EFFECT OF ETHANOL ON THE SOLUBILIZATION OF HYDROPHOBIC MOLECULES BY SODIUM CASEINATE
Open Access
- Author:
- Qu, Yanqi
- Graduate Program:
- Food Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- June 03, 2016
- Committee Members:
- John Neil Coupland, Thesis Advisor/Co-Advisor
Ryan John Elias, Committee Member
Federico Miguel Harte, Committee Member - Keywords:
- Drug Delevery
Casein
Milk protein
Solubilization Enhancement
Ethanol - Abstract:
- Many drugs and phytochemicals are insoluble in water, and therefore difficult to disperse in aqueous oral formulations. While some can be solubilized with surfactants or ethanol, there is a demand for alternative approaches. Casein has been shown to solubilize hydrophobic small molecules, and this might be an attractive option in some applications. Previous studies have shown that ethanol affects the conformation of the casein micelle, and in this work I consider if ethanol may facilitate the loading of sodium caseinate with lipophilic small molecules (i.e., pyrene and piperine). When ethanol and water are removed, I hypothesize the resultant powder can be readily rehydrated to produce an aqueous suspension of piperine- or pyrene-loaded caseinate with a total concentration in considerable excess to the aqueous solubility limit of the compounds. A large excess of pyrene or piperine was mixed with 2% (w/v) sodium caseinate in buffer (pH 6.7) and ethanol (0-40%, v/v), stirred for 24 h, and lyophilized to remove ethanol and water. The resultant powder was rehydrated in buffer to achieve a 2% casein suspension. The excess, insoluble pyrene or piperine was removed by gentle centrifugation and the concentration of solubilized fraction (i.e., dissolved in water, plus that which was bound to the the casein) was determined by UV-spectroscopy following extraction into acetonitrile or by fluorescence spectroscopy respectively. Pyrene is effectively insoluble in water (i.e., not fluorescing), however, in the presence of the caseinate, it was solubilized and showed a characterized fluorescence spectrum in buffer. The solubility of piperine in water was 49 mg/L, but in the presence of 2% casein (i.e., no ethanol used in preparation), the solubility increased to 134 mg/L (i.e.,50 moles of piperine per mole of caseinate). As the ethanol present at the solubilization step increased, the amount of solubilized piperine in the final product increased to a maximum of 342 mg/L (i.e., 175 moles of piperine per mole of caseinate) in 40% ethanol. I hypothesize that both pyrene and piperine were solubilized in water by binding to caseinate and that ethanol both facilitates mass transport to the protein from the insoluble powder by increasing the aqueous solubility of pyrene/piperine, and changes the conformation of the protein to increase binding. Next, the in vitro acid and proteolysis digestibility of the casein-pyrene complexes was tested over 48 hours. During the acid digestion, fluorescence dropped to zero immediately, because caseinate precipitated at the isoelectric point (i.e., 4.6) and pyrene remained the binding with precipitated protein. In the contrast, during the proteolysis, there was a gradual drop of fluorescence without protein precipitation, which meant pyrene were freed then stopped fluorescing. It suggests that the pyrene is able to be released in the gastrointestinal fluid during the enzymatic digestion. Finally the solubilizing capacity of piperine by various proteins (i.e., whey protein isolate, caseinate, native casein micelle extracts, and -lactoglobulin) were compared and caseinate performed the best.