Preparation of beef tallow injection matrix

The extraction and purification process of beef tallow by heat treatment and phase separation was performed according to a previously described lard extraction method with slight modifications⁵⁵. Fat around beef ribs was obtained from a butcher shop. The fat was placed on a stainless steel plate and heated at > 100 °C for 20 min to extract the tallow. In this process, the tissues were burnt and solid impurities were generated. To remove these, extracted beef tallow with impurities was passed through 50-µm mesh pores before solidification. Next, a phase separation method was performed at room temperature using distilled deionized water (DDW) to remove water-soluble impurities extracted with beef tallow. The extracted fat was bottled with boiled DDW, vortexed, and left at room temperature (23–25 °C) until solidified. The bottle cap was opened and water layer was removed. DDW was added to the solidified beef tallow, mixed, and layered at room temperature. When the beef tallow hardened, the DDW layer was removed. This process was performed five times. The obtained beef tallow was placed on a stainless steel plate and heated to > 100 °C to evaporate residual moisture. The beef tallow solution was then placed in a glass vial and stored at 4 °C.