Traceability And Identification Of Grass Carp Gelatin Based By Biological Mass Spectrometry

Due to religious beliefs,food safety,eating habits and other factors,fish gelatin as an excellent substitute for mammalian gelatin is gradually favored by consumers.With the gradual circulation of fish gelatin in the market,how to effectively identify the authenticity of fish gelatin in gelatin products has become an urgent problem to be solved.As the largest proportion of freshwater fish production,grass carp has great potential for use in the food industry.Grass carp gelatin can be obtained by heat extraction from various fish by-products such as fish skin,scales,bones,swim bladders and fish intestines produced during processing.Sources of different parts of grass carp,extraction temperatures,processing methods,and food matrices may have an impact on the traceability of grass carp gelatin.Therefore,based on the amino acid sequence of collagen type I and trypsin mimicry digestion of bulk freshwater fish,porcine and bovine,this paper determined that the characteristic peptides of bulk freshwater fish gelatin that can theoretically be detected by high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）and the unique characteristic peptides of specific fish gelatin were determined.Trypsin digestion and HPLC-MS/MS detection were used to identify the actual characteristic peptides of seven bulk freshwater fish gelatin,and the detected dcommon characteristic peptides of bulk freshwater fish gelatins and the unique characteristic peptides of grass carp gelatin were analyzed.Furthermore,taking different parts of grass carp,extraction temperature and different food systems as the influencing factors,the effects of each factor on the detection effect of the common characteristic peptides of bulk freshwater fish gelatins and the unique characteristic peptides of grass carp gelatin were studied,and the characteristicpeptides with high stability and good detection effect were screened to provide a basis for the accurate identification of grass carp gelatin in the food system.The main conclusions can be summarized as follows:（1）Taking the known type I collagen amino acid sequences of freshwater fishes（grass carp,crucian carp,silver carp）,porcine and bovine as the research object,multi-sequence alignment software and trypsin simulation digestion were used to study the characteristic polypeptides of freshwater fish that could theoretically be identified by HPLC-MS/MS,and further applied to the exploration of the actual characteristic peptides of seven bulk freshwater fishes gelatins.The results showed that whether it wasα₁ chain orα₂ chain,the similarity of type I collagen sequences of different bulk freshwater fishes（94.1%-98.5%）was significantly higher than that between freshwater fish and mammals（porcine,bovine）（70.9%-78.8%）,and the theoretical characteristic peptides of bulk freshwater fishes gelatins were 87,and the theoretical unique characteristic polypeptides of grass carp,crucian carp and silver carp were 26,78 and 32,respectively,which did not appear in porcine and bovine gelatins,and could be used to exclude typical mammalian gelatin（porcine,bovine gelatin）interferes with and gradually narrows the scope to accurately identify a certain species of bulk freshwater fish gelatin.Based on the above characteristic peptide information,7 actual common characteristic peptides of 7 bulk freshwater fishes gelatins（black carp,grass crap,silver carp,bighead,common carp,crucian carp,bream carp）（1 peptide without modification）were identified,of which 3 were derived fromα₁ chain and 4 were derived fromα₂ chain.In addition,based on the theoretical unique characteristic peptide of grass carp gelatin,44 detected unique characteristic peptides of grass carp gelatin were determined（7 peptides did not contain modification groups）,of which 20 were derived fromα₁ chain and 22 were derived fromα₂ chain.Combined common characteristic peptides bulk freshwater fish gelatin with unique characteristic polypeptides of grass carp gelatin,which can provide a reference for the identification of grass carp gelatin;（2）Gelatin was extracted from collagen-rich grass carp skin,scales,bones,swim bladder and intestines,combined with trypsinization and HPLC-MS/MS to explore the differences in the characteristic peptides of grass carp gelatin derived from different parts.The results showed that 545,560,451,568 and 591 characteristic peptides were detected from grass carp skin and scales,fish bones,swim bladders and fish intestine gelatin samples,respectively,of which 284 characteristic peptides were common（166 fromα₁ chain and 118 fromα₂ chain）.Based on the theoretical characteristic peptide information of bulk freshwater fishes gelatins,74 actual common characteristic peptides bulk freshwater fishes gelatins were screened that were not affected by the source of grass carp,of which 6 common characteristic peptides would stably appear in the actual detection of 7 bulk freshwater fishes gelatins.In addition,based on the theoretical unique characteristic peptide information of grass carp gelatin,35 detected unique characteristic peptides of grass carp gelatin that were not affected by the source of bodies site of the grass carp were screened and obtained,of which 24 unique characteristic polypeptides only existed in the actual detection of grass carp gelatin,and did not appear in the other six freshwater fishes gelatins.These common characteristic peptides of bulk freshwater fishes gelatins and unique characteristic peptides of grass carp gelatin that were not affected by the origin of different dodies site could help to continue to explore the influence of other different factors on the traceability and identification of grass carp gelatin.（3）Using grass carp scales as raw materials,gelatin was extracted at different temperatures（50-100℃）,and then HPLC-MS/MS technology was used to detect grass carp gelatin after trypsin enzymatic hydrolysis,so as to study the influence of different thermal extraction temperatures on the traceability and identification of grass carp gelatin.The results showed that the characteristic peptides identified in the grass carp gelatin extracted at 50℃,60℃,70℃,80℃,90℃ and 100℃ were 598,492,440,476,603 and 530,respectively,containing 268 common characteristic polypeptides（152 fromα₁ chain and 116 fromα₂ chain）.Among these peptides,75detected common characteristic peptides of bulk freshwater fishes gelatins and 41detected unique characteristic peptides of grass carp gelatin were obtained based on the theoretical common characteristic peptides of bulk freshwater fishes gelatins and theoretical unique characteristic peptides of grass carp gelatin,respectively.Furthermore,compared with the identification results of grass carp gelatin extracted from different parts,4 detected common characteristic peptides of bulk freshwater fishes gelatins and 19 detected unique characteristic peptides of grass carp gelatin were screened out respectively;（4）Five different grass carp gelatin food systems（gummies,marshmallows,ice cream,jelly and cheese）were prepared by using grass carp scale gelatin,and the grass carp gelatins in these food systems were extracted and enzymatically hydrolyzed and then detected and analyzed by HPLC-MS/MS to explore the influence of different food systems on the traceability and identification of grass carp gelatin,and to determine the characteristic polypeptides of grass carp gelatin that could be consistently detected.The results showed that 172,130,162,177 and 185characteristic peptides of grass carp gelatin were detected in gummies,marshmallows,ice cream,jelly and cheese,respectively,which may be related to the processing method,gelatin addition amount,food matrix and other variables in different food systems.In these five grass carp gelatin food systems,56 common characteristic peptides（25 fromα₁ chain and 31 fromα₂ chain）were detected.On this basis,based on the theoretical common characteristic peptide of bulk freshwater fishes gelatins and theoretical unique characteristic peptide of grass carp gelatin,17 detected common characteristic peptide of bulk freshwater fishes gelatins and 10 detected unique characteristic peptide of grass carp gelatin that were not affected by complex food systems were screened,respectively.Further compared with the identification results of grass carp gelatin extracted from different parts and different temperatures,2 detected common characteristic peptide of bulk freshwater fishes gelatins（all hydroxylated modified peptides）and 8 detected unique characteristic peptide of grass carp gelatin（3 polypeptides without modification）were detected constantly,which could provide a basis for the accurate identification of grass carp gelatin in the food system and a method reference for the traceability of other fish gelatin.