| 5’-Inosinic acid（IMP）is an important indicator of meat flavor.After animals are slaughtered,a certain amount of ATP is synthesized when oxygen supply is stopped,and it is rapidly degraded into IMP.IMP is mainly synthesized by denovo way,and finally adenylic acid（AMP）and guanylic acid（GMP）can be produced.AMP and GMP can remedy the formation of IMP.In this study,it was used as experimental material,and an efficient extraction and detection method of IMP in muscle of Lueyang black-bone chicken was established by optimizing chromatographic conditions The changes of IMP content in muscle at different ages（60,90,120,150,180,210 d）,different tissues（leg muscles and chest muscles）and different storage temperatures（4℃and-18℃）were analyzed by optimized method.We compared the differences of IMP gene expression by RT-q PCR in different sexes,different ages and different tissues,and discussed the correlation between IMP content and related gene expression.The analysis results show that:（1）A chromatographic method with high accuracy and good reproducibility was established in this experiment.With DIONEX Ulti Mate3 000 high performance liquid chromatograph,the chromatographic column,detection wavelength,mobile phase system and mobile phase ratio were controlled in turn.Through the detection and analysis of the standard substance,the best determination conditions were finally determined as follows:Cosmosil packed column C18（4.6 mm×250 mm,φ5μm）as chromatographic column;Methanol-triethylamine phosphate（4:96）was used as mobile phase,the column temperature was 25℃,the detection wavelength was 248 nm,and the injection volume was 20μL.The limits of detection were 0.02μg/m L and 0.08μg/m L,respectively,and the RSD of precision was 0.30%～4.78%.The average recovery rate was 99%and RSD was 2.71%.The linear range of IMP is 12.2～97.6μg/m L,R2=0.9999,and the linear relationship is good.In addition,the IMP in muscle was extracted by different treatments,and the IMP contents of fresh meat,freeze-dried samples,fresh meat degreased samples and freeze-dried degreased samples were0.014 g/kg,0.018 g/kg,0.023 g/kg and 0.029 g/kg,respectively.It is found that pretreatment can reduce fat interference and improve the extraction rate of IMP.（2）This experiment explored the factors affecting IMP content,such as age,sex,tissue and storage temperature.The results showed that the IMP content of Lueyang black-bone chickens with the same feeding conditions was significantly different between genders,and that of hens was larger than that of cocks（P<0.05）.With the increase of age,the content of IMP increased at first and then decreased（P<0.01）,with the highest content at 150 days old.IMP content in chest muscle was significantly higher than that in leg muscle in different tissues（P<0.01）.Storage temperature has a great influence on inosinic acid content.When stored in cold storage at 4℃,the IMP content increased significantly on the second day,which is the best time for eating,and then the IMP content gradually decreased.The loss rate was 9.36%on the fourth day and 19.46%on the sixth day,and the chicken deteriorated obviously on the eighth day.When frozen at-18℃,the content of IMP on the 8th day was higher than that on the 1st day,which was the best time for eating.The content of IMP changed slightly within 15 days,and lost 5.24%.The content of IMP was close to 2～4 days after 22 days and 4～6 days after 29 days.The best time for frozen storage was0～15 days,and the freezing effect was obviously better than that of cold storage.（3）By analyzing the expression laws of target genes related to IMP synthesis and degradation,it was found that the expression levels of GPATase,GARS-AIRS-GART,AMPD and GMPR genes in hens were extremely significant higher than those in cocks（P<0.01）,and the expression levels of pur H genes in hens were significantly higher than those in cocks（P<0.05）.There was no difference in the expression levels of ADSS and IMPDH genes between cocks and hens.With the increase of age,the expression of GPATase,GARS-AIRS-GART,pur H,AMPD and GMPR increased first and then decreased,while ADSS and IMPDH decreased first and then increased.The expression of GPATase,GARS-AIRS-GART,pur H,AMPD and GMPR genes in different tissues was extremely significant higher than that in leg muscles（P<0.01）,while the expression of ADSS and IMPDH genes had no significant difference between chest muscles and leg muscles（P>0.05）.The expression law of the abundance of IMP-related genes implies the reasons for the lasting flavor of Lueyang black-bone chicken:AMPD gene can transform AMP into IMP,and AMPD gene is expressed in large quantities,supplementing IMP content to maintain the flavor of meat;GMP is also one of the taste-presenting nucleotides,and the high expression of IMPDH gene is another important reason for maintaining the delicious taste of chicken.The above conclusions provide a basic basis for the gene regulation mode of meat flavor lasting.（4）Pearson correlation coefficient was used to analyze the correlation between IMP content and its synthetic degradation genes.It was found that IMP content was positively correlated with GPATase,GARS-AIRS-GART,pur H,AMPD,GMPR genes expression（P<0.05）,and there was a significant negative correlation with ADSS and IMPDH genes expression（P<0.01）.Therefore,IMP synthesized GPATase,GARS-AIRS-GART and pur H from scratch,degraded metabolic genes ADSS and IMPDH,and remedied synthesized genes AMPD and GMPR may be involved in the regulation of IMP content in muscle tissue of Lueyang silky fowl.It provides a theoretical basis for breeding high-quality Lueyang black-bone chicken.（5）From the content of IMP and the expression law of related genes,it was found that the content of IMP was the highest in the muscle of Lueyang Black Chicken at 150 d,while the expression of synthetic genes related to IMP was the highest and the expression of degradation genes was the lowest in the muscle of Lueyang Black Chicken at 150 d.Therefore,from the angle of flavor,150 d Lueyang black-bone chicken is suitable for slaughter,which provides a theoretical basis for the development of Lueyang black-bone chicken industry. |
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