Protein concentrations of three liquids, Kombucha tea, skim milk, and chicken soup were determined using a method developed by Bradford (1976). The amount of protein found in Kombucha tea was nearly half that of skim milk. It was suggested that this protein consists primarily of extra-cellular enzymes excreted by the micro-organisms in the tea in order to break down macro-molecules too large to penetrate the cell walls.
Proteins are high molecular-weight polymer compounds composed of a variety of amino acids which are joined together by peptide linkages. Proteins are build from 20 kinds of amino acids. Amino acids have a carboxyl and amino group in common and only differ in the side chains attached to the alpha carbon. Each type of protein has a unique sequence of amino acids (primary structure). This string of amino acids in turn is repeatedly coiled or folded (secondary structure). The coils and folds are arranged in a three-dimensional shape (tertiary structure) typical for each protein (Campbell, 1993).
Bradford (1976) has developed a fairly quick and sensitive method for the determination of small protein quantities in solutions. This method is based on the fact that the maximum light absorption of Coomassie Brilliant Blue dye at various wavelength experiences a shift from 465 to 595 nm when allowed to bind to protein. An increase in protein concentration results in a corresponding increase in optical density or light absorbency. According to Bradford the relationship is fairly linear with only a slight curvature.
Reagents: Bradford reagent consisting of Coomassie Brilliant Blue G-250 and phosphoric acid dissolved in ethanol and distilled water according to the method developed by M. Bradford (1976).
Protein and sample solutions: 1. Bovine serum albumin, 2. Kombucha tea 3. Skim milk, 4. Instant cream of chicken soup dissolved in 250 ml of cold water
Instruments: Test tubes, pipettes, cuvets, spectrophotometer.
Procedures for the establishment of a standard graph: Test tubes were filled with 0.1 ml of bovine serum albumin at the following concentrations: 0.2, 0.3, 0.5, 0.7 and 1.0 g/l and one with 0.1 ml of distilled water. 5 ml of the Bradford reagent was added to each of the solutions which were allowed to incubate for 10 minutes at room temperature to allow binding of the dye to the protein. Sufficient amounts of the individual solutions were transferred to cuvets for the individual readings. The spectrophotometer was set at 595 nm and the optical density indicator was zeroed using a cuvet with the solution containing the Bradford reagent and distilled water. Readings were taken for each of the remaining solutions. The entire procedure was repeated one more time and the averages between the two tests were recorded in table 1 and then plotted on a graph (chart 1). This graph was used as the standard reference for determining unknown protein concentrations.
Procedures for determining protein concentrations for sample solutions: Test tubes were filled with 0.1 ml of each of the solutions; skim milk, soup mix, and "Kombucha" tea, also one was filled with distilled water for establishing a base line. 5 ml of the Bradford reagent were added to each tube. The solutions were allowed to stand for 10 minutes at room temperature to allow binding of the dye to the protein. Cuvets were again filled with the solutions. The spectrophotometer was set to 595 nm and the optical density indicator reset to zero using the solution with the distilled water. After that, optical densities were determined for each of the solutions with the unknown concentration of protein. Using the standard graph established above, the protein concentration was determined.
Standard graph: As reported by Bradford, the graph showing the relationship between the standard protein solution (BSA) and optical density (chart 1) was fairly linear. The curvature for BSA concentrations greater than 0.7 g/l was, however, quite pronounced.
Protein Concentration of Three Solutions: At first, readings of optical densities were obtained for 1:1 solutions. Since the results were outside the linear range of the standard chart, a new series of tests were made with 1:50 solutions. To obtain these solutions, one part of the three samples was diluted with 50 parts of distilled water. Of this, 0.1 ml was mixed with the Bradford reagent and readings of optical densities were taken with the spectrophotometer and entered into table 2. Protein concentrations were read off the standard graph (chart 1) and multiplied by 50. The results were as follows:
|Sample||Optical Density||Protein g/l|
Table 1: Protein Concentration of Three Sample Solutions
Standard Graph: The results indicated a mostly linear relationship between optical density and protein concentration of BSA up to 0.7 g/l. There was a marked curvature beyond that which did not correspond to the literature (Bradford). This may have been caused by inaccuracy during the preparation of the solutions or the execution of the measurements.
Protein Concentration of Three Solutions: As expected, both the skim milk and the chicken soup contained significant amounts of protein (37.5 and 36.0 g/l respectively). The third solution, Kombucha tea, contained about half as much protein as skim milk (15.0 g/l). This result was surprising since the dye does not react to the proteins within microorganism cells. I concluded that these proteins found in the tea must consist of various enzymes secreted by the yeast and bacteria to break down the large molecules of different nutrients in the tea which usually cannot enter cells directly, for example sucrose (white sugar) and caffein. The tea was prepared following published instructions and contained , according to Frank (1994), black tea, sugar, several types of bacteria, yeast and compounds resulting from the fermentation and other metabolic processes, namely acetic acid, glucuronic acid, vitamins and ethanol. Since the culture continuously forms new layers of a zooglea on the surface of the liquid, a certain amount of cellulose would also be expected in the liquid. According to Bradford, none of the listed compounds should have had any significant influence on the indicator dye. Boyd (1984) stated that many microorganisms release extra-cellular enzymes into the liquid around them to break down larger molecules into smaller ones which then can cross the phospholipid membrane barrier for processing inside the cell.
Note: This research was carried out in the biology lab at St. Olaf College in Northfield, Minnesota.
First published on the Internet June 18, 1998