| The financial grant of this work has been supported by the R & D Special Fund or Public Welfare Industry from State Ocean Administration P.R. China (No.01305007).In this paper, to find the best harvest season for four species of clam, the method of detection of the bioactive and toxic component was established to study the dynamic change law of main components in these clams----Mactra veneriformis Reeve, Meretrix meretrix Linnaeus, Ruditapes philippinarum Adams et Reeve, and Cyclina sineusis Gmelin, which were harvested in different seasons. The quality standards of raw materials of these four clams were discussed, establishing scientific basis and theoretical fundament for the development of the deeply processed healthy food and the establishment of the quality standards of raw materials. Following sections constituted this paper.ReviewsThe related references in the domestic and overseas were checked about the bioactive and toxic components of shellfish Mollusca as well as the related methods of detection and analysis, offering the solid basis of references for further experiments.Law of dynamic change and the best harvest season of functional components in four shellfish MolluscaThe primary bioactive components of four species of clam--Mactra veneriformis Reeve, Meretrix meretrix Linnaeus, Ruditapes philippinarum Adams et Reeve, and Cyclina sineusis Gmelin----Were researched and discussed under the guides of the theory of Chinese Medicine and based on medicinal functions of low-value shellfish in Jiangsu coastal waters and the medicine food homology. The content of bioactive components of four species of clam in different harvest seasons was detected following the establishment and validation of the methods for the detection of bioactive components. The results showed that the content of polysaccharose and the water soluble protein, nucleoside, unsaturated patty acid EPA and DHA, amio acids, and taurine was associated with seasonal changes, laying the theoretical foundation for the best harvest time and the establishment of quality standards for the four species of clam.1. Analysis of polysaccharoseThe anthrone-sulfuric acid method for determination of polysaccharose with a high precise and easy process was established, and it proved good to determine the polysaccharose. In addition, the polysaccharose in four species of clams from LvSi, Jiangsu was detected. The results suggested that polysaccharose in these four clams was ample, among which Meretrix meretrix Linnaeus had the most polysaccharose with a content of 20.80 mg/g, followed by Cyclina sineusis Gmelin of 20.33 mg/g, Ruditapes philippinarum Adams et Reeve of 18.29 mg/g, Mactra veneriformis Reeve of 20.42 mg/g. The statistics were analyzed using ANOVA and the results showed that the content differed with seasons with the content in summer higher than that in winter, especially in June and July. Consequently, the best harvest time for these four clams were June and July in light of the content of poly saccharose.2. Analysis of water-soluble proteinsWater-soluble proteins in four species of clams were determined by the BCA method that was originally established here. Contrast to the traditional Lowry method, the BCA method showed a good specificity, easy procedure, high sensitivity, little difference of the reaction between different proteins and BCA regent, and little interference. The water-solube proteins in four species of clams from LvSi, Jiangsu was detected and results indicated that four clams all possessed substantial digestibly water-soluble proteins, especially when they were harvested in June and July. The content of water-soluble proteins in Meretrix meretrix Linnaeu was the highest, which was near 32.71 mg/g. As to other three clams, the content was similar:27.94 mg/g for Ruditapes philippinarum Adams et Reeve,26.70 mg/g for Cyclina sineusis Gmelin, and 26.23 mg/g for Mactra veneriformis Reeve. The harvest time were June and July when water-soluble proteins served as index.3. Analysis of nucleosideThe components of nucleoside were qualified by HPLC-Q-TOF-MS and 13 types of nucleoside were identified. In addition, the method of HPLC-UV was established to detect these 12 nucleosides in four species of clams and easy process, high sensitivity and good repeatability showed the method good.12 nucleosides were adenine, cytidine, hypoxanthine, xanthine, uridine, thymine,2-deoxyuridine, inosine, guanine,2-deoxyinosine,2-deoxyguanine, and thymidine, respectively. Dynamic changes of the components of nucleoside were analyzed using 1-way ANOVA, leading to a discussion of optimum harvest time for each clam. Total content of nucleosides significantly differed with seasons, which was lower in winter than in summer with a total content being the highest in June and July. The highest content was 4160.47 μg/g for Ruditapes philippinarum Adams et Reeve, when total content of nucleosides in the other three clams was approximate,2450.56 μg/g for Meretrix meretrix Linnaeus,2587.10 μg/g for Cyclina sineusis Gmelin, and 2850.91μg/g for 2850.91μg/g. In summary, the beat harvest time for four clams was June and July.4. Analysis of unsaturated fatty acid EPA and DHAThe constitution of fatty oil in four species of clams was analyzed by GC-MS. 17 fatty acids were identified, and 10 were unsaturated fatty acids including the wholesome DHA and EPA. Both the content of unsaturated fatty acids and DHA and EPA were the highest in Cyclina sineusis Gmelin, with the proportion of DHA and EPA being 25.42%. On the contrary, the content of unsaturated fatty acids were the lowest in Mactra veneriformis Reeve, as well as DHA and EPA with a proportion of 16.03%.Besides, the determination of EPA and DHA was easily performed using HPLC-UV and methodology indicated that the sensitivity was high and the repeatability good. Four species of clams from LvSi, Jiangsu were detected using this method. Results showed that four clams contained plenty of EPA and DHA, the total content being the highest in summer(June and July). The content of EPA and DHA was in the serials:Cyclina sineusis Gmelin(12023.06μg/g)> Ruditapes philippinarum Adams et Reeve (4565.38μg/g)> Meretrix meretrix Linnaeus(3115.78 μg/g)>Mactra veneriformis Reeve(2416.27 μg/g). As such depending on the content of EPA and DHA, and changes of total content, the best harvest time for four clams was June and July.5. TaurineTaurine in clams was determined by pre-column derivatization using OPA in conjunction with HPLC-UV, and methodology suggested that this method could be easily conducted and well repeated with a high sensitivity. Four species of clams from LvSi, Jiangsu were detected using this method. The results suggested that sufficient taurine was detected in these clams, and the content was the highest in June and July. The content of taurine in Meretrix meretrix Linnaeus was secondary to Ruditapes philippinarum Adams et Reeve, of which the content was 27.48 mg/g for the former and 42.79 mg/g for the latter respectively. The content of taurine in Cyclina sineusis Gmelin and Mactra veneriformis Reeve was at a relatively lower level,16.59 for the former and 15.01 mg/g for the latter. In combination of output and the content of taurine, the best harvest time of these clams was June and July.6. Analysis of free amino acidFree amino acids in four species of clams from LvSi, Jiangsu were analyzed by HPLC-MS, and 22 free amino acids were identified. By analysis of one-way ANOVA, the content of total free amino acids in Ruditapes philippinarum Adams et Reeve came first, about 130.26 mg/g in July. As with Mactra veneriformis Reeve and Meretrix meretrix Linnaeus, they are of about the same content (82.54mg/g and 91.77 mg/g), and Cyclina sineusis Gmelin however contained the least content of 54.58 mg/g but the higest content was also in June. In addition, γ-aminobutyric acid, ornithine and citrulline were also found in these clams. Hence, June and July were the best harvest time for these clams based on the total content of free amino acids.Law of dynamic change of toxic components in four shellfish Mollusca1. Heavy metal (As. Pb, Hg, Cd)In accordance with GB/T5009.12,11,17, and 15 in the National Food Safety Standards, content of AS, Pb, Hg, and Cd was determined using graphite furnace atomic absorption spectrophotometry. The results indicated that the content of four kinds of heavy metal was much lower in relation to the limitation of the food contamination, meeting the requirements of the National Food Safety Standards.The content of heavy metal showed a law of dynamic change with seasons. The content of Cd was the lowest in June and July, as well as As with exception of Mactra veneriformis Reeve of which the content of As was the lowest in March. Different from As and Cd, the lowest content of Pd was in August and Sepetember. As to Hg, the lowest content for Mactra veneriformis Reeve and Ruditapes philippinarum Adams et Reeve was in June and July whereas for Meretrix meretrix Linnaeus and Cyclina sineusis Gmelin, the highest content appeared in June and July and the lowest in April. 2. Shellfish Poison (DSP)The content of diarrhetic shellfish poison (DSP) were determined using UFLC-MS/MS. Methodology showed that the repeatability and accuracy of this UFLC-MS/MS method were satisfying, and a small amount of sample was needed for the determination. Subsequently, DSP in four species of clams from LvSi, Jiangsu was detected. The results indicated that the content of DSP significantly related with season change, and the highest content was detected in summer. Furthermore, YTX, AZA1, AZA2, and AZA3 were not detected. Ruditapes philippinarum Adams et Reeve contained four types of DSP---GYM, PTX-2, SPX, and OA, the most types of DSP among these four species of clams. Four types of DSP reached the highest in May, June and July, with a content of 0.06 ug/kg,0.48 ug/kg and 0.65 ug/kg. Content of GYM and PTX-2 of Meretrix meretrix Linnaeus was the highest in summer,4.74 ug/kg and 0.69 ug/kg, respectively. Only two types of DSP were detected in Meretrix meretrix Linnaeus, and the highest content were in June,5.29 ug/kg and 7.21 ug/kg respectively; for Mactra veneriformis Reeve, only PTX-2 was detected with the highest content of 0.40 ug/kg in July. The content of four types of DSP was all low, and even the highest one was less than the limitation of toxin of European Union, confirming to the requirements of the National Food Safety Standards.Quality standards research for raw materials in four species of clamTo rigorously control the quality of raw materials and to offer standard raw materials for the preparation of in-depth processed product, the quality of raw materials was preliminarily researched including original and characteristic identification, inspection and the determination of functional components. This work offered experimental basis and research foundation for establishing and drawing up the quality standards of raw materials. |
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