance at 460 nm) as compared to the pigment production in lactose (0.262 ± 0.317 absorbance at 460 nm).This was followed by supplementation of variable concentrations of starch for evaluating effect of starch concentration on pigment production.It was observed that with increase in starch concentration, there was an increase in pigment production as well.2%w/v and 2.5%w/v starch showed similar pigment production as can be seen in Figure S3(d) and therefore, 2% starch was taken as optimum starch concentration.In support to our observation, José et al. (2019) have also shown the usage of starch by Paracoccus sp, in their study.Likewise, (Kalathinathan et al., 2021) has also reported presence of genes encoding amylase in P. marcusii KGP, which indicate its ability to use starch as carbon source.Our results indicated that complex carbon sources (soluble starch and lactose) are more desirable than simple carbon sources (glucose and fructose) for the production of pigments (secondary metabolites).Similar phenomenon was also observed by Afra et al. (2017) in Arthrobacter sp., where they found significant increase in pigment production using complex carbon sources such as maltose.
Further, the effect of various nitrogen sources on pigment production was studied where yeast extract, malt extract and peptone were found to be the most beneficial sources.Our results were in comprehension with the findings of Valduga et al. (2009).They also observed increase in pigment production using peptone and malt extract.The addition of only 0.5% malt extract along with 2% starch, increased the pigment production by OD of 0.15 ± 0.042 than supplementing 2% starch only.As per the earlier reports, nitrogen sources are known to regulate influx of carbon sources into cells and therefore might be affecting the pigment production (Bren et al. 2016).This might be a reason why supplementation of only 0.5% of malt extract elevated the pigment production.Costa et al. (2002) also observed that use of soluble starch with organic nitrogen source like yeast extract provided good growth when compared to inorganic nitrogen sources.However, when malt extract concentration was increased; a constant decline was also observed.In reference to this, Minyuk et al., (2020) explained a valid reason, as they observed the production of ammonia increased in the medium upon utilization of nitrogen source by bacteria which shows detrimental effect on the growth and pigment production of bacteria.

Optimization of various physico-chemical parameters for pigment production
The high sensitivity in yield with respect to close variation in key parameters like temperature and pH have always been observed by different research groups in several past studies (De Carvalho et al. 2006;Orozco and Kilikian 2008;Kagliwal et al. 2009) Therefore the pigment was evaluated for pigment production using various physico chemical parameters as can be seen in Figure S4(a-h).Firstly, the pigment production was evaluated using the inoculum sizes in which the inoculum size of 10% was found to be most effective.This was followed by assessing the production of pigment using different pH within a range of pH 4 to 9. From this range, pH 7 was observed to be the most favourable for pigment production.Similar kind of results were also reported by Korumilli and Susmita (2014) where maximum production was obtained at 35 °C with pH 7. Further, on varying the temperatures such as 30, 37 and 45 °C; the highest pigment production was observed at 37 °C.While, Afra et al. (2017) observed maximum pigment production at 20 °C.The trial for pigment production using the oils as crude carbon sources showed no significant increase in production.To optimise the agitation speed for bacteria to maximize the pigment production, various agitation speeds were taken into account, As the P. marcusii are aerobic and non-motile bacteria, shaking conditions are necessary for them to produce significant amount of pigment.The different shaking conditions (60, 80, 100, 120 rpm) were examined where 100 rpm was observed to be the most effective.However, with further increase in rpm results in lesser pigment production.It may be due to the elevated aeration into the media which is responsible for developing stress condition for organism.Many a times, it is reported that inducing stress conditions may trigger enhancement of pigment production in certain species of bacteria, mostly in the organisms isolated from marine and desert environments.To evaluate if the isolated organism can tolerate the salt stress and enhance the pigment production, various NaCl salt concentrations were taken under study.It was noted that the pigment production was very negligible on addition of NaCl salt indicating that the organism cannot tolerate the salt stress.

Validation study
A validation study was performed for the production of carotenoids using the one factor at a time optimised parameters (2% starch, 0.5% malt extract supplementation at 37 °C with pH 7 at 100 rpm shaking) alongside basal nutrient broth.The carotenoid production with the basal media was found to be 4,865 ± 67 µg L −1 and the production with the optimised parameters (2% starch, 0.5% malt extract supplementation at 37 °C with pH 7 at 100 rpm shaking) was found to be 7,240 ± 41 µg L −1 the comparison between both the production can be seen in Figure S4(a).

Identification of culture
The biochemical characteristics, motility and sugar utilization tests for the isolate PO are mentioned in Table S2.This isolate was found to be Gram's negative rods.Further, this isolate was identified as Paracoccus marcusii RSPO1 using 16S rRNA identification as can be seen in Table S3. Figure S5 shows the phylogenetic correlation as found in Clustal ω phylogeny analysis.The isolate P. marcusii has the closest phylogeny with P. carotinifaciens.These two are the only organisms from Paracoccus sp. which can produce carotenoids (Kelly et al. 2006).Physiologically, the only difference between these organisms is motility; P. carotinifaciens is motile while P. marcusii is non-motile.

Characterization of pigment
The extracted pigment was characterized using different analytical methods such as UV-Visible spectroscopy, TLC, FTIR followed by LC-ESI-MS.The UV-Visible spectrum of the pigment showed maximum absorption peak at 460 nm which is in the range of wavelength corresponding to carotenoids (Sutthiwong et al. 2014).Four different mobile systems were utilized for TLC of carotenoid sample of isolate PO.The optimised system was found as Benzene: Glacial acetic acid: Methanol (8:1:1).Four bands were observed after running the TLC and their Rf values are shown in Table S4.Upon comparison of R f value with standard Table as shown in Table S4 (Jikasmita and Sahoo, 2015), probable compounds were found to be β-carotene, Echinone and 2 esters of Astaxanthin.Figure S6 shows IR spectrum of extracted pigment, where major peaks were found at 3247, 2959, 2926, 2855, 1633, 1455, 1407, 1242, 1115, 1056 and 575.Majority of peaks having close resemblance to β-carotene structure, the peaks at 2959 and 2855 are due to asymmetric and symmetric stretching vibrations of CH2 and CH3 group.The peaks at 1056 is for wagging vibrations of (RH)C = C(RH) groups of the synthesized pigment.1407 and 1455 are due to symmetric deformations of δ CH3 and deformation vibration of delta CH2 groups.3427 can be due to vibrational modes of water interference in the analysed pigment (Korumilli and Susmita 2014).Peaks at 2926 and 2855 can be assigned to the antisymmetric and symmetric C-H stretch from CH2 groups in lipids and this group is also contained in the carbon ring structure of astaxanthin.The peak at 1633 can be due to C = O group in astaxanthin due to its conjugation to a C = C group (Liu and Huang 2016).
The 1 H NMR of the purified extract as shown in Figure S7 suggests that the pigment possesses long chain alkane and alkene groups, which are present in the structure of carotenoids.
The HPLC separated four pigments obtained from the P. marcusii culture as shown in Figure S8.They were further identified using ESI-MS.The results are shown in Figure S9(a-f ).The peaks matched the database for following 6 pigments: apo-15-Astaxanthinal, apo-8′-astaxanthinol, Zeaxanthin, ζ-carotene and β-zeacarotene.The retention time in HPLC, UV-Visible absorbance maxima and ESI-MS peaks are as mentioned in Table S5.The ESI-MS peaks showed similarities to the peaks reported by several other researchers and organizations (Maoka et al. 2002;Weesepoel et al. 2014; https://spectrabase.com/).Beside carotenoids, the geranylgeranyl cysteine was found by LC-ESI-MS.It is a product formed on prenylation of cysteine residue using geranylgeranyl diphosphate.This compound acts as a precursor for carotenoid synthesis in methylerythritol 4-phosphate (MEP) pathway.
Based on this, it can be presumed that the isolate follows the MEP pathway for carotenoid production.

Anti-diabetic activity
Pancreatic α-amylase and α-glucosidase are two major enzymes that are present in human digestive system which catalyses the digestion of starch.Inhibition of these two enzymes decreases the breakdown and metabolism of carbohydrate and thereby curtailing postprandial blood glucose level can be managed in type II diabetic subjects.Therefore, here in this study anti-diabetic activity was determined by performing anti-α glucosidase and anti-α amylase tests where acarbose was taken as positive control.Here, the IC50 values of the carotenoid pigment were found to be 226 µg ml −1 and 0.8659 µg ml −1 for anti-α amylase and anti-α glucosidase tests respectively and that of acarbose for anti-α amylase and anti-α glucosidase tests were 49.82 µg ml −1 and 9.303 µg ml −1 respectively as shown in Figure S10(a,b).Similar in vitro anti-diabetic ability of lactucaxanthin carotenoid was tested against α amylase and α glucosidase in which their IC50 values were found as 434.5 µg ml −1 and 1840 µg ml −1 respectively (Gopal et al. 2017).The pigment possesses particularly good anti diabetic activity against α glucosidase enzyme while it has shown mild inhibition IC50 value against α amylase enzyme as shown in Figure S10(a,b).This can be because of a possibility that the isolate may be producing amylase enzyme itself as it has shown utilization of starch as carbon source.In support to that assumption, a strain of Paracoccus marcusii, Paracoccus marcusii KGP having very close resemblance with our isolate on 16s rRNA comparison, has also shown presence of genes for amylase and β-glucosidase enzymes (Pooja et al. 2021).Therefore, the pigment might not affect the amylase as good as it can act on α-glucosidase enzyme.

Anti-microbial activity
Anti-microbial activity of the pigment was conducted against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Bacillus cereus and Bacillus subtilis using agar well diffusion method.The extracted pigment possesses anti-microbial activity against gram negative bacteria E. coli ATCC 23716 and E. aerogenes ATCC 13048.as shown in Figure S11(a,b).However, it does not show any anti-microbial property against P. aeruginosa PA01 MTCC 3541 and gram-positive bacteria such as B. cereus ATCC 11778, B. subtilis MTCC 441 and Staph.aureus ATCC 6538.Carotenoids generally possesses antimicrobial properties against gram negative bacteria such as Salmonella sp. and are very weak inhibitors of gram positive bacteria especially of the Bacillus sp.(Konuray and Erginkaya 2015).Similar observation was observed in our study, it was found that the pigment is only able to inhibit Escherichia coli and Enterobacter aerogenes with MIC of 1000 µg ml −1 of carotenoid concentration.

Antioxidant activity
Anti-oxidant activity of carotenoid sample was determined by DPPH and ABTS radical scavenging assay.The antioxidant potential of extracted carotenoids for DPPH inhibition was 65 ± 06% at 20 µg ml −1 concentration as can be seen in Figure S12(a).Similarly, Mukherjee et al. (2017) have reported DPPH radical scavenging activity, methanolic extracts of carotenoid have shown 80.38 ± 0.64% and 73.16 ± 0.85% scavenging activity for isolates RP and YY, respectively.On the similar line, the antioxidant activity from carotenoid bacterial symbionts was also assessed by Masduqi et al. (2020) in which the percent inhibition by DPPH assay were 38.02% and 26.81% at 1000 µg ml −1 carotenoid concentration where the DPPH concentration was 0.01 mM with 30 min incubation time.In comparison to this result, the DPPH inhibitory effect of our bacterial pigment is more significant at the 0.5 mM concentration of DPPH with 10 min incubation.
Further, the ABTS radical scavenging activity was also performed for isolate P.marcussi where carotenoid has given 42 ± 07% scavenging activity at 20 µg ml −1 concentration as can be seen in Figure S12(b).Likewise, Mukherjee et al. (2017) have also reported the ABTS radical scavenging activity and 74.3 ± 0.2% and 95.5 ± 1% activity was reported for the methanolic extracts of carotenoid in case of bacterial isolates RP and YY.Thus, the ABTS scavenging activity can be vary organism to organism.Its value can be low because of the presence of hydroxyl groups in ring of xanthophylls while its value can be high because of the presence of apo-carotenoids as they provided the dimension of the conjugated double bond system (Müller et al. 2011).

Conclusions
The overview of these investigations and analysis suggests that the bacterial carotenoids extracted from the isolate P. marcusii can be a good alternative to the carotenoids from other conventional sources as they have shown good anti-bacterial, anti-diabetic activities, and antioxidant activities.Therefore, it suggests that these carotenoids can be used in food, pharmaceutical and therapeutic research and thus can be used in their respective industries.However, these are primitive studies and thus needs further investigations for commercial applications.It proves to be a necessary step towards understanding of bacterial carotenoids for their further exploitation.