Mechanism Of Naringenin In Promoting The Production And Maturation Of Porcine Myotubes And Its Application In Cultured Meat

Cultured meat is an emerging meat production technology formed on the highly interdisciplinary basis of cell biology,fermentation engineering,food science,etc.The poor differentiation efficiency of muscle stem cells in vitro leads to insufficient maturity of myotubes and protein(especially myosin)content,which is one of the technical bottlenecks limiting the commercialization of cultured meat.This study found that the natural compound naringenin(NAR)could promote porcine muscle stem cells to differentiate into mature myotubes,and analyzed the relevant molecular mechanisms.In addition,a serum-free differentiation medium based on NAR was developed and applied to myosin production at a bioreactor scale and preparation of high-quality diced cultured meat.The specific findings are as follows:(1)Bioactivity of NAR on porcine muscle stem cells.Firstly,the myogenic effects of five flavonoid compounds,including epicatechin,genistein,luteolin,quercetin,and NAR,were detected using model myoblasts.It was found that NAR had the best effect,with a myogenic fusion rate 5.70 times higher than that of ordinary differentiation medium(CM).Subsequently,the effect of NAR on the proliferation and differentiation of porcine muscle stem cells was investigated,and the results showed that up to 100 μM NAR had no cytotoxicity but no promoting effect on cell proliferation.Notably,20 μM NAR increased the myogenic fusion rate and the expression of myosin heavy chain(MYH)3 and MYH 8 of porcine muscle stem cells by 83%-178% and more than 50%.In the three-dimensional culture of porcine muscle stem cells,NAR induced the formation of "sarcomere" which represents myotube maturation.(2)The mechanism of NAR in promoting the generation and maturation of porcine myotubes.The results of immunoblotting indicated that NAR promoted the expression of estrogen receptor β(ERβ)and insulin-like growth factor-1(IGF-1),and significantly increased the phosphorylation levels of IGF-1R,AKT,and m TOR proteins.When the ERβ antagonist PHTPP was applied,the myogenic fusion rate induced by NAR was reduced from 57% to 43%,and the expression of IGF-1 and the phosphorylation levels of its downstream proteins were also decreased to less than 67% of the original level.The above results proved that NAR mediated ERβ to regulate the expression of endogenous IGF-1 and activated the anabolism pathway to promote the production and maturation of porcine myotubes.(3)Development of serum-free myogenic differentiation medium based on NAR and its application in bioreactors.Firstly,a serum-free basal medium(BMAI)that supported the survival and differentiation of myoblasts was developed by combining multiple serum substitutes.Subsequently,20 μM NAR was supplemented in BMAI to develop an efficient serum-free differentiation medium SFM.The survival rate of porcine muscle stem cells in SFM was similar to the serum-containing medium CM,but the myogenic fusion rate was significantly better than that of CM and BMAI.Furthermore,large-scale production of myosin was achieved in a bioreactor with the addition of SFM and the results showed that SFM increased the differentiation rate of porcine muscle stem cells to 62%(1.22 times that of the CM group),and the expression level of myosin was 1.26 times that of the CM group.These results indicated that the serum-free differentiation medium developed based on NAR supported efficient myogenesis and the large-scale production of myosin.(4)Preparation and quality assessment of diced cultured meat.The cultured meat samples with different myotube content and maturity were prepared in the proliferation medium(PM),CM,or SFM based on the texture vegetable protein scaffold,and their texture,chemical composition,and amino acids were analyzed.The results showed that the cultured meat sample prepared by SFM had a similar appearance and texture to commercial tenderloin,with water and protein contents of 80.24 ± 0.73 g/100 g and 15.22 ± 0.45 g/100 g,respectively,which were closest to commercial tenderloin.Moreover,the essential amino acid index(EAAI)of the SFMinduced cultured meat sample was 111.94,which was the highest in all samples and higher than the EAAI of standard high-quality protein(95).The above results indicated that SFM containing NAR promoted myotube maturation and myosin expression,thereby improving the quality of cultured meat.