INTRODUCTION:

Free radicals and reactive oxygen species (ROS) are highly reactive molecules that formed through different biochemical reactions by the cellular redox process^1–3. At moderate levels, ROS play an important role for immune function but at high concentrations, they generate oxidative stress that can damage cell, consecutive oxidation⁴. These radicals contribute to the degenerative diseases, such as cancer, diabetes and others^5,6. The human body can counteract this oxidative stress by producing antioxidants, which are either naturally produced or externally supplied through foods and supplements^7,8.

Natural antioxidants are widely distributed in food and medicinal plants, vegetables, spices and fruits^9–12. Beside plants, microorganisms are the potential source of natural antioxidant such as actinomycetes, bacteria, fungi, lichens and microalgae that produce several valuable bioproducts as a source of natural antioxidant^9-11.

In the last decades, various microalgae have been reported as a promising potential as antioxidant such as Chlorella Vulgaris¹⁴, Coelastrella sp¹⁵, Parachlorella kessleri GB1¹⁶, Scenedesmus subspicatus,¹⁷, Spiriluna Platensis¹⁸, Nannochloropsis sp., Synechocystis sp.¹⁹, Dunaliella salina, Scenedesmus dimorphus²⁰ and Haematococcus pluvialis²¹. Microalgae have different natural pigment compositions compared to other plants such as phycocyanin and carotenoid¹⁹. Carotenoids are a group of natural pigments synthesized by photosynthetic organisms including plants, bacteria, fungi and microalgae^22,23. Recently, the production of carotenoids from microalgae has become a very important field of research, with direct implications for well-established industries.

The growing interest in microalgae as an alternative feedstock for carotenoid production has inspired a new focus on valuable bioproducts of microalgae. Carotenoids have numerous potential health benefits, including neutralizing free radicals and reactive oxygen species. Most reports regarding the carotenoid content of microalgae are belongs to marine microalgae, there is still little information about the content of carotenoids from freshwater microalgae. There is some evidence suggesting that it freshwater microalgae also have potential as source of carotenoid such us Chlorella sorokiniana, Scenedesmus bijuga²⁴, Parachlorella kessleri²⁵ and Microcystis aeruginosa²⁶.

Nowadays, there are existing opportunities for microalgae to be used as a source of valuable bioproducts such as carotenoids for the food, nutraceutical, and cosmetic industries. The microalgal carotenoid market will continue to expand due to the increasing demand for natural products^27,28. Therefore, the main objective of this study was isolation and identification of freshwater microalgae from urban area in Padang City, Indonesia based on 18S rDNA and morphology analyses. Then, the antioxidant properties from carotenoids of naturally isolated microalgae were examined using DPPH(2,2-diphenyl-1-picrylhydrazyl) method; the profile carotenoid pigments were characterized using FTIR, and HPLC.

MATERIALS AND METHODS:

Microalgae Isolation:

Freshwater samples were collected from Andalas University Pond in Padang City, West Sumatera, Indonesia. Water samples filtered by the plankton net were placed into a bottle filled with Bol’d Basal Mediuam (BBM) medium and labeled for further identification and isolation of microalgae using a microscope. The micropipette washing technique method was used to isolate microalgae in this study. The pure isolated microalgae were cultivated in a 500mL flask containing sterile BBM and aerated 3.5L/min at 25℃ room temperature with light intensity ±2200 lux. Microalgae growth was evaluated daily from the optical density (OD) measurements at 680nm with UV-VIS Spectrophotometer (Genesys 20)²⁹^,³⁰.

Microalgae Identification:

Individual microalgae colonies were identified using a microscope with up to 1000x magnification. The microalgae samples were submitted for the DNA extraction and purification process using the Dneasy ® Tissue Kit (Qiagen Sciences, Valencia, Calif., Md, USA). After DNA purification, the 18S rDNA genomic region amplification was carried out using pairs of universal primers, named forward 5’-CCTGGTTAGTCCTGCCAG-3’ and reverse 5’-TTGATCCTTCTGCAGGTTCA-3’³¹. The Polymerase Chain Reaction (PCR) contained 50ng microalgal genomic DNA, along with 0.4μM of each primer, 1x Go Taq Green PCR master mix (Promega), and the final volume was adjusted to 25μL by nuclease-free water. The 18S rDNA amplification was carried out in a PCR BIO-RAD C1000 Thermal Cycler apparatus. PCR was performed as follows: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30s, primer annealing at 56.3°C for 30 seconds, primer extension at 72°C for 2 minutes, and final extension at 72°C for 7 minutes. PCR products were separated using electrophoresis on 1.5% agarose. The 18s rRNA was used for DNA sequencing, and all sequences were compared with the GenBank database using BLAST. The aligned sequences were submitted to distance estimation, and the phylogenetic tree was built applying Geneious Tree Builder according to Neighbor-Joining (NJ) method using MEGA 6.0 Software³².

Measurement of carotenoid pigment:

The total carotenoid pigment content was determined according to the method of Kumar³³ and Lichtenthaler³⁴, with slight modification. To determine concentrations of total carotenoids briefly dried biomass (20mg) was transferred to methanol (50mL) and kept in the dark then incubation in water bath at 40±5℃ for 30 minutes following by cooling in room temperature. The mixture was centrifuged at 3500rpm for 10 minutes. Aliquots of each extract were measured were measured by recording the optical densities at 470, 652 and 665 nm for optical density using a spectrophotometer UV VIS (Thermo Scientific Genesys 20) using the following equations³⁴:

Chlo a (µg/g) = (16.72 x A.665 nm) – (9.16 x A.652nm)

Chlo b (µg/g) = (34.09 x A.652nm) – (15.2 x A.665nm)

Tot Carotenoid (µg/g) = ((1000 ×A.470nm) - (1.63 ×Chlo a) - (104.96 ×Chlo b))/221

Extraction of carotenoids:

A modified macerations method was used for extraction³⁵³⁶, Briefly, 50mg of biomass sample was mixed with 1.5mL of water, 3mL of methanol, 1.5mL of dichloromethane, and followed by manual mixing. After a 10-min incubation at room temperature, the mixture was remixed with 1.5mL of distilled water and 1.5mL of dichloromethane. The organic layer was collected after centrifugation (10 min at 3500g). The extraction step was repeated twice more, each time with 3mL of dichloromethane. The solvent was dried with nitrogen at room temperature. The crude carotenoid extract was then saponified with 10% methanolic KOH and kept at room temperature in the dark for 1 hour before being washed with water to remove the alkali and kept at room temperature.

Identification and characterization of Carotenoid Extract:

An FTIR, UV-Vis spectrophotometer, and HPLC Shimadzu SPD-20A/SPD-20AV were used to characterize the crude carotenoid extract. A method based on³¹ was minor modified to separate the various carotenoids in microalgae by using a C30 column (4.6 × 150mm) and a gradient mobile phase of acetonitrile : methanol: dichloromethane (70:20:1, v/v/v). The column temperature was maintained at 25°C, the flow rate at 1 mL/min, and the detection wavelength at 450nm. The standard solution consisted of 100µg/mL each of canthaxanthin, astaxanthin, lutein, and β-carotene.

Antioxidant capacity assay:

The antioxidant capacity of the crude extracts was determined by DPPH assay. In brief, a fresh methanolic DPPH (stock solution (0.1mM) was prepared, followed by carotenoid extracts dissolved in methanol at a concentration of 1000µg/mL, then diluted into several variations of concentrations of 5, 10, 15, 20, 25, 50 and 100µg/mL. Then, 2mL of carotenoid extract was added with 1mL of 0.1mM DPPH. The mixture was incubated for 30 minutes in a dark room. Subsequently, the absorbance at 517nm was calculated, and its percentage inhibition was calculated using the formula:

DPPH rad sca. act (%) = [(A. Control - A. Sample)/A. Control] x 100%

Positive control was used for comparison. Ascorbic acid solution with concentrations from 0,5 to 4µg/mL was used as the standard³⁷. The IC₅₀ value was calculate based on a straight-line equation based on the calibration curve³⁸

RESULT:

Microalgae isolation and identification:

The shapes of unidentified microalgae were observed using an optical microscope which could show their features and purity. Microscope observation indicates that the AUP1 has a simple cellular structure with spherical in shape and green in color (Figure 1).The length of the 18S rRNA sequence of AUP1 microalgae after PCR amplification, electrophoresis, purification, and sequencing was found to be 1700 bp. The nucleotide sequence was submitted to GenBank. Homologous comparison with GenBank data revealed 99.50% similarity to Mychonastes racemosus CCAP 222. Homologous comparison using the phylogenetic tree (Figure 2) constructed by MEGA6.0 with the maximum likelihood method shows that AUP1 belongs to the same branch of Mychonastes racemosus CCAP 222. Thus, microalga AUP1 was identified as a species of the genus Mychonastes. These results were supported using the results for the construction of the phylogenetic tree (Figure 2).

Figure 1 Morphology of the microalgae AUP1

Figure 2. Molecular phylogeny of the microalgae AUP1

Microalgae Cultivation:

The growth profiles of the microalgae were determined by a UV-Vis spectrophotometer in the wavelength region of 680 nm. Figure 3 shows the growth curve of isolated microalgae in BBM medium of the isolated microalgae AUP1(Figure 3).

Figure 3 Growth curve for microalgae AUP1 in the medium BBM (ODλ=680 nm)

Chlorophylls and carotenoids content:

Total carotenoid and chlorophyll a and b contents in the isolated microalgae samples are given in Figure 4. The amount of total carotenoids content 3.4±0.2µg/g dry weight (d.w) biomass. The chlorophyll a and b con tents were 11.75±0.9µg/g d.w and 3.67±0.51µg/g d.w respectively. These pigments are essential for the photosynthetic system of microalgae especially used for the energy transfer and photochemical redox reaction during photosynthesis process²⁷.

Figure 4 Chlorophylls and carotenoids content microalgae AUP 1

Carotenoid extraction:

The carotenoid extract from microalgae AUP1 was yellow with 86 mg/g dry cell weight. The presence of a yellow carotenoid from the saponification extract indicates that saponification removes chlorophyll from carotenoid extracts. Chlorophyll is a pigment that hydrolyse easily in alkaline conditions³⁹. Solvent combination of water, methanol and dichloromethane mixture gave the effective for carotenoids extraction. In line with the present study, Priyanka et al³⁶ attained higher yield of carotenoid such zeaxanthin from Dunaliella tertiolecta using methanol and dichloromethane solvent mixture.

Identification and characterization of carotenoid extract:

The FTIR analysis of microalgae carotenoid extract is presented in Figure 5; the identified characteristic peaks related to the presence of functional groups of carotenoid compounds The peak around 1550–1600 cm⁻¹ corresponds to C=C double bond stretching vibrations of β-carotene⁴⁰. The FTIR resulted showed OH functional groups with peaks at 3500-3200 cm^-1 with medium intensity and widen⁴¹. In these spectra, it is possible to appreciate the same characteristic bands of the spectra of carotenoid microalgae AUP1 corresponding to the oscillation of groups with C–H bonds, 1636 cm⁻¹, associated with double bonds of β-carotene⁴²^,⁴³.

Figure 5. FTIR analysis of microalgae carotenoid extract

The presence of various functional groups in the metabolites detected by using UV and FTIR provided information about carotenoid characteristics such as the presence of conjugated double bonds and the presence of CH, C=C, and OH groups, indicating the presence of xanthophyll in microalgae carotenoid extracts^44,45. Figure 7 shows the HPLC chromatograms of the carotenoid extracts from the microalgae AUP1 recorded at 450 nm.

Figure 6. HPLC chromatogram of microalgae carotenoid extracts

Table 1: HPLC analysis that there are 7 peaks on the chromatogram microalgae carotenoid extract.

Peaks	Retention Times	% Area	Carotenoid Compounds ^a
1	3.356	7.988	-
2	4.261	43.325	lutein
3	4.745	7.419	-
4	5.143	2.369	-
5	5.585	29.759	-
6	6.057	4.823	-
7	16.861	4.317	β-carotene

Note: ^aComparing data with standard carotenoid, ^-unidentified

Anti-radical scavenging activity carotenoid extract of using the method of DPPH:

The carotenoid extract of microalgae AUP1 at different concentrations of 5, 10, 15, 20, 25, 50, and 100µg/mL showed significant antioxidant activity; the scavenging activities were 4.33, 6.82, 9.02, 11.01, 13.27, 32.45, and 63.11%, respectively, comparing with ascorbic acid at different concentrations 0.5, 1, 1.5, 2 and 4µg/mL showed significant activity; the scavenging activities were 10.2, 17.41, 30.06, 39.65, and 90.36%, respectively with half minimal inhibitory concentration (IC₅₀) was 2.36µg/mL(Data not shown). The half minimal inhibitory concentration (IC₅₀) of carotenoid extracts was calculated to be 78.61µg/mL. Our results demonstrated that the carotenoid extracts at different concentrations exhibited marked DPPH scavenging activities with antioxidant IC₅₀ value less than 100 µg/mL.

Figure 7. The results of the carotenoid extract antioxidant with DPPH method (average ± standard error represents the number of replications n = 3)

DISCUSSION:

Numerous studies have been done to use molecular identification methods using polymerase chain reaction (PCR) to identify microorganism such fungi^46,47 bacteria⁴⁸ and microalgae³⁰. The current study showed that microalgae isolated and observed under a microscope had single green colonies. This species features a particular regularly spherical shape and was identified as belonging to the genus Chlorophyta, for presenting cells with diameter between 2 and 10μm (Figure 1)⁴⁹. Morphological analysis is an important method for the identification of microorganisms, which is followed by DNA barcoding from the nuclear genes (18S rDNA) to identification based on sequence comparison against a DNA database^50,51 . By molecular analysis of the amplified sequences of the 18S rDNA genomic region using pairs of universal primers, microalgae AUP1 was molecularly identified as Mychonastes racemosus by BLAST/NCBI, with high similarity to species of the genus Chlorophyta. After 20 days of cultivation, optical density data of microalgae AUP1 reached the maximum. The growth of microalgae culture will be affected by light intensity, cell concentrations, and growth medium⁵². The growth of microalgae also influenced by the physicochemical characterization such as pH, temperature, and alkalinity of the medium⁵³.

Based on the results obtained from spectrophotometric determination of chlorophylls a and b, and total carotenoids (results of three replicates expressed as mean±SD) were 11.75±0.9µg/g biomass, 3.67±0.51 µg/g and 3.41±0.2µg/g dry weight (d.w) biomass respectively as shown in Figure 4. The highest values of chlorophyll a than chlorophylls b and total carotenoid content in microalgae Mychonastes racemosus AUP1 indicating that the process photosynthesis from this microalgae occur first before the carotenoid synthesis for life activities and growth⁵⁴. Previous studies showed total carotenoid content from several microalgae Chlorella sp.(0.3±0.4mg/g extract weight ), Navicula sp (0.374±0.41mg/g extract weight)⁵⁵. The carotenoid extract from microalgae AUP1 is a yellow in colour and weighs 86mg/g dry cell weight. FT-IR analysis revealed the presence of some functional groups like alcohols, alkanes and carbonyl groups in the carotenoid extracts⁵⁶. The FTIR analysis of microalgae carotenoid extract showed that a spectrum with medium intensity at the range 2839-2999 cm^-1 indicates the presence of C-H functional groups and one of the typical bands of β-carotene and lutein is also observed around 968-1100 cm⁻¹^42,57. A total of seven carotenoids were separated in the extracts of carotenoid microalgae AIP1; however, using the external standard, only two carotenoids lutein and β-carotene were identified^40,57. Recent studies have proven that the microalgae AUP1 contains of lutein and β-carotene from HPLC and indicated that microalgae AUP 1 has the potential to be a promising source of carotenoids.

Several publications stated that Mychonastes is very suitable for biodiesel production^58,59 and sources of natural and safe sources of antioxidants^60,61. These researchers described the antioxidant capacity of ethanol extracts Mychonastes homosphaera by the DPPH radical scavenging assay and expressed as IC₅₀ value as 80.08± 0.61μg/mL. Moreover, results revealed the presence of essential amino acids and essential fatty acids (omega 3, omega 6 and omega 9) which may differentially contribute to the antioxidant capacity. The IC₅₀ of carotenoid extracts was calculated to be 78.61μg/mL, whereas ascorbic acid showed an IC₅₀ value of 2.36 μg/mL (Data not shown). According to previous study, the strong antioxidant had IC50 value 50-100g/mL, moderate for 100-150μg/mL, and weak if the number of IC50 more than 150μg/mL⁶². Many researchers have used the DPPH method of analysis to determine the radical scavenging activity of carotenoids⁶³. Several studies have already been published on potential carotenoids as antioxidants such as β-carotene from Dunaliella salina, astaxanthin from Haematococcus pluvialis, and canthaxanthin from Coelastrella striolata⁶⁴. The results of this study provided a revelation microalgae AUP1 may be a potential feedstock for carotenoid production due to their relative faster growth rate and the carotenoid contents.

CONCLUSION:

In conclusion, the isolated microalgae from the freshwater of Andalas University Pond, Padang, was identified as Chlorophyta (Mychonastes racemosus) based on their 18S rDNA. The isolated microalgae species confirmed that the green microalgae AUP1 was strain Mychonastes racemosus, according to our findings. When the isolated microalgae AUP1 was cultured using medium BBM, it produced carotenoid pigments, with lutein and β-carotene being the major carotenoids based on FTIR, and HPLC analysis. The microalgae carotenoid extracts exhibited higher antioxidant activity with an IC₅₀ value of 78.61 µg/mL. This study highlights the potential of Mychonastes racemosus AUP1 as a potential carotenoid source for antioxidant and nutraceutical food applications.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The Authors thanks the Ministry of Research, Technology and Higher Education of the Republic of Indonesia for financial support through a PMDSU Scholarship [Grant number 146/SP2LH/LT/DPRM/2019].

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Received on 25.11.2021 Modified on 17.03.2022

Research J. Pharm. and Tech 2023; 16(1):404-410.

DOI: 10.52711/0974-360X.2023.00069