Identification Of Chicken Meat Quality Via Isoelectric Focusing

Beef,pork,mutton,and poultry products are the most sustainable and edible protein sources around the world.Due to their direct influence on health,meat quality has attracted public attention;still there is no general technique available for identifying and controlling meat quality.A novel isoelectric focusing(IEF)technique based on myoglobin(Mb)and hemoglobin(Hb)was proposed for the evaluation of chicken quality.The main design principles are as follows.We selected chickens as model animals for experiments.Generally,chickens are slaughtered by cutting the blood vessels in the neck;since the Hb content in the slaughtered and bloodletting chickens is very low,thus difficult to detect,and only myoglobin can be detected.However,if the chickens are died by other reasons(such as infectious,flood(drowning),heatstroke,freezing and other processing methods),Hb remains in the chicken;hence both Mb and Hb species can be detected at the same time.In theory,it is possible to distinguish normally slaughtered chicken from abnormally slaughtered based on the presence or absence of Hb.Since Hb species are only detected in the abnormally slaughtered chickens,thus,this technology has the potential to become a general method for the detection of chicken meat quality.To demonstrate the developed protocol,twenty-four chickens were divided into three groups;each had eight chickens.The chickens in Group 1 were slaughtered by exsanguination,Group 2 asphyxiated in water,and Group 3 were infected by new castle disease virus(NDV),respectively.Proteins were extracted from the meat samples by using pure water as an extractant,separated by IEF,Hb and Mb were verified by western blot and finally quantified via imaging analysis.The relevant experiments demonstrated that two myoglobin(Mb)bands were detected at pI 6.8 and 7.04 for all samples of Group 1,2 and 3,but there were additional hemoglobin(Hb)bands at pI 7.09 and 7.13(P < 0.05)for the samples of Group 2 and 3.The RSD values of two Mb bands(pI 6.8 and 7.04)in Group 1 were respectively,4.08% and 3.63%,the ones in Group 2 of two Hb bands(pI 7.09 and 7.13)were 3.66% and 2.10%,and these in Group 3 were 2.17% and 2.77%,respectively.All the RSD values indicate that the developed method has good stability and reliability.From the above comparison experiments,it could be simply evaluated that hemoglobin can be used to distinguish the quality of normal and abnormally slaughtered chickens.The results implied that Hb bands might be a potential biomarker for the determination of chicken meat quality.Additionally,the protocol had a direct readout of protein bands in IEF without staining.The developed general method and findings have the potential for screening of chicken meat quality.Although the developed method based on traditional isoelectric focusing(IEF)and protein extraction could be used for the determination of meat quality.However,it still suffered from some issues such as time-consuming,unavailability of online detection,laborious,and cost-effective performances,e.g.,3 hrs of protein extraction,more than 10 hrs rehydration time,5-12 hrs focusing time,and 5-10 hrs staining and imaging of protein band.To overcome these issues,a speedy extraction of colorful proteins was developed by controlling extraction and centrifugation of 0.2 g sample within 10 min and 15 min respectively.A rapid analytical method was designed by using a quick array IEF with 25 min rehydration,7 min focusing,2 min online scanning and imaging of focused proteins,and the total analytical time was controlled within 1 hr,significantly less than the original running time of 24 hrs.The experiments demonstrated that two Mb bands with pI 6.8 and 7.04 were present in normal group(slaughtered chickens),while four other bands with pI 6.83,6.95,7.09,and 7.13 were observed in abnormal group chickens.The additional four proteins were verified by western blot(WB)as hemoglobin proteins.Furthermore,array IEF has high sensitivity for protein detection of chicken processed in different ways;(absolute LOD of Mb 1.3ng and Hb 5.5 ng),fair stability(RSD values of 2.32%,2.27%,and 1.69 %)for slaughtered,drowned,NDV-infected chickens for intra-day and(2.94%,1.66%,and 1.07%)for inter-day,and good recovery(99%,99% and 100%).Finally,the developed method could be used for the identification of chicken meat quality with less time and small volume reagents consuming.In conclusion,we first proposed a general IEF detection method to identify the quality of chicken meat.A new method for chicken quality identification using hemoglobin and myoglobin as markers was established.However,traditional IEF analysis methods have some disadvantages(such as high cost,long operation time and inability to detect online).Therefore,we further solve the problem of traditional IEF based on our laboratory developed microarray IEF chip.Furthermore,the method was used to identify the meat quality of three different chicken breeds,also considering the effect of different storage time.Experimental results show that microarray IEF provides a new method for meat quality detection,and has important guiding significance for meat quality identification.Obviously,this design principle could be extended to general methods for quality identification of other meat products such as pork,beef,and mutton in the future.