13J Contemp Med Sci | Vol. 1, No. 4, Autumn 2015: 13–15 Research Objectives Because of the less studies in this field in Iraq, this study aimed to use local Streptococcus pyogenes isolates to produce hyluronic acid. Methods The quantitative estimation of hyaluronic acid (HA) produced from eight local S. pyogenes isolates at different pH (6.3, 6.6, 6.9, 7.2, and 7.5) and glucose concentration (4%, 6%, 8%, and 1%) were done using the HA ELISA kit. Results This study showed that the maximum yield of HA was obtained at pH 7.5, and it was found that the differences in pH of the production media of HA enhanced the HA production, but the glucose concentration has no beneficial effect for HA production. Conclusion There were differences in HA production among local isolates at the same pH. Keywords Streptococcus pyogenes, hyaluronic acid, HA ELISA kit, pH, glucose concentration Production and optimisation of hyaluronic acid extracted from Streptococcus pyogenes Kawkab Abdulla Al-Saadiaaa, Hassan Fadhil Najib, & Ali Hmood Al-Saadib Introduction Hyaluronic acid (HA), also known as hyaluronan, is a linear polysaccharide (up to 10 MDa) consisting of alternating units of (1,4)-glucuronic acid and (1,3)-N-acetyl-D-glucosamine (GlcNAc).1 Additionally to retaining and modulating the flow of water, it also helps as the backbone for proteoglycan gath- ering by binding aggrecan monomers via link protein.2 Fur- thermore, HA carries numerous functions in altering cellular functions during the development of human, specifically during the development of diarthrodial joints. In developed cartilage, HA has a great significance of ligand for cell-matrix interactions with pericellular matrix via the CD44 cell receptor.3 The physical, chemical, and biological attribution of the HA includes lubricity, visco-elasticity, holding of water, biocompatibility, cell multiplication, morphogenesis, inflam- mation, and wound repair as well as specific signal transduc- tion and cellular interactions through cell surface receptors.4,5 HA is highly hygroscopic, biocompatible, and decompos- able biopolymer, real attractive for biomaterials fabrication. It is intensively used in cosmetics, surgery, and delivery of drugs.6 HA has been normally extracted from rooster combs and bovine vitreous humor. However, it is difficult to isolate high molecular weight HA at industrially practicable rate from these sources, because it makes a complex with proteoglycans exist in animal tissue. It is presently impractical to manipulate the molecular weight of the biopolymer while it is synthesized in animal tissue. Moreover, the use of animal-derived bio- chemicals for human therapeutics has embossed ethical out- comes, and is met with growing resistance. To overcome these disadvantages, the recent tendency involves the usage of Lancefield’s group A and group C Streptococci, which natu- rally produce a mucoid capsule of HA.7 The requests for HA products from bacterial fermentation have fundamentally expanded because of both their increased use as medical devices and the immune issues that happened from the use of animal-based HA5. Because of both the high prices of HA and the high standard requirements of its appli- cations in medical products, high-quality HA products rather than high quantity have been the essential criteria utilized when selecting the bacterial strains utilized for HA generation. Streptococci are ideally meant and adapted for studying the biosynthesis of HA due to the abundant availability of hyaluro- nate and since in this organism the hyaluronate is the only pol- ymer into which glucuronic acid is comprised.8 In Streptococci, HA is created as a secondary metabolite and the production is affected by different agents that involve genetic as well as nutritional. Streptococci produces HA both under aerobic and anaerobic condition.9 Materials and Methods Bacterial Isolates Eight local isolates of Streptococcus pyogenes were isolated from ENT infectious, and identified depending on traditional methods as described by Macfaddin,10 in addition to use the strepto-system 9R according to the manufacture’s instructions and molecular methods by amplification of universal and specific species genes. These isolates are given numbers 1, 2, 3, 4, 5, 6, 7, and 8. Isolation of HA HA creating bacteria were chosen based upon their hemolytic character. Best hemolysis producing colonies were picked from every blood agar plate and streaked on Todd Hewitt agar plates. The plates were incubated at 37ºC in a 5% CO2 atmos- phere for 24 hours.9 pH Effect on the Production of HA The effect of pH on the production of HA was measured by the following technique. Ten milliliters of Tood Hewitt broth (THB) medium kept up at five different pH (6.3, 6.6, 6.9, 7.2, and 7.5) were inoculated with a loopful of S. pyogenes isolates, and incubated at 37ºC for16 hours. The overnight cultures were inoculated into 10 ml of fresh THB medium and inocu- lated at 37ºC for 24 hours under shaking condition. The resulting suspensions were centrifuged, washed with 10 ml of 10 mM Tris HCl (pH 7.5) and vortexed for 10 seconds.9 Glucose Concentration Effect in the Production of HA The effect of glucose concentration in the production of HA was measured by the following technique. Four conical flasks ISSN 2413-0516 aUniversity of Karbala, College of Science, Department of Biology, Iraq. bUniversity of Babylon, College of Science, Department of Biology, Iraq. Correspondence to Kawkab Abdulla Al-Saadi (email: kowkab_abdalla@yahoo.com). (Submitted: 30 August 2015 – Revised version received: 26 September 2015 – Accepted: 9 October 2015 – Published online: Autumn 2015) 14 J Contemp Med Sci | Vol. 1, No. 4, Autumn 2015: 13–15 Production and optimisation of HA extracted from Streptococcus pyogenes Research Kawkab Abdulla Al-Saadiaa et al. containing 10 ml of THB medium, each was supplemented with different glucose concentration (0.4, 0.6, 0.8, and 1%) were inoculated with 15–30 colonies of S. pyogenes isolates, and incubated at 37ºC for 16 hours. The overnight culture were sub-cultured in to 10 ml of fresh THB media and incu- bated at 37ºC for 24 hours under shaking condition. The resulting suspensions were centrifuged, washed with 10 ml of 10 mM Tris HCl (pH 7.5), and vortexed for 10 seconds.9 HA Extraction Bacterial cells cultivated under different condition were pel- leted out and the cell pellets were resuspended in 1.5 ml of water and vortexed for 10 seconds. This washing sequence was repeated twice and the cell pellets were resuspended in water to a final volume of 1.5 ml. The HA capsule was extracted by adding 1.5 ml of chloroform and shaking for 1 minute. Cell remained at room temperature for 1 hour and then was pelleted and the aqueous phase was used for estimation.9 Quantitative Estimation of HA by HA ELISA Kit The quantitative estimation of HA according to the manufac- ture’s instructions of the HA ELISA kit (Elabscience/China) was carried out. Results Quantitative Estimation of HA Eight different isolates of S. pyogenes with pathogenic characters were used for HA production. After the cultivation of S. pyo- genes isolates in Todd Hewitt agar plates, the large white mucoid colonies were selected for HA production. The HA content of these isolates, which were incubated at different pH, were ana- lyzed according to HA ELSA kit protocol. Based on the results obtained in Fig. 1 the maximum HA yield was observed with isolated numerated one at pH 7.5 (67.9 ng/ml). While the same pH (7.5) shows no HA production in isolated number 5, 6, 7, and 8. Discussion The ELISA procedure is sensitive, simple, and is based on a microtitre plate format. The assay involves competition between HA absorbed to the plate and HA free in solution for binding to biotinylated cartilage proteoglycan binding region. The range of the assay is 10–2500 ng/ml with 50% inhibition at 0 10 20 30 40 50 60 70 1 2 3 4 5 6 7 8 HA concentration (ng /ml ) S. pyogenes isolates pH 7.5 pH 7.2 pH 6.9 pH 6.6 pH 6.3 Fig. 1 HA yield (ng/ml) generated from S. pyogenes under different pH. about 200 ng/ml. This technique involves fewer experimental steps and is simpler to perform than other methods.11 Production media were maintained at an optimised pH of some isolates estimated at four different glucose concentrations (0.4, 0.6, 0.8, and 1%) according to the ELSA kit protocol. Based on the results shown in Fig. 2, all of selected isolates undergo an acute decline in HA production. The study by Saranraj et al.9 showed gradually increase of HA yields with an increase of glucose concentration. The decline of HA yields may be due to the decrease of the pH of the medium resulted from the high consumption of the carbon sources, which led to the production of organic acid and reduce the pH of the medium.12 The other reason may be its agitation speed. The increase of agitation speed led to a sig- nificant decrease of cell growth and HA production.12 Fig. 2 HA yield (ng/ml) generated from some S. pyogenes isolates under different glucose concentration. 0 2 4 6 8 10 12 HA  concentration   (ng)    0.4%          0.6%               0.8%           1%   glucose  concentration    1- 7.2 1-7.5 2-6.6 6-7.2 7-6.6 References 1. Boeriu CG, Springer J, Kooy FK, van den Broek LA, Eggink G. Production methods for hyaluronan. International Journal of Carbohydrate Chemistry. 2013;2013:1–14. doi: http://dx.doi.org/10.1155/2013/624967 2. Mankin H, Mow V, Buckwalter J, Iannotti J, Ratcliffe A. Form Function of Articular Cartilage. Orthopaedic Basic Science. Rosemont, Ill: American Academy of Orthopaedic Surgeons; 1994. pp. 1–44. 3. Knudson CB. Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix. J Cell Biol. 1993 Feb;120(3):825–834. doi: http://dx.doi. org/10.1083/jcb.120.3.825 PMID: 7678838 4. Burdick JA, Prestwich GD. 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