Intermuscular fat is essential for enhancing the flavor and texture of cultured meat. Mesenchymal stem cells derived from intermuscular adipose tissues are a source of intermuscular fat. Therefore, as a step towards developing a platform to derive intermuscular fat from mesenchymal stem cells (MSCs) for insertion between myofibrils in cultured beef, an advanced protocol of intermuscular adipose tissue dissociation effective to the isolation of MSCs from intermuscular adipose tissues was developed in cattle. To accomplish this, physical steps were added to the enzymatic dissociation of intermuscular adipose tissues, and the MSCs were established from primary cells dissociated with physical step-free and step-added enzymatic dissociation protocols. The application of a physical step (intensive shaking up) at 5 minutes intervals during enzymatic dissociation resulted in the greatest number of primary cells derived from intermuscular adipose tissues, showed effective formation of colony forming units-fibroblasts (CFU-Fs) from the retrieved primary cells, and generated MSCs with no increase in doubling time. Thus, this protocol will contribute to the stable supply of good quality adipose-derived mesenchymal stem cells (ADMSCs) as a fat source for the production of marbled cultured beef.

Acanthopanax species is known commonly as Siberian ginseng, touch-me-not, devil’s shrub, prickly eleutherococc, eleutherococc and wild pepper. A diverse group of chemical compounds isolated from Acanthopanax species was named ‘eleutherosides’. Among eleutherosides, eleutherosides B and E were widely known in Acanthopanax species. Acanthopanax species are cultivated and grow wild in a various area of Korea and have a variety of pharmacological effects. But, there are a lot of difficulties on producing excellent Acanthopanax species, according to the cultivated method is different pharmacological ingredients. This study, therefore, analyzed eleutherosides B and E in A. divaricatus and A. koreanum by different fertilizer ratio using HPLC. We will be investigated a high content of eleutherosides B and E by different fertilizer ratio and suggest an efficient fertilizer ratio of A. divaticatus and A. koreanum. All samples of A. divaricatus and A. koreanum were collected at Yeongcheon Agricultural Technology & Extension Center, Yeongcheon, Korea. The sample was prepared by upper and lower parts. The fertilizer ratio are N-P-K(10.5-8.5-8.5: 50 kg/10a), 2N-P-K (21-8.5-8.5: 50 kg/10a), N-2P-K (10.5-17-8.5: 50 kg/10a), N-P-2K (10.5-8.5-17: 50 kg/10a), and 2N-2P-2K (21-17-17: 50 kg/10a), respectively. To analyze eleutherosides B and E, 5 g of A. divaricatus and A. koreanum was extracted with 50% MeOH (3 × 100 ml) by reflux and evaporated in vacuo. The residue was dissolved in 1 ml of MeOH. The resulting solution was used for HPLC analysis. HPLC separation of eleutherosides B and E for qualitative and quantitative analysis was performed using a reverse phase system. A Discovery®C18 (4.6 × 250 mm, 5 μm) column was used with a mobile phase that consisted of water and acetonitrile. A gradient solvent system of water and acetonitrile (90:10 to 70:30 for 20 min) was used for the elution program. UV detection was conducted at 350 nm. The injection volume was 10 μl and the flow rate was 1 ml/min. All injections were performed in triplicate. The different fertilizer ratio yielded total eleutherosides B and E contents of 4.417-6.905 and 3.652-7.227 mg/g in the upper and lower parts of A. divaricatus, respectively. In A. koreanum, the total eleutherosides B and E contents were 4.591-10.108 and 3.834-9.079 mg/g in the upper and lower parts, respectively. The best conditions to increase eleutherosides B and E content in A. divaricatus was determined to be with N-2P-K fertilizer ratio, on the other hand, in A. koreanum was 2N-2P-2K fertilizer ratio.