| Chicken liver protein, which contains balanced amino acids, is considered to be a complete and high-quality protein. The protein content in chicken liver is about 20% and is common higher than other protein sources. In China, the annual yield of chicken liver is more than 500,000 tons, and most of which is sold fresh or used as a resource for animal and aquatic feeds. The utilization value of chicken liver has not been optimal, as further processing is few. The reason. is that the processing characteristics of the chicken liver have not been fully understood, especially that only a few studies focused on high protein content and many kinds of complex chicken proteins. Thus,extracting high-quality protein from the chicken liver of low value is of great significance to clarify its main processing properties and mechanisms for applications in the food industry.Isoelectric solubilization/precipitation (ISP) technology ensures a high extraction rate,safety, and no-pollution. Moreover, its use in the extraction of aquatic products, chicken,and mechanically-separated turkey meat has been very popular, with its applications in aquatic products being observed in industrial manufacturing. However,we should also determine how ISP technology could improve the extraction and emulsifying properties of chicken liver protein as well as format the mechanisms of its emulsifying properties.Label-free liquid chromatography tandem mass spectrometry quantitative proteomics(Label-free LC MS/MS) has advantages in the analysis of complex proteins. This paper explores the extraction yield and physical and chemical properties of chicken liver proteins obtained by ISP, focusing on emulsification and its formation mechanism. Combined with proteomic technology, it can further help explain the emulsion formation mechanism of chicken liver proteins obtained by ISP, which can provide theoretical basis and technical support for the application of chicken liver proteins obtained by ISP in the food industry. In this paper, the main research contents and results are as follows:1. Evaluation of the physico-chemical and nutritional qualities of normal- and abnormal-colored fresh chicken liverThis study evaluates the differences between the physico-chemical and nutritional qualities of abnormal-colored chicken livers (ANCCLs) and normal-colored chicken livers(NCCLs) as well as their levels of safety to provide scientific best before dates and to improve food manufacturing processes. Two types of fresh chicken livers from the same species were obtained in different batches to determine proximate composition, pH, total pigment content, fatty acid levels, amino acid levels, mineral elements, and heavy metal content. Compared with NCCLs, ANCCL is lower in protein, water content (P < 0.01), pH,and pigment content (P < 0.05), and NCCLs contain higher polyunsaturated fatty acids(PUFA) and saturated fatty acids (SFA) (P < 0.05). The PUFA/SFA ratio of NCCLs is 0.453, which is higher (P < 0.05) than that of ANCCLs. The levels of alanine, valine,tyrosine, lysine, and histidine in NCCLs are higher (P < 0.05) than in ANCCLs. Moreover,NCCLs have higher (P < 0.05) contents of K, Na, P, Cu, Fe, and Se, with the exception of Ca, which is lower (P < 0.05) than that of the heavy metals in ANCCLs. Heavy metal contents (As, Hg, Pb, and Cd) of both types of livers complied with food safety requirements. Although NCCLs have a higher nutritional value than ANCCLs,both are acceptable for human consumption.2. The relationship between protein structural changes and water- and fat- binding capacity during chicken liver emulsifying in different oil mediumProtein structural changes and water mobility properties in chicken liver paste-batters prepared with plant oil (sunflower and canola oil combinations) substituting 0-40% pork back-fat combined with pre-emulsification were studied by Raman spectroscopy and low-field nuclear magnetic resonance (NMR). Results showed that pre-emulsifying back-fat and plant oil, including substituting higher than 20% back-fat with plant oil increased the water- and fat-binding (P < 0.05) properties, formed more even and fine microstructures,and gradually decreased the NMR relaxation times (T21a, T21b and T22),which was related to the lower fluid losses in chicken liver paste-batters. Raman spectroscopy revealed that compared with control, there was a decrease (P < 0.05) in a-helix content accompanied by an increase (P < 0.05) in β-sheet structure when substituting 20%-40% back-fat with plant oil combined with pre-emulsification.Pre-emulsification and plant oil substitution changed tryptophan and tyrosine doublet hydrophobic residues in chicken liver paste-batters.3. Comparative study on extraction efficiency and composition of protein recovered from chicken liver by ISPThis study focuses on the extraction efficiency and composition of proteins recovered from chicken liver after extraction by acid (pH 2.0, 2.5, 3.0, and 3.5) and alkaline (pH 10.5,11.0, 11.5, and 12.0) solubilization and, afterward, a precipitation in pH 5.5. The highest extracted protein yield was obtained at pH values of 2.0, 11.5, and 12.0 (77.9%, 78.4%, and 79.3%, respectively), and the lowest yield (58.5%) was obtained at pH 3.5. Extraction of salt-soluble proteins was attributed to pH 3.0 and 3.5, whereas pH 10.5, 11.0, and 11.5 extracted more water-soluble proteins. Both acidic and alkaline processing extracted many insoluble proteins. The total pigment contents achieved by acid processing were significantly higher (P < 0.05) than those by alkaline processing. The highest total pigment content was observed at pH 2.5. The particle sizes (D10, D50 D90,D3,2, and D4,3) of protein recovered from chicken livers by alkaline processing all significantly increased with the increase of pH value (P < 0.05) and were significantly higher than those by acid processing,except for pH 2.0 treatment (P < 0.05). The profiles of other amino acids were not affected by ISP except for lysine and cysteine (P > 0.05). TEAA/TAA values were also not significantly changed among ISP groups (P > 0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles indicated higher concentrations of myosin heavy chains (MHCs) and actin by alkaline extraction than by acid extraction. Concisely, this method of recovering valuable protein components by ISP helps to produce innovative products and leads to an increased consumption of chicken liver by humans.4. Study on emulsifying properties and its conformation changes of chicken liver protein extracted by ISPThe emulsion stability, activity, viscosity, and optical microstructures of chicken liver protein extracted with acid (pH 2.0, 2.5, 3.0, and 3.5) and alkaline (pH 10.5, 11.0, 11.5, and 12.0) and raw material chicken liver emulsified with plant oil respectively were determined.Afterward, they were compared according to their emulsifying properties. The influence mechanism of the conformational changes in emulsifying properties in chicken liver protein extracted with ISP emulsified with plant oil by Raman spectroscopy was also studied. The results showed that, compared with acid extraction,chicken liver protein with alkaline extraction had significantly higher emulsion stability, viscosity, and emulsifying activity (P< 0.05). The sample extracted at pH 11.0 had more preferable maximum emulsion stability,viscosity, and emulsifying activity (P < 0.05) than those of the raw chicken liver protein,followed by pH 11.5. Samples extracted with pH 2.5 and 3.0 treatments showed significantly minimum emulsion stabilities, emulsion activities, and viscosities (P < 0.05).Except for pH 2.0 treatment,the emulsion particle sizes of D3,2, D4,3, Dio, D50, and D90 values of all alkaline extraction groups (pH 11.0, 11.5, and 12.0) were greater than the acid treatment, which resulted from the alkaline treatment being able to extract larger grains of protein. Optical micrographs of chicken liver proteins extracted with acid- and alkaline-processing and emulsion of raw material chicken liver showed that the sample by acid extraction processing had relatively large irregular droplets and distribution. Moreover,agglutination of connection was evident between droplets, with the observation in pH 3.0 treatment being significant. Chicken liver proteins extracted with the alkaline extraction showed distinct and relatively small spherical drops distributed evenly in the continuous phase; this phenomenon is most observable in the pH 11.0 treatment. It was benefited to form the stable emulsion by the droplets with uniform distribution. After emulsification,compared with raw chicken liver, the Raman spectra of emulsion layers of the acid and alkaline extraction involved in secondary structure changes of protein, in which the a-helical content of the alkali processing was significantly reduced (P < 0.05), accompanied by β-folding increase. The a-helix content of the acid-extracted treatment also decreased significantly (P < 0.05); the β-sheet content also reduced; and the content of irregular or random coil structures increased significantly (P < 0.05). Increased β-sheet content makes it possible to form a stable emulsion and was related to improve water and oil holding capabilities. Acid-processing increased the gauche-gauche-trans content of disulfide bonds.The content of g conformation in alkaline extraction treatments was also significantly increased (P < 0.05). Emulsions of protein extracted with acid- and alkaline-processing converted the residue state of tryptophan from an "exposed" to a "buried" position of polarity. All the tyrosine residues were exposed or were able to participate in moderate or weak hydrogen bonding during emulsification in each treatment. The protein side-chains of the alkaline extraction treatment showed strong emulsifying activity during the emulsion processes, including methyl and methylene entrapped and hydrophobic amino acid exposure, resulting in hydrophobic interaction enhanced, thereby increasing the emulsion stability.5. Label-free LC MS/MS proteomics quantitative research of chicken liver protein extracted by ISP emulsified with plant oilThe kinds and quantities of protein that were extracted from the emulsion layer formed by chicken liver protein (extracted with acid-alkali processing) and emulsified with plant oil were studied by label-free LC MS/MS proteomic quantitative technology. Functional annotations of all proteins were also made. Proteomic quantity of emulsion layer proteins showed that 2662 kinds of proteins (after protein merging) were present in the emulsion layer of protein samples across 9 treatments (pH 2.0, 2.5, 3.0, 3.5,10.5,11.0, 11.5,12.0,and raw chicken livers treatments). These were 1729, 1724, 1783, 1728, 1923, 2218, 2110,2092, and 1477. Among the 9 treatments, pH 11.0 treatment revealed the most protein kinds, followed by pH 11.5 and 12.0, and with raw chicken liver treatment obtaining the least. The kinds and contents of high-abundance proteins among treatments were different,and the number of unique proteins in the first 20 high-abundance proteins in each treatment are as follows: 3 (pH 2.0 treatment), 3 (pH 2.5 treatment), 6 (pH 3.0 treatment), 2 (pH 3.5 treatment), 3 (pH 10.5 treatment), 1 (pH 11.0 treatment), 1 (pH 11.5 treatment), 3 (pH 12.0 treatment), and 0 (raw chicken liver treatment). Compared with the raw chicken liver treatment, the different multiples of the expression levels of the first 20 high-abundance proteins were completely different to other treatments. Compared with acid extraction treatments (pH 2.0, 2.5, 3.0, 3.5) and alkaline extraction treatments (pH 10.5, 11.0, 11.5,12.0), the number of significantly different contents of protein in the emulsion layer was increased between alkaline-and acid-extracted treatments; and the clustering analysis of differential proteins also showed a consistent trend. |
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