Growth Factors for a Primary Chick Muscle Cell Culture from Shochu Distillery By-products

An unidentified growth factor (UGF) was separated from shochu distillery by-products (SDBP) and its effect on the growth of a primary chick muscle cell culture was investigated. Chick muscle cells were isolated from fertile eggs (13-day-old embryos) of commercial broilers. UGF was separated on Sephadex LH-20 with a solvent system of water–methanol–ethylene dichloride (10: 90: 20, v/v), and the fraction eluted between 136 and 164min was collected (fraction I). Fraction I was further purified by HPLC with an Inertsil ODS-2 column using a solvent system of methanol-butanol (80: 20, v/v). Three fractions having retention times of 3.76, 4.57, and 5.12 min were collected and are referred to as fraction A, B, and C, respectively. In experiment 1, chick muscle cells were cultured in an m-199 medium containing 0.001, 0.01, or 0.1% of fraction I. In experiment 2, chick muscle cells were cultured with 0.01 or 0.005% of each fraction A, B, and C. Creatine kinase (CK) activity, protein and DNA contents were measured as indices of myotube growth, cell growth and cell proliferation, respectively. Nτ-methylhistidine (Nτ-MH) release from the muscle cell was also measured to observe the effect on proteolysis. In experiment 1, the protein content was significantly (p < 0.05) increased by fraction I, despite the low dose level. CK activity was significantly (p < 0.05) higher than the control when 0.001% of fraction I was added to the medium. However, increasing the level beyond 0.01% did not further increase the CK activity. The DNA content was not significantly changed. In experiment 2, the protein content, CK activity, and DNA content were significantly (p < 0.05) higher when fractions A and B were added to the medium. However, this was not the case when fraction C was added. Nτ-MH release was significantly (p < 0.05) higher when fraction A was added, but, was significantly (p < 0.05) lower when fraction B was added, while fraction C had no effect on Nτ-MH release. The present results show that SDBP contained two growth-promoting factors for a primary chick muscle cell culture, although their modes of action may be different.