Methods for detection and quantification of gelatin from different sources.

Gelatin is a water-soluble protein obtained from the collagen of various animal origins (porcine, bovine, fish, donkey, horse, and deer hide) and has diverse applications in the food, pharmaceutical, and cosmetics industries. Porcine and bovine gelatins are extensively used in food and non-food products; however, their acceptance is limited due to religious prohibitions, whereas fish gelatin is accepted in all religions. In Southeast Asia, especially in China, gelatin obtained from donkey and deer skins is used in medicines. However, both sources suffer from adulteration (mixing different sources of gelatin) due to their limited availability and high cost. Unclear labeling and limited information about actual gelatin sources in gelatin-containing products cause serious concern among societies for halal and fraud authentication of gelatin sources. Therefore, authenticating gelatin sources in gelatin-based products is challenging due to close similarities between the composition differences and degradation of DNA and protein biomarkers in processed gelatin. Thus, different methods have been proposed to identify and quantify different gelatin sources in pharmaceutical and food products. To the best of our knowledge, this systematic and comprehensive review highlights different authentication techniques and their limitations in gelatin detection and quantification in various commercial products. This review also describes halal authentication and adulteration prevention strategies of various gelatin sources, mainly focussing on research gaps, challenges, and future directions in this research area.

Development and characterisation of 3D collagen-gelatin based scaffolds for breast cancer research.

While 2D culture presents a useful tool for cancer research, it fails to replicate the tumor microenvironment as it lacks proper three-dimensional cell-cell/cell-matrix interactions, often resulting in exaggerated responses to therapeutic agents. 3D models that aim to overcome the issues associated with 2D culture research offer a new frontier for cancer research with cell growth, morphology and genetic properties that more closely match in vivo cancers. Herein, we aim to develop a collagen-based scaffold that supports the attachment and proliferation of breast cancer (BC) cells as a 3D culture model. Scaffolds were produced on a repeatable basis using a freeze-drying procedure. The constructs were highly porous (>99%) with homogenous pore sizes (150-300 µm) and an interconnected structure. The application of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) crosslinking resulted in scaffolds with elastic moduli in the range of 1-2 kPa, mimicking cancerous breast tissue stiffness. Furthermore, the incorporation of gelatin into the scaffolds enabled the porosity, pore size and mechanical properties to be tailored, resulting in scaffolds with stiffness values that accurately replicate the stiffness of human BC extracellular matrix (ECM) (1.3-1.7 kPa). Scaffolds displayed high in vitro stability with 90% of mass remaining after 14 days of culture. The scaffolds were shown to be highly biocompatible, and capable of supporting the attachment, infiltration and proliferation of MCF7 breast cancer (BC) cells over +14 days. These results confirm the suitability of these scaffolds as culture models for BC cells. These collagen-based scaffolds offer significant potential for the exploration of aspects of BC, such as gene expression profiles and patterns, and for the assessment of the efficacy of therapeutic agents in treating BC.

Qualitative and Quantitative Analysis of Ejiao-Related Animal Gelatins through Peptide Markers Using LC-QTOF-MS/MS and Scheduled Multiple Reaction Monitoring (MRM) by LC-QQQ-MS/MS.

Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes.

Bovidae-based gelatin: Extractions method, physicochemical and functional properties, applications, and future trends.

Gelatin is one of the most important multifunctional biopolymers and is widely used as an essential ingredient in food, pharmaceutical, and cosmetics. Porcine gelatin is regarded as the leading source of gelatin globally then followed by bovine gelatin. Porcine sources are favored over other sources since they are less expensive. However, porcine gelatin is religiously prohibited to be consumed by Muslims and the Jewish community. It is predicted that the global demand for gelatin will increase significantly in the future. Therefore, a sustainable source of gelatin with efficient production and free of disease transmission must be developed. The highest quality of Bovidae-based gelatin (BG) was acquired through alkaline pretreatment, which displayed excellent physicochemical and rheological properties. The utilization of mammalian- and plant-based enzyme significantly increased the gelatin yield. The emulsifying and foaming properties of BG also showed good stability when incorporated into food and pharmaceutical products. Manipulation of extraction conditions has enabled the development of custom-made gelatin with desired properties. This review highlighted the various modifications of extraction and processing methods to improve the physicochemical and functional properties of Bovidae-based gelatin. An in-depth analysis of the crucial stage of collagen breakdown is also discussed, which involved acid, alkaline, and enzyme pretreatment, respectively. In addition, the unique characteristics and primary qualities of BG including protein content, amphoteric property, gel strength, emulsifying and viscosity properties, and foaming ability were presented. Finally, the applications and prospects of BG as the preferred gelatin source globally were outlined.

Hydroxyproline determination for initial detection of halal-critical food ingredients (gelatin and collagen).

Gelatin and collagen are considered halal-critical ingredients as they are typically derived from either bovine or porcine animals. Current analytical methods for determining the sources of gelatin and collagen suffer from limitations in terms of robustness and false positives in peptide matching. Thus, the aim of this study was to investigate the utility of monitoring hydroxyproline, a signature amino acid for gelatin and collagen, for identifying potentially haram foodstuffs. To determine the hydroxyproline profiles among animal- and plant-based samples, one-way univariate analysis of variance followed by pair-wise comparison was used to establish statistical significance. Multivariate chemometric analysis through principal component analysis revealed a discrete distribution pattern among 59 samples due to hydroxyproline variability. Finally, inter- and intra-laboratory comparisons demonstrated the validity and robustness of hydroxyproline determination according to ISO 17025. Thus, this preliminary identification technique will aid the identification of potentially haram foodstuffs.

Multi-target detection of egg-white and pig gelatin fining agents in Nebbiolo-based aged red wine by means of nanoHPLC-HRMS.

The presence of residues from fining agents in wines may represent a risk for allergic consumers and a source of discomfort for others, such as vegans. Even though ELISA is the official detection method for such residues, this technique may be hindered by cross-reactivity issues, or by matrix-molecule interference due to a high polyphenol content, especially in red wines. An HRMS-based method has been developed to detect pig gelatin and egg white in experimental five-year aged Nebbiolo-based red wine. Biomarker peptides were selected, after tryptic digestion, and quantified by multitarget nanoHPLC-HRMS analysis. The method resulted in an LLOQs of 5 µg/mL in the experimental wine, and between 1 and 2 µg/mL in the buffer. This method allowed both gelatin and egg white proteins to be detected and quantified in aged red wine, while whereas the commercial ELISA kit was instead unable to detect egg white in the same samples.

Microstructure and characteristic properties of dogfish skin gelatin gels prepared by freeze/spray-drying methods.

The effects of two pretreatments (microwaves or oven-drying) on the dogfish (Squalus acanthias) skin as well as two drying processes (freeze-drying or spray-drying) on the extracted gelatins were studied. Thus six types of gelatins were obtained, three of which were freeze-dried (FG) and the others were spray-dried (SG), from the untreated skin (US), microwaves-pretreated skin (MS) and oven-pretreated skin (OS). The highest yield (8.67%) was obtained for the OSFG, while the lowest one (3.06%) was measured for the OSSG. Interestingly, all gelatins exhibited relatively high protein (84.02-89.53%), and low lipid (0.50-1.71%) and ash (3.05-7.17%) contents. In addition, gelatins were analyzed by the Fourier transform infrared and the spectra displayed important differences in some specific peaks, particularly in the amide I, amide II and amide III. The gelatins extracted from the untreated skin, regardless the drying method, presented the highest foaming capacity. The textural profile analysis showed that USSG was the hardest (213.6 g) and the chewier (23.8 N × mm) gelatin. Moreover, analysis of thermal properties showed that USSG also has the highest glass-transition temperature. The interesting properties of gelatin extracted from dogfish skin encourage their future use as a functional ingredient in industrial food formulations.

Moisture sorption and desorption properties of gelatin, HPMC and pullulan hard capsules.

The moisture sorption and desorption properties of hard capsules have a great influence on the quality of capsule products. However, studies on them have rarely been reported. Herein, we studied the moisture sorption and desorption properties of three kinds of hard capsules (gelatin, hydroxypropyl methyl cellulose (HPMC) and pullulan capsules) in terms of hygroscopicity, crystallinity, thermal behaviors and so on. It is found that HPMC capsules have weaker moisture sorption ability and moisture keeping ability than pullulan or gelatin capsules with lower moisture sorption rates, equilibrium moisture contents, moisture keeping rates and higher critical relative humidity. In comparison with gelatin capsules, pullulan capsules have weaker moisture sorption ability and comparable moisture keeping ability. HPMC or pullulan capsules can more effectively protect high, moderate and low hygroscopic capsule contents (chitosan, potato starch or ethyl cellulose) from outside moisture absorption. The diffraction peaks of the moisture equilibrated gelatin, HPMC and pullulan capsules are much smaller than those of their dried ones. The dried and the moisture equilibrated gelatin, HPMC or pullulan capsules all have smooth surface morphology. HPMC or pullulan capsules can be an attractive alternative to animal gelatin capsules due to their appropriate moisture sorption and desorption properties.

Determination of Chloramphenicol, Thiamphenicol and Florfenicol in Chinese Gelatin Medicines using Dispersive Solid-Phase Extraction Coupled with Ultra High-Performance Liquid Chromatography-Mass Spectrometry.

This study established a rapid and reliable method to determine chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) residues in Chinese gelatin medicines. CAP, TAP and FF were extracted from medicine samples using 2% (v/v) ammonium hydroxide in acetonitrile. Trypsin was used to eliminate the matrix effect caused by protein components in gelatin medicines, whereas anhydrous sodium sulfate, C18-N and NH2-PSA adsorbents were applied to reduce matrix effect induced by other components. The analytical method of these drugs was optimized on ultra high-performance liquid chromatography-mass spectrometer (UHPLC-MS/MS) through the analysis of their standard linearity and regression. The optimized extraction and analytical method were validated in one Chinese gelatin medicine sample (Colla corii asini, E Jiao) with three fortification levels (2, 5 and 10 µg/kg), and the recoveries of these drug residues ranged of 87.6-102.7%. The limit of detection and quantification of CAP, TAP and FF in the sample were 0.2 and 0.5 µg/kg, 0.4 and 1.5 µg/kg, and 0.5 and 1.5 µg/kg, respectively. A total of 30 Chinese gelatin medicine samples were analyzed using the established method. No drug residues were found in these samples except for one Testudinis Carapacis et Plastri (1.67 µg/kg FF) and one turtle shell glue (2.55 µg/kg FF).

Materials Characterization of Cranial Simulants for Blast-Induced Traumatic Brain Injury.

INTRODUCTION: The mechanical response of brain tissue to high-speed forces in the blast and blunt traumatic brain injury is poorly understood. Object-to-object variation and interspecies differences are current limitations in animal and cadaver studies conducted to study damage mechanisms. Biofidelic and transparent tissue simulants allow the use of high-speed optical diagnostics during a blast event, making it possible to observe deformations and damage patterns for comparison to observed injuries seen post-mortem in traumatic brain injury victims. METHODS: Material properties of several tissue simulants were quantified using standard mechanical characterization techniques, that is, shear rheometric, tensile, and compressive testing. RESULTS: Polyacrylamide simulants exhibited the best optical and mechanical property matching with the fewest trade-offs in the design of a cranial test object. Polyacrylamide gels yielded densities of ~1.04 g/cc and shear moduli ranging 1.3-14.55 kPa, allowing gray and white matter simulant tuning to a 30-35% difference in shear for biofidelity. CONCLUSIONS: These materials are intended for use as layered cranial phantoms in a shock tube and open field blasts, with focus on observing phenomena occurring at the interfaces of adjacent tissue simulant types or material-fluid boundaries. Mechanistic findings from these studies may be used to inform the design of protective gear to mitigate blast injuries.

Chicken Heads as a Promising By-Product for Preparation of Food Gelatins.

Every year, the poultry industry produces a large number of by-products such as chicken heads containing a considerable proportion of proteins, particularly collagen. To prepare gelatin is one of the possibilities to advantageously utilize these by-products as raw materials. The aim of the paper was to process chicken heads into gelatins. An innovative method for conditioning starting raw material was using the proteolytic enzyme. Three technological factors influencing the yield and properties of extracted gelatins were monitored including the amount of enzyme used in the conditioning of the raw material (0.4% and 1.6%), the time of the conditioning (18 and 48 h), and the first gelatin extraction time (1 and 4 h). The gelatin yield was between 20% and 36%. The gelatin gel strength ranged from 113 to 355 Bloom. The viscosity of the gelatin solution was determined between 1.4 and 9.5 mPa.s. The content of inorganic solids varied from 2.3% to 3.9% and the melting point of the gelatin gel was recorded between 34.5 and 42.2 °C. This study has shown that gelatin obtained from chicken heads has a promising potential with diverse possible applications in the food industry, pharmacy, and cosmetics.

Postmarket Laboratory Surveillance for Forbidden Substances in Halal-Certified Foods in Thailand.

Limited information exists regarding adulteration of Halal-certified food by substances forbidden under Islamic law (Haram substances). This study was conducted using forensic laboratory testing to investigate the prevalence of this type of adulteration. In this large-scale survey of Halal-certified food products randomly collected from markets in Thailand, 4,829 food samples from 10 food groups were tested in the laboratory for four potentially Haram substances: porcine DNA, porcine fatty acids, ethanol, and hydroxyproline (gelatin). No samples were adulterated with porcine DNA or fatty acids. However, 62 samples (approximately 1.3%) were positive for ethanol (>0.5% for non-naturally fermented products and >1% for naturally fermented products). The hydroxyproline concentration in the samples was compared with that of a negative control. Gelatin, as indicated by the presence of hydroxyproline, was the major suspicious substance found in these products. Further investigations are required to determine whether the gelatin is of Halal origin. These results from this first large-scale postmarket surveillance of Halal-certified food products for forbidden substances reveals the important role of forensic laboratory testing for supporting Halal supervision and certification. These findings provide useful information for government agencies seeking to encourage Halal compliance by food enterprises and for Muslim consumers and Halal food importers and exporters.

Potato Protein Fining of Phenolic Compounds in Red Wine: A Study of the Kinetics and the Impact of Wine Matrix Components and Physical Factors.

Producing wines within an acceptable range of astringency is important for quality and consumer acceptance. Astringency can be modified by fining during the winemaking process and the use of vegetable proteins (especially potato proteins) as fining agents has gained increasing interest due to consumers' requirements. The research presented was the first to investigate the effect of a potato protein dose on the kinetics of tannin and phenolic removal compared to gelatin for two unfined Cabernet Sauvignon wines. To further understand the results, the influence of the wine matrix and fining parameters (including pH, ethanol concentration, sugar concentration, temperature, and agitation) were tested according to a fractional 25-1 factorial design on one of the Cabernet Sauvignon wines using potato proteins. The results from the factorial design indicate that potato protein fining was significantly influenced by wine pH, ethanol concentration, fining temperature as well as an interaction (pH × ethanol) but not by sugar content or agitation. Insights into the steps required for the optimisation of fining were gained from the study, revealing that potato protein fining efficiency could be increased by treating wines at higher temperatures (20 °C, rather than the conventional 10-15 °C), and at both a lower pH and/or alcohol concentration.

A strategy for identifying species-specific peptide biomarkers in deer-hide gelatin using untargeted and targeted mass spectrometry approaches.

Deer-hide gelatin (DHG) is an important animal-derived traditional Chinese medicine (TCM), which has been applied in TCM for over 400 years. However, it is extremely difficult to distinguish DHG with adulteration or made with other animal skins due to the highly processing procedure. Therefore, a simple strategy for identifying species-specific peptide biomarkers in deer-hide gelatin (DHG) is needed. In the present study, untargeted and targeted mass spectrometry approaches were implemented to analyze comprehensive peptidomic profiles of trypsin-digested animal gelatins. Mathematics set theory was then used to interrogate the relationship between different samples and peptides in the target species set, while the peptides were not considered as species-specific biomarkers in other sets. Two peptides were identified as DHG-specific peptides. Targeted mass spectrometry approach was then used to verify these two peptides. It showed that these two peptides could be used for distinguishing DHG from other animal hide gelatins. The present strategy provides a simple method for peptide biomarker discovery, which can be applied in the identification of specific peptides in some highly processed animal derived traditional Chinese medicines (TCMs). Thus, the present work provides an effective strategy for rapid, simple discovery and application of species-specific peptide biomarkers to ensure animal derived TCMs quality and make them authenticable and traceable.

Assessment of the denaturation of collagen protein concentrates using different techniques.

The use of collagen and gelatin in the field of regenerative medicine is widely extended. However, most of the studies in this topic are focused on the scaffolds' properties, but only a few are related to the properties of the raw material used. The raw material analysis not only consists of a study of the composition, but also of the denaturation degree that can influence the processing and properties of the structure of the scaffold. Thus, the denaturation degree analysis of different collagen proteins was performed and assessed by the comparison of four different methods: differential scanning calorimetry (DSC), Fourier transform Infrared Spectroscopy (FTIR) and circular dichroism (CD) spectra and sulfhydryls content analysis. DSC measurements put forward a glass transition between 88°C and 95°C as well as from the FTIR measurements; the characteristic peaks for proteins are evidenced. However, from the sulfur content, only a small proportion of free sulfhydryls are present with respect to their total amount. In addition, CD spectra allow to estimate the secondary structure of the protein by the analysis of the α-helix and ß-strand and also quantify the denaturation degree with the 'positive/negative ratio' (RPN) from the CD profiles, obtaining values in the range between 25% and 100%.

Immunological Methods for the Detection of Binders in Ancient Tibetan Murals.

Tibetan mural samples from the Jiazhaer mountain cave were studied using enzyme-linked immunosorbent assays (ELISA) and immunofluorescence microscopy (IFM). Samples containing protein binders were first identified using ELISA, and then IFM was used to determine the location of protein binders. Using these methods, we discovered gelatin and casein in samples from wall murals, distributed in both red and black pigments. We excluded the possibility of contamination by conducting further experiments where simulated samples were spiked with milk. We conclude that both gelatin and casein were used as binders in the pigments of the Tibetan Buddhist murals in the Jiazhaer (Transliteration from Tibetan) mountain cave. This is the first evidence of casein being used as a binder in Chinese mural pigments.

Interference Effect of Silica Colloidal Crystal Films and Their Applications to Biosensing.

With the aim to develop better and more reliable interference effective substrates, silica colloidal crystal films with different sphere diameters and film thicknesses were successfully made by an improved vertical deposition method and a systematic investigation of their reflectometric interference spectroscopy (RIfS) properties are presented in this work. The influence of silica sphere diameter and film thickness on the RIfS signals was studied. The results showed that the film thickness is the key factor of RIfS signals. An RIfS system was set up by using a silica colloidal crystal film as an interference effective substrate. The influence of film thickness on the response to refractive index changes of the proposed system was also investigated. When the influence of film thickness on RIfS signals and refractive index response we considered together, silica colloidal crystal films with a thickness between 4 and 6 µm were chosen for sensor construction. Monitoring the digestive process of gelatin with trypsin was also demonstrated by combining gelatin-modified silica colloidal crystal films with RIfS. The system showed excellent sensitivity with a wide linear range and could achieve real-time measurement of each process. It has been proved that this is a promising method to construct biosensors using silica colloidal crystal films as interference-sensitive substrates.

Advances in LC-MS/MS Methods for Allergen Testing, Meat Speciation, and Gelatin Speciation.

Background: Food authenticity is demanded by the consumer at all times. The consumer places trust in the manufacturer that the food product is genuine in terms of what is recorded on the packaging label. Objective: Recent advancements in LC-tandem MS methodology in the detection of allergens, meat, and gelatin speciation in raw food products and processed foods are detailed in this paper. Method: For each of the three methods, initial proteome analysis and the screening leading to the determination of unique tryptic peptides were conducted using a high-resolution, accurate tandem mass spectrometer. Having identified the unique markers, the method was transferred to a tandem quadrupole mass spectrometer for a higher-sensitivity quantitative study, multiple reaction monitoring transition analysis. Results: For the allergens method a detection limit of at least 10 ppm was attained across the 12 allergen peptides in this workflow. In the gluten workflow the resulting chromatograms show good detection down to 5 ppm, with no interference from the food matrices. The meat speciation method details that signature peptides could be readily identified at 1% w/w with no matrix interference. Conclusions: These single-injection workflows with cycle-time optimization enable wide coverage of analytes to identify multiple species within challenging matrix samples.

Method for Determining Gelatinolytic Activity in Tissue: In Situ Gelatin Zymography.

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present an in situ gelatin zymography method that allows for a precise localization of active gelatin-degrading enzymes in a tissue section. In this method, dye-quenched gelatin is put on top of a tissue section. During an incubation period, active gelatinolytic enzymes will degrade the substrate and fluorescent signals are emitted from the locations of these enzymes.

Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography.

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin-degrading proteases in tissue extracts. This method uses fluorescence-labeled gelatin and therefore we also present an easy, fast, and cheap method for labeling gelatin with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF).