A Review of Gelatin Source Authentication Methods <s> Electrophoretic Analysis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) <s> Gelatin is a mixture of polypeptides obtained by hydrolysis of collagen primarily from bovine and porcine skin and bones. The similarity between different gelatins makes it difficult to trace their species origin. In this work, a new method for differentiation between bovine and porcine gelatin was developed based on detection and identification of marker peptides in digested gelatins. Sequence alignment analysis indicates that bovine and porcine Type I collagen contain differential sequences. The gelatins were digested by trypsin, and the resulting peptides were analyzed by high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). The marker peptides specific for bovine and porcine were successfully detected in the digested bovine and porcine gelatin, respectively. Comparative analysis indicated that more marker peptides could be detected in gelatin with a higher mean molecular weight. It was found that proline hydroxylation was a key factor affecting the peptide identification. For peptides such as GPPGSAGSPGK and GPPGSAGAPGK detected in digested bovine and porcine gelatin, respectively, the sequence should be verified manually since the mass shift caused by proline hydroxylation can be confused with the mass difference between Set and Ala residues. The results indicate that detection of marker peptides in the digested gelatin sample using HPLC-MS/MS is an effective method to differentiate between bovine and porcine gelatin. (C) 2009 Elsevier Ltd. All rights reserved. <s> BIB001 </s> A Review of Gelatin Source Authentication Methods <s> Electrophoretic Analysis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) <s> Food and pharmaceutical industries all over the world are witnessing an increasing demand for collagen and gelatin. Mammalian gelatins (porcine and bovine), being the most popular and widely used, are subject to major constraints and skepticism among consumers due to socio-cultural and health-related concerns. Fish gelatin (especially from warm-water fish) reportedly possesses similar characteristics to porcine gelatin and may thus be considered as an alternative to mammalian gelatin for use in food products. Production and utilization of fish gelatin not only satisfies the needs of consumers, but also serves as a means to utilize some of the byproducts of the fishing industry. This review focuses on the unique features, advantages, constraints, and challenges involved in the production and utilization of fish gelatin in order to provide a comprehensive look and deeper insight on this important food ingredient, as well as prospects for its future commercial exploitation and directions for future studies. <s> BIB002 </s> A Review of Gelatin Source Authentication Methods <s> Electrophoretic Analysis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) <s> This study was conducted to explore the differentiation of bovine and porcine gelatins before and after pepsin hydrolysis based on peptide pattern from spectroscopic and electrophoretic analysis due to development of the halal food products analysis. In this study, pepsin (EC 3.4.23.1) was used to hydrolyze the two sources of gelatin with consideration to its ability to digest up to 20% of ingested amide bonds by cleaving preferentially after the N-terminal of aromatic amino acids such as phenylalanine , tryptophan , and tyrosine . In this study, we expect to produce the fragment of gelatins with differentiation in relative molecular weights. Gelatins fragments then analyzed by UV-Vis and FTIR spectroscopy to characterize the functional groups on each source of gelatins, followed by SDS-PAGE ( Sodium Duodecylsulphate Polyacrylamide Gel Electrophoresis ) to identify the molecular weight of the resulting fragments. In UV-Vis spectroscopy, both gelatin source before and after hydrolysis had different absorbance at 229 nm and 240 nm showing the proportion of C=O amida and differences in two -dimensional conformation of the peptide . In terms of FTIR spectra, both gelatin have wavenumber at 3300-3400 cm -1 (NH stretching), 1600 cm -1 (C=O stretching, amida), 1500 cm -1 (C-N stretching), and at 620-767 cm -1 (OCN bending). This indicates that the relative amino acid compositions from two sources of gelatins were relatively different. In contrast, SDS-PAGE analysis does not give a real differentiation, except for porcine gelatin, that fragments which on 2 hour incubation show two peptide fragments with molecular sizes below 36,2 kDa and 28.6 kDa. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ ::: table.MsoNormalTable ::: {mso-style-name:"Table Normal"; ::: mso-tstyle-rowband-size:0; ::: mso-tstyle-colband-size:0; ::: mso-style-noshow:yes; ::: mso-style-priority:99; ::: mso-style-qformat:yes; ::: mso-style-parent:""; ::: mso-padding-alt:0in 5.4pt 0in 5.4pt; ::: mso-para-margin-top:0in; ::: mso-para-margin-right:0in; ::: mso-para-margin-bottom:10.0pt; ::: mso-para-margin-left:0in; ::: line-height:115%; ::: mso-pagination:widow-orphan; ::: font-size:11.0pt; ::: font-family:"Calibri","sans-serif"; ::: mso-ascii-font-family:Calibri; ::: mso-ascii-theme-font:minor-latin; ::: mso-fareast-font-family:"Times New Roman"; ::: mso-fareast-theme-font:minor-fareast; ::: mso-hansi-font-family:Calibri; ::: mso-hansi-theme-font:minor-latin; ::: mso-bidi-font-family:"Times New Roman"; ::: mso-bidi-theme-font:minor-bidi;} <s> BIB003 </s> A Review of Gelatin Source Authentication Methods <s> Electrophoretic Analysis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) <s> The study was aimed to differentiate between porcine and bovine gelatines in adulterated samples by utilising sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with principal component analysis (PCA). The distinct polypeptide patterns of 6 porcine type A and 6 bovine type B gelatines at molecular weight ranged from 50 to 220 kDa were studied. Experimental samples of raw gelatine were prepared by adding porcine gelatine in a proportion ranging from 5% to 50% (v/v) to bovine gelatine and vice versa. The method used was able to detect 5% porcine gelatine added to the bovine gelatine. There were no differences in the electrophoretic profiles of the jelly samples when the proteins were extracted with an acetone precipitation method. The simple approach employing SDS-PAGE and PCA reported in this paper may provide a useful tool for food authenticity issues concerning gelatine. <s> BIB004 </s> A Review of Gelatin Source Authentication Methods <s> Electrophoretic Analysis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) <s> Pharmaceutical dosage forms containing gelatin are recently of high attention in regard to its porcine content. In Muslim countries, halal products assurance is strictly regulated, not only for food but also for pharmaceutical products. This study aimed to develop gelatin identification and verification method in capsule shells by performing simple simultaneous protein- and nucleic acid based-identification methods. Identification and verification performing polyacrylamide gel electrophoresis (PAGE)—coupled with principle component analysis, and polymerase-chain-reaction (PCR)-restriction fragment length polymorphism (RFLP) were carried out. Gelatin was obtained by extracting the capsules using cold acetone; the precipitate was used directly for PAGE, and was used as well for DNA extraction. The results showed that the porcine gelatin reference showed 12 major bands of peptide profile, with predominant bands at ±200 and ±100–135 kDa on 8 % Tris-glycine gel, while bovine gelatin only showed four major bands. Further, PCR-RFLP result showed two specific bands of the porcine gelatin DNA at 228 and 131 bp, whereas the bovine gelatin DNA bands occurred at 316 and 44 bp. The results demonstrated that the capsule shell samples used in this study contained the following: sample 1 was verified contain porcine gelatin, whereas the three other samples identified contain bovine gelatin. Although the simultaneous approaches seemed need to be improved, it is crucial to maintain their low cost and simple protocol. <s> BIB005
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique is used for separation of proteins with different molecular weight  . Molecular weight is among the factors that affect the functional quality of gelatin. The variation in molecular weight distribution of protein/peptide fractions among different sources of gelatin can be used as a basis for authentication of gelatin source (G. BIB001 . Commercial gelatins contain different protein fractions including α (100 kDa), β (200 kDa) and g-chains (300 kDa) (Z.  . Polypeptide molecular weight distribution is also affected by processing methods and the level of hydrolysis that take place BIB002 ). BIB003 studied the differentiation of bovine and porcine gelatin and its peptides using SDS-PAGE techniques. Although the molecular weight distribution of gelatin and its fragments was successful, no real differentiation was achieved except for gelatin from porcine after 2 h of pepsin hydrolysis in which two peptide fragments were observed with molecular sizes below 36.2 and 28.6 kDa. BIB004 used SDS-PAGE combined with densitometry (principal component analysis) for differentiation of gelatin sources (bovine and porcine). Polypeptides with a molecular weight ranging from 50 to 220 kDa exhibited differing intensities when separated using SDS-PAGE. Principal component analysis of molecular weight distribution of electrophoretic polypeptide patterns was able to detect 5% porcine gelatin in bovine gelatin mix. Another study by BIB005 , SDS-PAGE coupled with densitometry analysis was also used to differentiate porcine and bovine gelatin in capsule shells. Twelve major bands of peptide profile were observed in porcine gelatin as against bovine gelatin that only showed four major bands.