Princely, Gnanakani (2008) Beta-Galactosidase Production from Streptococcus thermophilus Isolated from Milk Yoghurt. Masters thesis, Nandha College of Pharmacy, Erode.
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Abstract
Beta-Galactosidase was an intracellular enzyme isolated from Streptococcus thermophilus grown in whey. The Beta-Galactosidase from Streptococcus thermophilus exhibited twice the activity of enzyme from Saccharomyces fragilis being stable at 4 °C and having high heat stability. Also whey utilization for the production of many valuable products has been extensively studied. One of the major obstacles to the whey utilization is lactose content, which causes crystallization at low temperatures, low sweetness, and poor digestibility when used as food. These problems can be solved if whey lactose is hydrolyzed to glucose and galactose. Nine samples from commercial brand yoghurts namely, Amul (A1, A2, A3, A4, A5, A6, A7, A8, A9), Nilgiris (N1, N2, N3, N4, N5, N6, N7, N8, N9), and Nestle (NE1, NE2, NE3, NE4, NE5, NE6, NE7, NE8, NE9) were collected. From Amul, the samples A1, A4, A5, and A6 showed clear white colonies and were isolated and subcultured in MRS broth for further studies. From Nilgiris, the samples N5 and N6 showed clear white colonies and were isolated and subcultured in MRS broth for further studies. From Nestle, the samples NE4 and NE5 showed clear white colonies and were isolated and subcultured in MRS broth for further studies. Microscopical identification of the sample isolates were done. Five sample isolates A4, A5, A6, N6, and NE5 were identified as spherical or cocci shaped, grampositive, and non-motile, having characteristics of Streptococcus species. Three sample isolates A1, N5, and NE4 were identified as rod shaped, gram-positive, and non-motile, having characteristics of Lactobacillus species. The five sample isolates A4, A5, A6, N6, and NE5, which are having characteristics of Streptococcus species were used for further studies. Biochemical characterization was done for the sample isolates A4, A5, A6, N6, and NE5, which gave negative results for catalase test and mannitol fermentation test and positive results for glucose fermentation test, lactose fermentation test, and ST agar test. ST agar test was the identification test for Streptococcus thermophilus, so the five isolates A4, A5, A6, N6, and NE5 were subcultured in MRS broth and used for further studies. Production of Beta-Galactosidase enzyme were done for the five isolates A4, A5, A6, N6, and NE5, examined synthesized beta-galactosidase with yields ranging from 5.07 to 8.36 U/ml. But the sample isolate A5 was selected for further studies because of high productivity (7.76 U/ml). Partial purification of Beta-Galactosidase were done by Ammonium sulphate precipitation method (70%), dialysis, and gel filtration chromatography on Sephadex G-100. The fractions 5, 14, 15, 20 gave high peaks. In these, peaks 14, 15 showed enzyme activity. So these fractions were pooled and used for further studies. The protein estimation was performed by Lowry’s method and the protein concentration for the sample isolate A5 was found to be 67 μg/ml. Purification of Beta-Galactosidase enzyme was performed by SDS-PAGE. The comparison was done with standard Beta-Galactosidase, the partially purified Beta-Galactosidase bands was shown near to standard Beta-Galactosidase band. The assay of Beta-Galactosidase was performed by constructing ONP standard curve. The enzyme activity for sample A5 was found to be 7.76 U/ml. The units of enzyme for sample A5 was found to be 3.6 milliunits by constructing standard curve using standard Beta-Galactosidase enzyme for determination of enzyme activity. The ONPG confirmatory test for Beta-Galactosidase was done. The disc changed to yellow colour, which indicated the presence of Beta-Galactosidase enzyme. The optimization of substrate concentration was performed using ONPG as substrate. The optimum substrate concentration was found to be 24 mM. The active sites of enzyme were saturated with the substrate molecules and blocked further action. From the Lineweaver-Burk plot, Km and Vmax values were found to be 3.05 mM and 2.8 U/ml respectively. The effect of pH and temperature was performed using ONPG as substrate. The optimum pH and temperature were found to be 7.2 and 40 °C. Finally, it can be concluded that Beta-Galactosidase enzyme is a major source of Streptococcus thermophilus and also whey is utilized, which could otherwise be an environmental pollutant. The results presented here indicate that S. thermophilus grown in whey may represent a good source for the production of commercial lactase. The S. thermophilus strain contained plasmids, which can be used as a vector in recombinant DNA technology. Also Beta-Galactosidase enzyme is used as a reporter gene in recombinant DNA technology and immunology.
| Item Type: | Thesis (Masters) |
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| Additional Information: | Reg No. 26063826 |
| Uncontrolled Keywords: | Beta-Galactosidase Production ; Streptococcus thermophilus ; Isolated ; Milk Yoghurt. |
| Subjects: | PHARMACY > Pharmaceutical Biotechnology |
| Depositing User: | Ravindran C |
| Date Deposited: | 21 Jun 2017 09:44 |
| Last Modified: | 16 May 2018 09:00 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/183 |
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