Mass spectrometric detection of marker peptides in tryptic digests of gelatin: A new method to differentiate between bovine and porcine gelatin

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 Ser 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.     

阿胶制品中典型明胶特征性多肽的鉴定

为了研究不同类型阿胶制品中典型明胶的来源,本文选取9种市面上常见的阿胶制品（阿胶糕、阿胶口服液和阿胶颗粒各3种）为研究对象,分别添加质量分数为1%的牛明胶和猪明胶,采用高效液相色谱-高分辨率质谱联用技术（high-performance liquid chromatography-mass spectrometry,HPLC-MS/MS）鉴定这9种阿胶制品中典型明胶的特征性多肽。结果表明,不同数量的典型明胶特征性多肽被检出于9种阿胶制品。进一步分析发现,所有阿胶制品共有的阿胶、牛明胶、猪明胶的特征性多肽分别为25、3、14条,其中,未经修饰的特征性多肽依次为7、2、3条。在上述共有特征性多肽中,源自α1链的分别有22、2、5条,源自α2链的分别为3、1、9条。这些共有特征性多肽可作为HPLC-MS/MS鉴定复杂体系中明胶源的依据,对不同阿胶制品中明胶来源的鉴定具有指导意义。     

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.     

高效液相色谱-高分辨率质谱联用技术鉴定阿胶块中不同胶类的特征性多肽

为研究阿胶块中不同明胶的来源,本文以4种品牌的阿胶块为原料,向其中分别加入低含量(1 mg/mL)的牛明胶和猪明胶,采用高效液相色谱-质谱联用技术(high-performance liquid chromatography-mass spectrometry,HPLC-MS/MS)分别鉴定阿胶、牛明胶和猪明胶的特征性...     

Gelatin controversies in food,pharmaceuticals, and personal care products: Authentication methods,current status,and future challenges

Gelatin is a highly purified animal protein of pig, cow, and fish origins and is extensively used in food, pharmaceuticals, and personal care products. However, the acceptability of gelatin products greatly depends on the animal sources of the gelatin. Porcine and bovine gelatins have attractive features but limited acceptance because of religious prohibitions and potential zoonotic threats, whereas fish gelatin is welcomed in all religions and cultures. Thus, source authentication is a must for gelatin products but it is greatly challenging due to the breakdown of both protein and DNA biomarkers in processed gelatins. Therefore, several methods have been proposed for gelatin identification, but a comprehensive and systematic document that includes all of the techniques does not exist. This up-to-date review addresses this research gap and presents, in an accessible format, the major gelatin source authentication techniques, which are primarily nucleic acid and protein based. Instead of presenting these methods in paragraph form which needs much attention in reading, the major methods are schematically depicted, and their comparative features are tabulated. Future technologies are forecasted, and challenges are outlined. Overall, this review paper has the merit to serve as a reference guide for the production and application of gelatin in academia and industry and will act as a platform for the development of improved methods for gelatin authentication.     

Fish gelatin: properties,challenges, and prospects as an alternative to mammalian gelatins

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.     

Conventional and real-time PCR-based approaches for molecular detection and quantitation of bovine species material in edible gelatin

The majority of edible gelatin in Europe is derived from pigskin, but a significant portion is extracted from bovine tissue. Because of the bovine spongiform encephalopathy crisis, consumers might be concerned about the gelatin used in various products. To assure consumers of the quality and safety of edible gelatin, European Union directive 1999/724/EC described general guidelines for gelatin production, including requirements for documentary proof confirming that raw materials are from animals fit for human consumption. Analytical methods to confirm gelatin documentation or raw material animal species source in the finished product are lacking. In this study, several published species-specific PCR systems were evaluated as potential molecular methods for determining the origin of the raw material used in making gelatin. A recently validated bovine species-specific PCR primer set targeting the ATPase 8 subunit gene in bovine mitochondrial DNA was suitable for detection of bovine material in gelatin. This PCR primer set was optimized using conventional and real-time PCR approaches. An evaluation of these two PCR methods confirmed the high specificity for the adopted primer set in various gelatin matrices of known origin. The inclusion of bovine gelatin in pork or fish gelatin can be detected at 0.1 to 0.001%. These PCR assays are potential molecular detection tools that can be used to routinely detect bovine gelatin either alone or as an inclusion in gelatin made from other species.     

Novel multiplex PCR-RFLP assay discriminates bovine,porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus’ consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.     

Thermal characterisation of gelatin extracted from yellowfin tuna skin and commercial mammalian gelatin

Glass transition and other thermal characteristics of gelatin from different sources were studied by differential scanning calorimetry (DSC) and modulated DSC (MDSC). The initial glass transition temperatures of equilibrated gelatin samples at 11.3% relative humidity, determined from reversible heat flow thermogram of MDSC, were 23, 75 and 59 °C, respectively, for tuna skin, bovine and porcine gelatin. When gelatin samples were equilibrated at higher relative humidity of 52.9%, glass transition temperature of fish skin and bovine gelatin decreased to −3 and 57 °C, respectively. Further increase of equilibration relative humidity to 75.3% showed increased value in the case of tuna skin, whereas bovine and porcine did not show any significant change. DSC and MDSC results indicated that tuna gelatin showed lower glass transition compared to mammalian source gelatin equilibrated at the same constant relative humidity. In general glass transition measured by DSC was found lower than the values measured by MDSC. The results in this study showed that the degree of plasticization varied with the source of gelatin as well as their extraction methods.     

Gelatin: The Paramount Food Additive

Of all the hydrocolloids in use today, surely none has proven as popular with the general public and found favor in as wide a range of food products as gelatin. A sparkling, clear dessert jelly has become the archetypal gel and the clean melt-in-the-mouth texture is a characteristic that has yet to be duplicated by any polysaccharide. Despite its apparently unfashionable status, more gelatin is sold to the food industry than any other gelling agent. It is relatively cheap to produce in quantity, and there is a ready supply of suitable raw material. The traditional sources of gelatin include bovine and pig skins and demineralized bones and hooves. However, recent studies have shown that there are viable new sources of gelatin such as marine fish skins and bones. Researchers have further sought to develop gelatin derivatives or modified gelatins like coldwater soluble gelatin, hydrolyzed gelatin and esterified gelatin.     

Folate-binding protein in milk: a review of biochemistry, physiology, and analytical methods

Folate-binding protein (FBP) was discovered in cow's milk around 40 years ago. Bovine FBP belongs to a family of several folate-binding proteins. In milk, it is a soluble whey protein with the ability to sequester folate from blood plasma. Bovine FBP is a well-characterized protein in terms of amino acid sequence and binding characteristics. Affinity and binding kinetics towards various folate forms have been intensively studied because they are crucial in using bovine FBP as an analytical tool. Shortly after the identification of bovine FBP, a competitive protein-binding assay for measuring serum and blood folate concentrations was introduced. Another analytical application of bovine FBP is in affinity chromatography, as a clean-up/concentration step for analysis of folates in foods and biological samples by liquid chromatographic methods. Concentrations of FBP in milk and dairy products have been determined by ELISA and Surface Plasmon Resonance-biosensor techniques. Since the initial reports of FBP in cow's milk, its physiological role has been discussed, especially regarding its effects on folate absorption from milk and dairy products. This review summarizes recent biochemical, analytical, food science, and nutritional advances regarding folate-binding protein in milk.     

Gelatin: The Paramount Food Additive

Abstract

Of all the hydrocolloids in use today, surely none has proven as popular with the general public and found favor in as wide a range of food products as gelatin. A sparkling, clear dessert jelly has become the archetypal gel and the clean melt‐in‐the‐mouth texture is a characteristic that has yet to be duplicated by any polysaccharide. Despite its apparently unfashionable status, more gelatin is sold to the food industry than any other gelling agent. It is relatively cheap to produce in quantity, and there is a ready supply of suitable raw material. The traditional sources of gelatin include bovine and pig skins and demineralized bones and hooves. However, recent studies have shown that there are viable new sources of gelatin such as marine fish skins and bones. Researchers have further sought to develop gelatin derivatives or modified gelatins like coldwater soluble gelatin, hydrolyzed gelatin and esterified gelatin.     

Analysis of Porcine Gelatin DNA in a Commercial Capsule Shell Using Real-Time Polymerase Chain Reaction for Halal Authentication

The presence of pig derivatives, such as porcine gelatin, in any products is prohibited to be consumed by Muslim community. This study is intended to develop a specific primer from mytochondrial D-loop capable of amplifying DNA from porcine gelatin in commercial capsule shells. Two pairs of primers designed from mitochondrial D-loop region were tested in order to confirm the primer specificity in gelatin sources (pork, beef, and catfish) and fresh tissue (pig, cows, goat, chickens, and rat). Primers were then used to perform sensitivity test of six dilution series (1000, 200, 100, 10, 5, and 1 pg/µL) of porcine gelatin and porcine capsule shell. The amplification was also performed on capsule shell from porcine-bovine mixture gelatin at 0, 10, 20, 30, 40, 50, and 100% concentration. The repeatability test was performed by measuring amplification capsule shells from porcine–bovine gelatin mixture. Real time polymerase chain reaction method using primers designed was further applied to analyze capsule shells purchased from markets. From two primers have been designed specifically, only primer D-Loop 108 (forward: 5’-CGT ATG CAA AAA ACC ACG CCA-3’; reverse: 5’-CTT ACT ATA GGG AGC TGC ATG-3’) had the capability to identify the presence of porcine DNA in fresh tissue and gelatin sources at optimum annealing temperature of 58.4ºC. Sensitivity of the developed method expressed as limit of detection of DNA in gelatin and capsule shells is 5 pg.     

Analysis of Pig Derivatives for Halal Authentication Studies

Pig derivatives such as lard and pork in any food system are prohibited for consumption by Muslims and Jews. For this reason, analytical methods offering accurate and reproducible results are needed to assure the halalness, kosherness, and wholesomeness of food. This article describes some analytical techniques, namely Fourier transform infrared (FTIR) spectroscopy, chromatography-based techniques, differential scanning calorimetric (DSC), and electronic noses for detection and quantification of pig derivatives (lard, pork, gelatin) in food products.     

Species identification and animal authentication in meat products: a review

Meat species identification and animal authentication in meat products is a significant subject, attention to which would contribute to fair-trade, and would enable consumers to make informed choices. Analytical methods are often based on protein or DNA measurements. Methods based on protein fractions include electrophoretic, chromatographic and immunological techniques and are often not suitable for compound food products, nor are they sensitive in processed products to differentiate closely related meat species. Advances in DNA technology have led to the rapid development of alternative approaches to species identification. Recently, application of polymerase chain reaction in food analysis has increased in the light of their simplicity, specificity and sensitivity. This review discusses a wide range of analytical methods with a focus on their ability to quantify meat and authentication of meat products.     

Utilization of Fish Processing By-Products in the Gelatin Industry

Since the bovine spongiform encephalopathy crisis, there has been a growing interest for finding an alternative source of raw materials for gelatin production. Gelatin produced from fish processing by-products is a potential alternative to mammalian gelatin. Fish processing generates solid wastes that can be as high as 50-80% of the original raw material. These wastes are an excellent raw material for preparation of high protein foods. About 30% of the wastes consists of skin and bone with a high collagen content. Fish gelatin can be obtained by hydrolysis of collagen the principal protein found in skin and bone. Fish skin and bone gelatin can be prepared with bloom strength similar to that obtained from mammalian sources. Fish gelatin has numerous applications, particularly, in the food, pharmaceutical, and photographic industries due to its unique chemical and physical properties. This review presents how fish processing by-products can be utilized in the manufacture of gelatin.     

Utilization of Fish Processing By-Products in the Gelatin Industry

Since the bovine spongiform encephalopathy crisis, there has been a growing interest for finding an alternative source of raw materials for gelatin production. Gelatin produced from fish processing by-products is a potential alternative to mammalian gelatin. Fish processing generates solid wastes that can be as high as 50–80% of the original raw material. These wastes are an excellent raw material for preparation of high protein foods. About 30% of the wastes consists of skin and bone with a high collagen content. Fish gelatin can be obtained by hydrolysis of collagen the principal protein found in skin and bone. Fish skin and bone gelatin can be prepared with bloom strength similar to that obtained from mammalian sources. Fish gelatin has numerous applications, particularly, in the food, pharmaceutical, and photographic industries due to its unique chemical and physical properties. This review presents how fish processing by-products can be utilized in the manufacture of gelatin.     

Physical, mechanical, and barrier properties of carp and mammalian skin gelatin films

Films of 0.11 to 0.13 mm thickness were prepared using gelatins from the skins of cultured freshwater carp species and mammalian gelatins viz., porcine and bovine skin gelatin. A comparative study was made on the physical, mechanical, and barrier properties of these films. The amino acid composition, gel strength, clarity, and gel setting point of the gelatins were also determined. Carp skin gelatins had a lower imino acid content (19.16% to 20.86%) than mammalian skin gelatins (22.91% to 23.7%). Grass carp gelatin had gel strength of 230.2 B that is comparable to the reported value for bovine skin gelatin (227.2 B). The bloom values of rohu and common carp skin gelatins were 188.6 B and 181.3 B, respectively, which were significantly lower than mammalian gelatins. Mammalian gels have significantly higher (P < 0.05) setting temperatures (23.7 to 24.2 °C) than carp skin gelatins. Tensile strength (TS) was lowest for films from common carp and rohu skin gelatin (490 and 497 kg/cm(2), respectively) and highest for porcine skin gelatin film. The degree of transparency (L*) was significantly higher for films from grass carp, bovine hide, and pork skin gelatin films. Carp skin gelatin films had significantly lower water vapor permeability (WVP) and oxygen permeability (OP) than mammalian skin gelatin films, which indicated that carp skin gelatin based films have superior barrier properties than mammalian skin gelatin films.     

Advanced analytical tools for bovine lactoferrin identification and quantification in raw skim milk to finished lactoferrin powders

As the use of bovine lactoferrin (bLF) has increased, greater regulatory and commercial interest is being placed on bLF identification and quantitation and quality. Several methods can be used to quantitate bLF, but a robust accurate quantification method applicable as a standardised tool for feed milk, process monitoring, and evaluation of end products has been less of a concern. bLF concentrations increase through the bovine lactation cycle, so accurate and timely information is required that can increase both processing plant performance and product quality and output. In this article we evaluate analytical cation exchange, reverse phase high-performance chromatography and size exclusion chromatography methods for the analysis of bLF identification and quantification from whole milk, process intermediates, ingredients, milk and infant formula products. However, the methods described in this article to identify and quantify the bLF may not be directly assimilated to the biological activities of the bLF.     

Functional and bioactive properties of collagen and gelatin from alternative sources: A review

The rising interest in the valorisation of industrial by-products is one of the main reasons why exploring different species and optimizing the extracting conditions of collagen and gelatin has attracted the attention of researchers in the last decade. The most abundant sources of gelatin are pig skin, bovine hide and, pork and cattle bones, however, the industrial use of collagen or gelatin obtained from non-mammalian species is growing in importance. The classical food, photographic, cosmetic and pharmaceutical application of gelatin is based mainly on its gel-forming properties. Recently, and especially in the food industry, an increasing number of new applications have been found for gelatin in products such as emulsifiers, foaming agents, colloid stabilizers, biodegradable film-forming materials and micro-encapsulating agents, in line with the growing trend to replace synthetic agents with more natural ones. In the last decade, a large number of studies have dealt with the enzymatic hydrolysis of collagen or gelatin for the production of bioactive peptides. Besides exploring diverse types of bioactivities, of an antimicrobial, antioxidant or antihypertensive nature, studies have also focused on the effect of oral intake in both animal and human models, revealing the excellent absorption and metabolism of Hyp-containing peptides. The present work is a compilation of recent information on collagen and gelatin extraction from new sources, as well as new processing conditions and potential novel or improved applications, many of which are largely based on induced cross-linking, blending with other biopolymers or enzymatic hydrolysis.