HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATIONFACULTY FOR HIGH QUALITY TRAINING GRADUATION PROJECT Thesis code: 2022-18116020 PREPARATION OF FERMENTED SOYBEAN MEAL BY SOLID-STAT
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
SDS PAGE or Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis is a technique used to separate proteins based on their molecular weight.
SDS-PAGE is a technique that utilizes polyacrylate gel electrophoresis with SDS as a denaturing agent, allowing for the determination of protein molecular weight, composition, and purity In this method, proteins are treated with SDS detergent and a reducing agent, such as mercaptoethanol or DDT, which alters their secondary structure to a primary form and imparts a negative charge Consequently, during electrophoresis, the migration of protein molecules through the gel is solely dependent on their size, with larger molecules moving more slowly than smaller ones.
Conducting: Prepare the Resolving gel at the anode (12,5%)
Mix the mixture according to the following table:
Buffer Tris-HCl 1.5M pH 8.8 Distilled water 2 times 30% acrylamide solution 10% SDS solution APS 10%
To create a homogeneous solution, gently shake the mixture and use a micropipette to inject it into the mold, ensuring no air bubbles form, until the height reaches approximately 5-8 cm Next, carefully add a layer of distilled water on top of the gel and allow it to harden for 15-20 minutes.
Prepare the stacking gel at the cathode (4%).
Mix the mixture according to the following table:
Buffer Tris-HCl 1.5M pH 6.8 Distilled water 2 times 30% acrylamide solution 10% SDS solution APS 10%
Gently shake the mixture to achieve a homogeneous solution Once the separation gel has fully solidified, remove the upper layer of water Use a micropipette to carefully add the solution onto the solidified gel layer, ensuring no air bubbles are present, and insert the comb to create wells After the gel has set properly, carefully remove the comb.
To perform electrophoresis, carefully place the gel mold into the electrophoresis unit and fill the chamber with electrophoresis buffer Sequentially add 5-15 μl of the sample solution to the wells, then close the electrophoresis box and connect the circuit at 100V Run the process for approximately 2 hours at room temperature, maintaining a constant current of 30mA The electrophoresis buffer consists of 14.4 g/L of Glycine, 1 g/L of SDS, and 3.02 g/L of Tris-base, prepared with double distilled water to make a total volume of 1 liter.
After performing SDS-PAGE electrophoresis, carefully remove the gel from the plate and immerse it in a protein staining solution, gently shaking for 30 minutes to 1 hour Next, transfer the gel to a washing solution, changing it several times until the gel becomes transparent, revealing blue bands that indicate the presence of proteins The staining solution consists of 10% (v/v) acetic acid, 0.25% CBB – R250 or G250, and 50% (v/v) methanol, while the de-staining solution includes 10% (v/v) acetic acid, 25% (v/v) methanol, and 65% water.
Gel Permeation Chromatography (GPC) analysis
High Performance Liquid Chromatography (HPLC) is a method used to separate mixtures into individual components by utilizing the interactions between the analyte, the mobile phase (typically liquids), and the stationary phase (usually solids) In this process, the mobile phase transports the analyte through the stationary phase, causing components that have stronger interactions with the stationary phase to move more slowly compared to those with weaker interactions.
HPLC operates on a fundamental principle that involves separating a mixture's components into individual parts This separation is achieved by exploiting the varying affinities of different molecules for the mobile and stationary phases utilized during the process.
GPC analysis was conducted using an Agilent Technologies 1100 series UV probe coupled Liquid Chromatography (HPLC-UV) instrument equipped with an autosampler and a variable wavelength detector to assess molecular weight Each sample, with a volume of twenty microliters, was analyzed on two analytical columns: Ultrahydrogel 2000 (7.8x300 mm, 12 µm particle size) and Ultrahydrogel 250 (7.8x300 mm, 6 µm particle size) The analytical solvent comprised 70% acetic acid trifluoride solution (0.10%) and 30% acetonitrile, with eluted compounds detected by UV absorbance at 220 nm.
All samples were collected in triplicate, and the data were analyzed using one-way analysis of variance (ANOVA) Statistical significance was assessed with SPSS software, employing Duncan's Multiple Range Test at a significance level of p ≤ 0.05.
RESULTS
Selection of microorganisms with intense protease activity
Figure 3 1 Growth curve of strain LM 1.
Figure 3 2 Growth curve of strain LM 2. nm 60 0 A b s
Figure 3 3 Growth curve of strain LM 3 Figure 3 4 Growth curve of strain LM 4.
Assessing the growth capacity of bacterial strains involves surveying their growth curves, while evaluating the casein hydrolysis ability requires measuring the diameter of the proteolytic zone These two experiments are essential for selecting the most suitable strains for fermentation processes.
The growth rates of four microbial strains in NBC medium at 37°C show significant differences, as evidenced by the four growth curve plots Notably, LM 1 exhibits a brief log phase duration with a relatively low peak, leading to a rapid transition into the death phase.
The LM 4 and 2 strains exhibit a prolonged lag phase, leading to a gradual decline in growth rate and a subsequent rapid death phase In contrast, the LM 3 strain begins to grow at the 3rd hour, stabilizes at 15 hours, and maintains this growth for 8 hours before entering the death phase, resulting in a bacterial content of 2.7 × after 24 hours of culture.
The LM 2 and LM 4 strains exhibited a significantly higher bacterial density during the stationary phase compared to the LM 1 and LM 3 strains All four strains, LM 1, LM 2, LM 3, and LM 4, reached the stationary phase in 12 hours.
Table 3 1 Diameter size (mm) hydrolysis of 4 microbial strains on YNB agar plate at 37˚C.
Different values printed in the same row indicate a statistically significant difference (p