Research On The Water-soluble Metabolites Profiles Of Takifugu Flavidus

Pufferfish（Takifugu flavidus）is regarded as one of the most cost-effective fish species in China because of its high economic value and palatable taste.The distribution of Takifugu flavidus is mainly in the coastal waters of the East China Sea,yellow sea and Bohai Bay.This species is described as one of the most famous foods in Asia for the unique delicious taste of the non-toxic muscle part.Pufferfish has special taste properties and their taste-active components were identified as amino acids and nucleotide-related compounds,such as glycine,lysine,alanine and inosine5’-monophosphate（5’-IMP）,which were the predominant compounds in pufferfish meat.Compared with artificially cultured pufferfish,the wild pufferfish meat is unforgettable to eat.The pufferfish is rich in collagen,which is famous for its beauty function.The scientific consumption of pufferfish is not only a delicious enjoyment,but also beneficial to human health.For safety reasons,the currently cultured pufferfish has reached muscle non-toxic levels.The pufferfish muscle has a strong taste characteristic of the amino acid sequence of the taste peptide,and these taste peptides make an important contribution to the delicious taste of the pufferfish.As a new active peptide flavoring agent,umami peptide is easily absorbed by the human body and has good biological activity,which is the focus of current research.In this paper,water-soluble metabolites in Takifugu flavidus were studied.The nuclear magnetic resonance（NMR）and water-soluble metabolites in the raw pufferfish meat and boiled pufferfish meat of Takifugu flavidus were determined and analyzed.The changes of water-soluble metabolites in pufferfish muscles of different processes were obtained.At the same time,using modern purification and identification technology of peptides,such as nano-liquid chromatography-mass spectrometry（NanoLC-Q-TOF-MS）to identify the amino acid sequence of five novel umami peptides.Combined with sensory evaluation,the taste characteristics were further explored.Finally,the taste characteristics and quantitative umami intensity of umami peptides in the muscles of Takifugu flavidus were verified by synthetic peptide and receptor biosensor.The main research contents are as follows:（1）The difference of metabolite profile between raw and cooked pufferfish（Takifugu flavidus）meat was explored by ¹H-NMR technique and multivariate statistical methods.The orthogonal projection to latent structure-discriminant analysis（OPLS-DA）results showed obvious separation between two samples.There were 24 dominated metabolites in the water-soluble extraction of pufferfish muscle,of which 11 metabolites changed significantly（p<0.05）during different processing methods,including 9 amino acids（alanine,leucine,methionine,tyrosine,glutamate,glycine,arginine,lysine,,taurine）,1 organic acids（lactate）and 1 alkaloid（betaine）.Moreover,Student’s t test was performed to identify the significant change of metabolites.The sensory evaluation was conducted to compare the taste difference between two samples.Sensory intensity experiments showed that there was a significant difference in the umami taste between raw and cooked pufferfish meat（p<0.05）.The aim of this study was to provide essential information for the taste difference between raw and cooked pufferfish meat.（2）Pufferfish（Takifugu flavidus）was known for its delicious taste,and the most praiseworthy was umami,the aim of this study was to identify and verify the taste characteristics of novel umami peptides in Takifugu flavidus.In this study,three flavor components（F1,F2,F3）with different molecular weights were obtained by ultrafiltration and nanofiltration,according to the results of sensory evaluation,the F2 component presented a unique umami,so the F2（200-3000 Da）component was separated and purified by gel filtration chromatography（GFC）.The dilution analysis（TDA）and the molecular weight distribution experiment of the peptides in the muscle of Takifugu flavidus showed that the F2-1 fraction contributed the most to the umami taste.Five novel peptides were found by identifying the F2-1fraction with nano-liquid chromatography-mass spectrometry（Nano-LC/Q-TOF-MS）.The amino acid sequences of five peptides were Asp-Val-Ile-Leu-Pro-Val-Pro-Ala-Phe（DVILPVPAF）,Thr-Val-Ala-Gly-Gly-Ala-Trp-Thr-Tyr-Asn-Thr-Thr-Ser-Ala-Val-Thr-Val-Lys （TVAGGAWTYNTTSAVTVK）,Ala-Met-Leu-Glu-Gln-Val-Ala-Met-Thr-Asp-Lys（AMLEQVAMTDK）,Gly-Gly-Lys-Leu-Val-Val-Asp-Gly-His-Ala-Ile-Thr（GGKLVVDGHAIT）and Ile-Gly-Ala-Glu-Val-Tyr-His-Asn-Leu-Lys（IGAEVYHNLK）.According to the amino acid composition of the taste peptides and the contribution of different amino acids to the taste,it can be inferred that the hydrophilic amino acid（Asp,Glu,Lys）and the sweet amino acid（Ala,Gly,Thr）contained in the peptides may be the the key factor of umami taste in Takifugu flavidus.（3）Through the principle of the biosensor,the quantitative determination of the umami intensity of the five synthetic umami peptides was carried out,the quantitative detection of the umami peptide was completed in combination with the sensory analysis results.Five umami peptides（DVILPVPAF,TVAGGAWTYNTTSAVTVK,AMLEQVAMTDK,GGKLVVDGHAIT and IGAEVYHNLK）were identified by mass spectrometry.Five kinds of umami peptides were synthesized by solid phase synthesis,and the taste characteristics of five peptides were analyzed by sensory technique.The sensory results showed that the binding constant（K_a）of DVILPVPAF,TVAGGAWTYNTTSAVTVK,AMLEQVAMTDK,GGKLVVDGHAIT,IGAEVYHNLK were:K_DF=4.541×10^-13g/L,K_TK=8.337×10^-13g/L,K_AK=7.916×10^-13 g/L,K_GT=11.242×10^-12 g/L,K_IK=6.675×10^-12 g/L,and the sensitivity of T1R1 to five umami peptides was as follows:DVILPVPAF>AMLEQVAMTDK>TVAGGAWTYNTTSAVTVK>IGAE VYHNLK>GGKLVVDGHAIT,the threshold of five peptides DVILPVPAF,TVAGGAWTYNTTSAVTVK,AMLEQVAMTDK,GGKLVVDGHAIT and IGAEVYHNLK were 0.258 mmol/L,0.799mmol/L,0.273 mmol/L,0.437 mmol/L and 0.858 mmol/L,respectively,it showed that DVILPVPAF has the strongest umami intensity,followed by AMLEQVAMTDK,GGKLVVDGHAIT,TVAGGAWTYNTTSAVTVK and IGAEVYHNLK.Because of the synergy between umami peptide and umami-enhancer,when adding flavor enhancers MSG and NaCl,the umami taste of the five synthetic peptides showed an upward trend.Combining the binding constant of five peptides with sensory thresholds,it was found the binding constant of the sensor can verify the results of sensory intensity and quantify the intensity of umami.