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Unlocking the Potential of β-galactosidase: From its Source to a Modern Application Beyond Lactose Hydrolysis

Ruiz-Ramirez, Silvette Melissa
http://orcid.org/0000-0002-9257-9966
http://rave.ohiolink.edu/etdc/view?acc_num=osu1712567452031791

Abstract Details

2024, Doctor of Philosophy, Ohio State University, Food Science and Technology.
This dissertation investigates the enzymatic parameters of a β-galactosidase derived from the probiotic candidate, Lactobacillus helveticus OSU-PECh-4A (Lb. helveticus), with a focus on its potential for galacto-oligosaccharide (GOS) production. β-galactosidase is a hydrolytic enzyme that catalyzes the breakdown of lactose into its simpler sugar, glucose, and galactose. Simultaneously with lactose hydrolysis and under specific conditions, β-galactosidase can also produce highly demanded prebiotics known as galacto-oligosaccharides (GOS). In this dissertation, a thermostable β-galactosidase from Lb. helveticus has been isolated through diafiltration and size-exclusion chromatography and characterized through enzymatic assay and differential scanning fluorimetry (DSF). The isolated enzyme consists of a heterodimer with a molecular mass of 110 kDa, with a small and large subunit of 36 kDa and 74 kDa, respectively. The Km and Vmax values for lactose hydrolysis were 29.87 ± 1.05 mM and 1.88 ± 0.02 μmol D-glucose released per min per mg of protein, respectively. The enzyme is stable under a wide range of pH and high temperatures in terms of activity (≥ 40 °C). Once characterized, the enzyme capacity to produce GOS was evaluated through a 2x2 factorial design using substrate concentration and temperature as variables. The GOS produced were quantified and identified using high-performance liquid chromatography coupled with a charged aerosol detector (HPLC-CAD) and liquid chromatography with electrospray ionization mass spectroscopy (LC-ESI-MS/MS), respectively. To provide a perspective of the many possible applications of the enzyme, the enzyme's capacity to use different sugar acceptors to produce fucose-containing GOS was also evaluated by comparison. A maximum GOS yield of 12% was obtained at an initial lactose concentration of 200 g/L under 45 °C for 12 h. The GOS mixture consisted of 82% GOS, from which 6’galactosyl-lactose (trisaccharide), lactulose (disaccharides), and potential trehalose (disaccharides) were identified. In the presence of fucose, fucose-containing oligosaccharides were detected, demonstrating the enzyme's capacity to use different glycosides as sugar acceptors. Lastly, the effect of Lb. helveticus as the carrier of a β-galactosidase during the manufacture and storage of a functional yogurt was investigated. The yogurt's pH, lactic acid content, composition, rheology, and volatile profile were monitored through 28 days of storage. Lb. helveticus OSU-PECh-4A showed remarkable probiotic properties, allowing its incorporation as a safe starter in the development of functional whole milk stirred yogurt. The supplementation of this strain does not affect fermentation, post-acidification, composition, or volatile profile when compared to traditional yogurt. However, the yogurt supplemented with Lb. helveticus produced a thicker and more viscous gel than traditional yogurt. This dissertation underscores the practical and consumer advantages of using the β-galactosidase derived from Lb. helveticus not only for GOS formation as food ingredients but also for fucosylated sugars and its in-situ formation in dairy products with probiotic benefits, presenting a significant contribution to the future of probiotic-derived enzymes and functional food products.
Rafael Jiménez-Flores (Advisor)
Valente Álvarez (Committee Member)
Monica Giusti (Committee Member)
Osvaldo Campanella (Committee Member)
153 p.
Food Science
Lactic Acid Bacteria, Galactooligosaccharides, Beta-galactosidase, Lactobacillus helveticus, lactose hydrolysis

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Citations

  • Ruiz-Ramirez, S. M. (2024). Unlocking the Potential of β-galactosidase: From its Source to a Modern Application Beyond Lactose Hydrolysis [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1712567452031791

    APA Style (7th edition)

  • Ruiz-Ramirez, Silvette. Unlocking the Potential of β-galactosidase: From its Source to a Modern Application Beyond Lactose Hydrolysis . 2024. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1712567452031791.

    MLA Style (8th edition)

  • Ruiz-Ramirez, Silvette. "Unlocking the Potential of β-galactosidase: From its Source to a Modern Application Beyond Lactose Hydrolysis ." Doctoral dissertation, Ohio State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=osu1712567452031791

    Chicago Manual of Style (17th edition)

osu1712567452031791
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