A Review of Gelatin Source Authentication Methods <s> Spectroscopic Methods (UV-Vis and Fourier-transform infrared spectroscopy, FTIR) <s> Quantitation of the amount of protein in a solution is possible in a simple spectrometer. Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). Therefore the A280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 [for some tyrosine-rich wool proteins], although most values are in the range 0.5–1.5 [1]). The advantages of this method are that it is simple, and the sample is recoverable. The method has some disadvantages, including interference from other chromophores, and the specific absorption value for a given protein must be determined. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption. <s> BIB001 </s> A Review of Gelatin Source Authentication Methods <s> Spectroscopic Methods (UV-Vis and Fourier-transform infrared spectroscopy, FTIR) <s> In order to classify unknown gelatin into their species of origin, a simple and rapid method for the qualitative determination was developed using Fourier transform infrared (FTIR) in combination with attenuated total reflectance (ATR) and discriminant analysis. The spectra were analysed using a chemometric method, principal component analysis (PCA), to classify and characterise gelatin compounds using regions of the FTIR spectra in the range of 3290–3280 cm−1 and 1660–1200 cm−1 as calibration models. Results from PCA, which were subsequently represented by the Cooman’s plot showed a clear distinction between gelatin samples of bovine and porcine origins. This qualitative approach, besides providing a rapid determination of the source of gelatin, may also be established based on a second derivative study of the FTIR spectrum to alleviate any doubt of the gelatin source for applications in the food and pharmaceutical industries. <s> BIB002 </s> A Review of Gelatin Source Authentication Methods <s> Spectroscopic Methods (UV-Vis and Fourier-transform infrared spectroscopy, FTIR) <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. 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In this study, the method was performed in two steps. In the first step, gelatin was extracted from these products before the MS-sample preparation. In the second step, tryptic gelatin peptides were separated and analyzed with ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (nanoUPLC-ESI-q-TOF-MS(E)). The novelty of this setup was that it functioned in a data independent acquisition mode and that alternate low and elevated collision energy was applied to acquire precursor and product ion information. This enabled accurate mass acquisition on the peptide level to identify the gelatin peptides. The marker peptides specific for porcine and bovine could be successfully detected in the gelatin added to the dairy products analyzed, revealing that the detection of marker peptides in the digested gelatin samples using nanoUPLC-ESI-q-TOF-MS(E) could be an effective method to differentiate porcine and bovine gelatin in the dairy products. <s> BIB004
Spectroscopic measurements are very sensitive, nondestructive and require a small amount of sample with little or no sample preparation. UV-Visible spectroscopy is used to measure the absorption of high-energy light (wavelengths of 200-800 nm) that causes excitation by molecules containing conjugated pi-electron system in a covalent bond. Biological macromolecules contain delocalised electron in aromatic systems that often absorb light in the near-UV (150-400 nm) or the visible (400-800 nm) region BIB001  . Molecular origin of protein absorbance includes the peptide groups in the far-UV (180-230 nm), the aromatic side chains of Tyr, Trp and Phe absorb light in the far-UV (180-230 nm) region and the near-UV (240-300 nm) region and the disulfide in near-UV (260 nm) BIB001  . Infrared spectroscopy is one of the most powerful spectroscopic methods to generate infrared spectrum which contained details of the functional group and chemical composition of food compounds  BIB003 . Infra-Red (IR) spectroscopy measures the amount of light absorbed as a result of molecular vibrations over a range of frequencies of the incident light. FTIR spectroscopy has been used to study the changes in secondary structure of collagen and gelatin, collagen crosslinking, thermal self-assembly and gelatin melting BIB003 . Spectra changes in amide A, amide I, amide II and the amide III regions have been observed in collagen secondary structures. FTIR spectroscopy is a valuable technique for determination of adulteration in food products including gelatin  . Use of FTIR for differential analysis of intramolecular structures of gelatin from different sources is a viable approach for Halal authentication practice BIB003 . BIB002 has established the potential use of FTIR in combination with attenuated total reflectance and principal component analysis for differentiation of bovine and porcine gelatins. FTIR spectra in the range of 3290-3280 cm −1 and 1660-1200 cm −1 were identified and used as calibration model. A clear difference was obtained from the result for PCA represented by the Cooman's plot. Information from FTIR spectra provide rapid determination of the gelatin source and serves as a base for second derivative study. BIB003 studied the difference between pepsin hydrolysates of bovine and porcine gelatin using UV-Vis and FTIR spectroscopy. In UV-Vis spectroscopy, both gelatin sources had a different absorbance at 229 nm and 240 nm showing the proportion of C=O amide and differences in the twodimensional conformation of the peptide. The three differences in FTIR spectra of the hydrolyzates after 1 h of hydrolysis that were observed are the region 2800-3000 cm-1 (aliphatic C-H stretching), the region 1543 cm −1 (C-N-H bending) and in the frequency of 1450-1300 cm −1 (C-H bending). The third difference was associated with a difference in the amino acid profile of the two samples. Although, spectroscopic methods were reportedly suitable for gelatin source authentication, data should be repeatable in order to obtain reliable information BIB004 . Likewise, differentiation of processed-transformed gelatin from different sources is still a challenge.