Isolation and Characterization of Potential Microorganism from Dhanyamla - An...

[Full title: Isolation and Characterization of Potential Microorganism from Dhanyamla - An Ayurvedic Formulation with Therapeutic Properties]

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Asian Journal of Pharmaceutic s • Jul-Sep 2021 • 15 (3) | 385 Isolation and Characterization of Potential Microorganism from Dhanyamla - An Ayurvedic Formulation with Therapeutic Properties J. Prarthana 1 *, Reshmi Pushpan 2 , Smitha Jain 2 , M. R. Hamsa 1 , S. Jagadeep Chandra 3 , Ramith Ramu 4 1 Department of Biotechnology, Sri Dharmasthala Manjunatheshwara College (Autonomous), Ujire, Karnataka, India, 2 Department of Agada Tantra, Sri Dharmasthala Manjunatheshwara Institute of Ayurveda and Hospital, Bengaluru, Karnataka, India, 3 Department of Microbiology, School of Life Sciences, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India, 4 Department of Biotechnology and Bioinformatics, School of Life Sciences, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India Abstract Objectives: Ayurveda is one of the oldest codified medical knowledge systems with equal emphasis on curative, preventive, and promotive aspects of health. Ayurveda pharmaceutics was developed from the quest to administer plants, animals, or metals and minerals products in a palatable and longer shelf life modification. The study aims to evaluate and characterize the microbial sp. and their biochemical properties in extrapolating its use as a potent probiotic formulation with multifaceted use. Materials and Methods: The microbial diversity of the formulation was evaluated by isolation followed by its microbial characterization using Gram’s staining, biochemical characterization using catalase assay, and molecular characterization by sequencing the internal transcribed spacer (ITS) region. Results: The study revealed that the bacteria isolated in the present study were Gram-positive, rod-shaped organism that exhibited catalase-positive test. Further, molecular characterization studies using the ITS sequence analysis revealed that the isolated organism showed similarity with that of Bacillus species. Conclusion: Therapeutic efficacy of any formulation depends on the process of its preparation, the kind of microflora that is established during aging or fermentation, and the kind of bioactive compounds released during fermentation. The present study identifies the microorganism that plays a pivotal role in this fermentation process and renders therapeutic properties to Dhanyamala formulation. This study can form the basis for further investigations on formulating this as a promising probiotic supplement Keywords: Ayurveda, Dhanyamla, Evaluate, microbial and biochemical properties Address for correspondence: Dr. J. Prarthana, Department of Biotechnology, Sri Dharmasthala Manjunatheshwara College (Autonomous), Ujire, Karnataka, India. E-mail: prarthanarao 6@gmail.com Received: 18-07-2021 Revised: 22-09-2021 Accepted: 30-09-2021 INTRODUCTION A yurvedic pharmaceutics is formulated through the transference of active ingredients by different manufacturing processes [1,2] Sandhana Kalpana (fermented preparations) is a unique form in which acidic and alcoholic fermented formulations are prepared [3,4] The term Sandhana is used to denote the fermentation process. This processing method gained popularity since ancient times owing to its higher preservation, pharmaceutical, and therapeutic value. As per the information collected from Ayurveda texts and electronic sources, “Dhanyamla” is an acidic and alcoholic “‘fermented cereal” (Suktakalpana) widely used in India for nutritional and therapeutic use [5] According to Sushruta, Dhanyamla is a fermented product prepared from cereals such as rice, barley, and kodo millets [6] Acharya Vagbhata mentioned that Dhanyamla is prepared by fermenting the water in which rice and such other grains and pulses have been slightly cooked or merely washed and kept for fermentation ORIGINAL AR TICLE

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Prarthana, et al .: Probiotics from Dhanyamla - an ayurvedic formulation Asian Journal of Pharmaceutic s • Jul-Sep 2021 • 15 (3) | 386 [7] Sahasrayoga, a popular Ayurvedic text from Kerala describes the ingredients, procedure of preparation, and shelf life of Dhanyamla [8] Dhanyamla is recommended internally for appetite, fatigue, exhaustion, obesity, urinary calculi, and fistula in ano. Dhanyamla is profusely used for Panchakarma and other external therapies, especially in inflammatory musculoskeletal disorders, muscular dystrophies, stroke, and headaches [9] There is a paucity of studies to support that the process of fermentation imparts greater therapeutic potential to the formulation [10,11] However, phytochemicals present in formulations at the time of fermentation are shown to promote the growth of probiotics. Triphala is found to promote the growth of Bifidobacteria and Lactobacillus species while inhibiting the growth of undesirable gut residents such as Escherichia coli owing to the presence of polyphenols such as quercetin and gallic acid [9] Ranasinghe and Ediriweera 2015 [12] reported constituents in Dhanyamla include starch, globulin, albumin, oryzagenin, vitamin B, trigonelline, trigonelline falvonoides, urease, glycosides, lenoleic acid, polyphenols, beta sitosterol, smino acidsglycine, alanine, cysteine, serine, isoflavones genistein, isoferririn, cumesterol, alkaloid, phenol, tannins, alkaloids, falvonoids, saponins zingerone, shogaol, camphene, phellandrene, zingiberene, cineol, borneol, gingerol, gingerin, resins, geraniol citric acid, malleic acid, phosphoric acid, volatile oil, hesperidin. This study aims to evaluate the type of microorganism is this formulation by microbial, biochemical, and molecular characterization to assess the potential of the formulation to be used as a probiotic supplement [13,14] MATERIALS AND METHODS Preparation of dhanyamla In a large earthen pot, water was filled in the required quantity [Table 1] and boiled. Meanwhile, all the aforesaid drugs [Table 1] were coarsely crushed and tied separately in nine bundles loosely tied in cotton cheesecloth bags. These bundles of drugs were immersed into the boiling water and the vessel loosely covered by an earthen lid. The boiling was stopped on the 1 st day and on each consecutive day the earthen pot was subjected to heat only till the liquid started boiling for the next 6 days. On the eighth day, Dhanyamlawas filtered and collected in aseptic glass bottles Determination of pH The pH of Dhanyamla was determined using the pH meter at 30ºC Isolation of probiotics by selective media DeManRogosa and Sharpe (MRS) 1 ml of sample was taken in 9 ml of saline, serial dilution tubes were prepared up to 10 -8 , pour plate method was followed on MRS agar. Plates were incubated at 37°C for 24 h. Colony-forming Unit was enumerated. Procedure is repeated every week to understand microbial load [15] Gram staining Gram staining test was performed for all isolated strains according to the standard procedure. A smear of single colony was prepared on a clean glass slide and the smear was allowed to air-dry and then heat fixed. The heat-fixed smear was flooded with crystal violet solution and after 1 min, it was washed with water and flooded with mordant Gram’s iodine. The smear was decolorized with 95% ethyl alcohol and rinsed with water. Finally, safranin was used as counterstains for 60–80 s and washed with water, and examined under oil immersion (100×) Catalase test A drop of 3% hydrogen peroxide was added to a fresh culture on a sterile glass slide and mixed well. Producing bubble or froth indicated catalase-positive and no bubble or froth indicated catalase-negative Molecular characterization of bacteria Genomic DNA extraction Bacterial isolates were sub-cultured on MRS medium and incubated at 30°C for 48–72 h; The DNA of isolates was extracted and purified using the standard method [16] Single colony was inoculated in nutrient broth and grown for overnight at 37°C. Cells were harvested from 5 ml of the culture and to this 100 µ l of lysozyme was added and incubated at room temperature for 30 min, followed by the addition of 700 µ l of cell lysis buffer (Guanidiumisothiocyanate, SDS, Tris-EDTA). The contents were mixed by inverting the Table 1: Composition of dhanyamla Identity of the ingredient Part used Quantity used Tandula ( Oryza sativa ) Seed 750 g Pruthuka ( Oryza sativa ) Pressed seed 750 g Kulattha ( Macrotyloma ) Seed 200 g Laja ( Oryza sativa ) Puffed seed 200 g Kangubeeja ( Panicum sumatrense ) Seed 300 g Kodrava ( Paspalum scrobiculatum ) Seed 300 g Nagara ( Zingiber officinalis ) Rhizome 150 g Nimbuka ( Citrus aurantifolia ) Fruit 600 g Deepyaka ( Trachyspermum involucratum ) Seed 150 g Jala RO purified water 20 L

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Prarthana, et al. : Probiotics from Dhanyamla - an ayurvedic formulation Asian Journal of Pharmaceutic s • Jul-Sep 2021 • 15 (3) | 387 vial for 5 min with gentle mixing till the suspension looked transparent. 700 µ l of isopropanol was added on top of the solution. The two layers were mixed gently till white strands of DNA were seen. The DNA extracted from the aqueous layer was ethanol precipitated. The DNA pellet was dried and dissolved in 50 µ l of 1× TE buffer Amplification of 16 S rDNA of bacterial isolates Fragments of the 16 S rRNA genes of each bacterial isolate were separately amplified using the universal primers 27 F 5’-AGAGTTTGATCCTGGCTCAG-3’ and 1492 R 5’-GGTTACCTTGTTACGACTT-3’. For amplification of 16 S rDNA genes of each bacterial isolate, polymerase chain reaction (PCR) reaction mixtures 1 µ l DNA template (25 ng), 2 µ l 10× reaction buffer, 0.5 µ l MgCl 2 (50 pM), 1 µ l dNTPs mix (10 mM), 1 µ l forward primer (10 pM), 1 µ l reverse primer (10 pM), 0.5 µ l Taq polymerase (5 U/pi) and the final volume 25 µ l will be adjusted with molecular grade water. The temperature program and the cycle of reactions were as initial denaturation step at 95°C for 2 min Final denaturation 95°C for 30 s. Annealing 50°C for 30 s Elongation 72°C for 1 min. Final Elongation 72°C for 10 min. Thermal cycle is executed for 30 cycles and finally held at 4°C until evaluated Sequencing of 16 S rDNA bacteria Sequence of the PCR amplified product was analyzed through Applied Biosystems 3130/3130 × l Genetic Analyzers. The bacterial 16 S-rDNA sequences obtained were then aligned with known 16 S-rDNA sequences in Genbank using the basic local alignment search tool (BLAST), National Center for Biotechnology Information, and percent homology scores were generated to identify bacteria, and get accession numbers shown in Tables 2 and 3. The deduced sequence was aligned using BLAST pairwise alignment using which a phylogenetic tree was drawn Ultraviolet (UV) spectroscopic analysis Spectra scan of Dhanyamla was determined using Systronics double beam spectra 2205 between range of 200 and 800 nm to identify bioactive compounds, analysis was carried out every week [16] RESULTS AND DISCUSSION Dhanyamla is the fermentation of rice, millets, pulses, and few spices in water. There are several methods available for its preparation. Therapeutic values of Dhanyamla vary greatly depending upon combinations of ingredients used and method adopted for preparation. Presently Dhanyamla is a much preferred external application for Panchakarma therapies addressing ailments such as tender and painful joints, body ache, and obesity. The present study aims to find out the occurrence of any probiotics property of the formulation by assessing its microbial content [17,18] Dhanyamla is an acidic formulation that was proved with pH determination assay routine carried out. There was a gradual drop in pH from initial value of 4.7 to 3.6 over the weeks, probably due to mixed acid produced during fermentation. Ranasinghe and Ediriweera, 2015 [12] in their study have reported the pH to be 4.13 for Dhanyamla at 30°C [ Figure 1], which may be probably due to mixed acid produced during fermentation. MRS formulation is widely used for the isolation of probiotics and similar organisms. Serial dilution Table 2: Sequence similarity for amplified regions of primer 27 F Strain name E-value Percentage of similarity Accession number Bacillus subtilis strain GuanMX 8 e -108 96.33 MN 473282.1 Bacillus subtilis strain OTPB 4 8 e -108 97.07 KT 265082.1 Bacillus subtilis strain M-1 3 e -107 96.68 MN 538261.1 Bacillus subtilis strain N 402 3 e -107 95.62 MK 629804.1 Bacillus subtilis strain CTPRKVG 18-3 3 e -107 95.95 MK 392043.1 Bacillus subtilis strain NA 3 3 e -107 95.95 MH 187647.1 Bacillus subtilis strain X 502 3 e -107 95.62 KU 240496.1 Uncultured Bacillus sp clone N 98 3 e -107 95.60 KP 704278.1 Bacillus tequilensis strain S 521 B-7 3 e -107 95.95 HQ 238440.1 Bacillus subtilis strain 1 e -106 95.93 MN 84775.1 0 1 2 3 4 5 1 week 2 week 3 week 4 week 5 week 6 week Figure 1: pH studies of Dhanyamla performed weekly

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Prarthana, et al .: Probiotics from Dhanyamla - an ayurvedic formulation Asian Journal of Pharmaceutic s • Jul-Sep 2021 • 15 (3) | 388 Figure 2: Isolation of microbes from Dhanyamala on DeManRogosa and Sharpe media Figure 3: Gram positive rods Figure 4: Catalase positive reaction Table 3: Sequence similarity for amplified regions of 1492 R Strain name E-value Percentage of similarity Accession number Bacillus tequilensis strain NJ 6 1 e -115 98.75 KT 58868.1 Bacillus methylotrophicus PO 7 1 e -115 98.75 JN 700153.1 Bacillus subtilis strain NS 6 1 e -115 97.98 HQ 834863.1 Bacillus licheniformis CRN 3 5 e -115 98.74 MK 8635666.1 Bacillus velezensisi AAUBCT 5 e -115 98.74 MK 801267.1 UnculturedDZGS 22 5 e -115 98.74 MH 267236.1 Bacillus sp strain 3 BJ 5 5 e -115 98.75 MG 062834.1 Bacillus subtilis inaquosorum STRAIN YN 32 5 e -115 98.74 KC 511536.1 Bacterial environmental culture clone W 141 (2011) 5 e -115 98.74 HQ 731030.1 and pour plate method yielded confluent growth of creamish pink colony repeatedly [Figure 2] Gram staining of confluent grown colonies yielded slender rods, purple in color, indicating Gram-positive reaction [Fig ure 3 ] Catalase, an extracellular enzyme secreted by several microorganisms, helps in degradation of hydrogen peroxide produced during carbohydrates utilization by aerobic bacteria, thereby its presence or absence in a microbial cell can be used as a significant indicator that the isolates could belong to the Bacillus species that was further characterized through molecular means for confirmation [Fig ure 4 ] Molecular approach is used in bringing about preferential amplification of 16 srRNA genes by using universal primers 27 F and 1492 R [ Fig ures 5 and 6]. The amplified segments are sequenced, sequence similarity assessed by BLAST and corresponding dendrogram was constructed. From the data, the isolate found in Dhanyamla had 98% similarity with Bacillus sp. There were reports of non-pathogenic mainly aerobic and mesophilic organisms and a few counts of Staphylococcus aureus that were reported previously (Ranasinghe and Ediriweera 2015) [10] CONCLUSION Ayurvedic formulation, resultant of aging is said to be rich in several bioactive compounds, some may be prebiotics or unusual amino acids having special therapeutic values. In our study, we could successfully isolate Bacillus sp. There are few species of Bacillus cereus , Bacillus clausii , and Bacillus pumilus currently being used as probiotics. The occurrence of Bacillus sp in Dhanyamla could be purely associated with the preparation method. Kumar et al . (2012) [19] reported the occurrence of Gallic acid, Ellagic acid, and its derivatives through. High-performance liquid chromatography–mass spectrometry analysis and presence of Lactobacillus sp., Acinetobacter sp., Alcaligenes sp., and Methylobacterium sp in Kutajarista through 16 S rRNA gene clone library approach

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Prarthana, et al. : Probiotics from Dhanyamla - an ayurvedic formulation Asian Journal of Pharmaceutic s • Jul-Sep 2021 • 15 (3) | 389 in Kutajarista an Ayurvedic fermented herbal formulation prescribed for gastrointestinal disorders. The herbal formulation undergoes a gradual fermentative process and takes around 2 months for production with physicochemical changes in terms of alcohol percentage between 9% and 11% and a pH range from 3.6 to 3.8. A similar study was carried out by Ranasinghe and Ediriweera 2015 and reported Dhanyamla is to be light brown liquid with acidic odor and sour taste. Specific gravity and the pH of Dhanyamla was 1.0068 and 4.13 at 30°C. There was also reports of study was carried out by Elmahood and Doughari [20] for Kunun-zaki, an indigenous fermented non-alcoholic beverage which is widely consumed in Nigeria. The pH of the tested samples of Kunun-zaki was in a range of 3.34–4.42 Although probiotics and prebiotics in isolation offer significant health benefits, multi-strain formulations boost their effectiveness combinatorically as the gut microbiota is a large dynamic system that requires intricate crosscommunication to have significant clinical benefit. Hartmann et al ., 2015 reported a group of more than 20 small water soluble compounds Mycosporine-like amino acids (MAAs) with absorption maxima between 309 and 360 nm primarily identified through UV spectralstudies and confirmed through hydrophilic liquid chromatography. Geraldes and Pinto 2021 [21] suggested existence of MAAs are present especially in organisms that live in environments with high levels of UV radiation. Their composition varies according to the taxonomic group with the frequent coexistence of several MAAs with different absorption maxima allowing a more effective protective filter. Wittenberg (1960) isolated, for the 1 st time, compounds with high UV absorption from a siphonophore, Physalia physalis [22] In 1965, mycosporines were discovered in fungal sporulating mycelia [23] A few years later, MAAs have also been detected in corals and cyanobacteria from the Great Barrier Reef [24] Since then, MAAs have been identified in a wide variety of organisms, such as heterotrophic bacteria, [25] fungi, [26] cyanobacteria, [27] microalgae, [28,29] macroalgae, [30] lichens, [31] invertebrates (e.g., dinoflagellates, sponges, corals, sea urchins, and crustaceans) [32] and vertebrates (e.g., fishes) [33,34] In our study, there was absorption maxima of 3.078 between 300 and 350 nm indicating the probability of MAAs as preliminary data this has to be validated further, strikingly this compound is neither produced by plant-based ingredient nor by micro-organisms alone but here the aging process during fermentation forces MAAs synthesis, which gradually develops moreover week confirmed by spectral analysis. So far MAAs are attributed as antioxidant, but they also act as immunomodulators. There needs more research to expedite further medicinal properties of Dhanyamla ACKNOWLEDGMENT Authors would like to immensely thank S.D.M College, Ujire and S.D.M Institute of Ayurveda and Hospital, Bengaluru for Figure 5: Dendrogram of nearest sequence similarity matching with amplified sequences for primer 27 F Figure 6: Dendrogram of nearest sequence similarity matching with amplified sequences for 1492 R

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Prarthana, et al .: Probiotics from Dhanyamla - an ayurvedic formulation Asian Journal of Pharmaceutic s • Jul-Sep 2021 • 15 (3) | 390 providing laboratory infrastructure and teaching pharmacy facility to carry out the work REFERENCES 1. Chaudhary A, Singh N, Dalvi M, Wele A. A progressive review of Sandhanakalpana (Biomedical fermentation): An advanced innovative dosage form of Ayurveda. Ayu. 2011;32:408 2. Patil SM, Shirahatti PS, Ramu R, Prasad N. Azadirachta indica A. Juss (neem) as a contraceptive: An evidence-based review on its pharmacological efficiency. Phytomedicine 2021 a;88:153596 3. Patil SM, Shirahatti PS, Ramu R. Azadirachta indica A. Juss (neem) against diabetes mellitus: A critical review on its phytochemistry, pharmacology, and toxicology. J Pharm Pharmacol 2021 b;2021:rgab 098 4. Joshi D, Jha CB. Critical study of the asavaishta preparations of brhatirayee. Anc Sci Life 1990;9:125-33 5. Sharma PV, editor. Sushruta Samhita. 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Prarthana, et al. : Probiotics from Dhanyamla - an ayurvedic formulation Asian Journal of Pharmaceutic s • Jul-Sep 2021 • 15 (3) | 391 et al . Distribution, contents, and types of mycosporinelike amino acids (MAAs) in marine macroalgae and a database for MAAs based on these characteristics. Mar Drugs 2020;18:43 32. Shukla V, Kumari R, Patel DK, Upreti DK. Characterization of the diversity of mycosporine-like amino acids in lichens from high altitude region of Himalaya. Amino Acids 2015;48:129-36 33. Carreto JI, Carignan MO, Montoya NG, Cozzolino E, Akselman R. Mycosporine-like amino acids and xanthophyll-cycle pigments favour a massive spring bloom development of the dinoflagellate prorocentrum minimum in Grande bay (Argentina), an ozone hole affected area. J Mar Syst 2018;178:15-28 34. Chioccara F, Gala AD, De Rosa M, Novellino E, Prota G. Mycosporineaminoacids and related compounds from the eggs of fishes. Bull Soc Chim Belges 2010;89:1101-6 Source of Support: Nil. Conflicts of Interest: None declared.

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