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研究生:陳筠芸
研究生(外文):Chen, Yun Yun
論文名稱:進口飼料用魚粉之組織胺相關衛生品質與三聚氰胺檢測方法之探討
論文名稱(外文):Histamine Related Hygienic Quality and Determination of Melamine in Fish Meals
指導教授:蔡永祥博士
口試委員:蔡永祥博士林家民博士江啟銘博士陳重文博士
口試日期:2011-07-25
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:水產食品科學研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:137
中文關鍵詞:魚粉組織胺
外文關鍵詞:Fish MealsHistamine
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本研究將不同國家進口之飼料用魚粉與魚溶漿進行一般組成分分析、化學品質、微生物品質、組織胺含量與組織胺生產菌之分離,以了解進口魚粉及魚溶漿之衛生品質;另外並以高效能液相層析儀(HPLC)探討魚粉中三聚氰胺含量檢測方法。研究結果顯示,魚溶漿樣品之平均水活性(0.80)、水分(39.9%)、鹽分(7.32%)、總揮發性鹽基態氮(150.7 mg/100g)與鹽酸不溶物(0.84%)均高於魚粉,且兩者間存在顯著性差異(p<0.05);但粗脂肪(3.94%)及粗蛋白(40.7%)含量平均值則顯著(p0.05)。所有樣品之總生菌數介於<1.0~6.14 log CFU/g之間,另有2件魚粉檢出大腸桿菌群,含量分別為2.85與1.60 log CFU/g;所有樣品均未檢出大腸桿菌與沙門氏菌。檢測8種生物胺中以Putrescine、Cadaverine、Histamine、Tyramine含量偏高;而2-phenylethylamine、Spermidine、Spermine、Tryptamine則較低,所檢測的魚粉及魚溶漿樣品組織胺含量分別有10% (4/40) 與20% (1/5) 高於20 mg/100g。另外,從魚粉樣品中分離出5株組織胺生產菌於含1% histidine之TSB培養液中可產生1.31~6.21 ppm之組織胺,經以PCR法增幅菌株之16S rDNA序列分析,得知5株組織胺生產菌分別為Bacillus licheniformis (3株)、B. amyloliquefaciens (1株)以及B. subtilis (1株)。
三聚氰胺含量的HPLC檢測方法則使用HILIC SILIC管柱,偵測器為光電二極體矩陣檢測器(Photodiode Array Detector, PDA),檢測波長為225 nm。結果顯示以HPLC-PDA檢測三聚氰胺之濃度於0.25~4 ppm呈線性關係,相關係數為0.9999,偵測極限值(LOD)為0.5 ppm,偵測限量值(LOQ)為1.5 ppm;添加三聚氰胺於魚粉樣品(No.10)之檢測回收率介於84.0%~91.9%,同日內精密度與異日間準確度於濃度0.5~4 ppm之變異係數皆小於5%。另外,在探討不同萃取條件對魚粉中三聚氰胺之回收率時,發現蛋白質沉澱劑(亞鐵氰化鉀、醋酸鋅、冰醋酸)可降低三聚氰胺HPLC層析圖譜雜訊;但樣品成分似乎會影響其回收率。並得知25% 乙腈(Acetonitrile)萃取液可取代原先使用50% 乙腈萃取液,且不影響樣品中三聚氰胺之回收率;其次,省略樣品純化過程中二氯甲烷(CH2Cl2)處理步驟,亦不影響其回收率,故可減少ACN及CH2Cl2等毒性物質之使用,並能確實有效檢測出魚粉中三聚氰胺含量。最後,以本研究建立之HPLC檢測法分析國外進口之45 件魚粉樣品(其中包含5件烏賊內臟粉),並以LC/MS/MS加以確認,得知只有一件進口烏賊內臟粉檢出7.3 ppm三聚氰胺。
The purpose of this study is to understand the histamine related quality of imported fish meal and fish soluble concentrates (FSC). Forty fish meals and five FSCs imported from foreign country were collected and tested to determine the biogenic amine and histamine-forming bacteria. The average levels of water activity (0.8), water content (39.9%), salt content (7.32%), total volatile basic nitrogen (TVBN) (150.7 mg/100g) and hydrochloric acid insoluble content (0.84%) in FSC samples were significantly higher than those of fish meal samples, whereas the average levels of crude fat (3.94%) and crude protein (40.6%) in FSC samples were significantly lower than those of fish meal samples. None of these samples contained Escherichia coli and Salmonella spp. The average contents of putrescine, cadaverine, histamine and tyramine in tested samples were more than 1.40 mg/100g. Four tested fish meal samples (10%, 4/40) and 1 tested FSC sample (20%, 1/5) had histamine levels greater than 20 mg/100g. Five histamine-producing bacterial strains isolated from fish meal samples producing 1.31~6.21 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH) were identified as Bacillus licheniformis (three strains), B. amyloliquefaciens (one strain) and B. subtilis (one strain).
Moreover, a method for the determination of melamine in fish meal by high performance liquid chromatographic with photodiode array detector (HPLC-PDA) was developed. The correlation coefficient for the standard curve of melamine between 0.25 and 4 ppm was 0.9999. The coefficients of variation of melamine in intraday and interday analysis were less than 5 %. Recovery of melamine from fish meal sample ranged from 84.0% to 91.9%. The detection of limit (LOD) and detection of quantitation (LOQ) of melamine were 0.5 ppm and 1.5 ppm, respectively. To evaluate the recovery effects of different extraction methods for melamine in fish meal, it was found that mixed protein precipitant, including potassium ferrocyanide, zinkacetat-dihydrate and acetic acid glacical, could reduce the background signal of the chromatography for melamine. In addition, the application of 25% ACN solution substituted for 50% ACN and dichloromethane for extraction in purification steps did not affect the recovery of melamine in HPLC analysis. Only one fish meal sample (squid viscera powder) had melamine content of 7.3 ppm in 45 imported fish meal samples by HPLC analysis and LC/MS/MS confirmation analysis.
中文摘要 I
英文摘要 III
誌謝 V
目錄 VII
表目錄 IX
圖目錄 XI
壹、研究動機 1
貳、文獻整理
一、魚粉、魚溶漿類飼料原料介紹 4
二、魚粉加工及流程 6
三、食品中之生物胺 9
四、組織胺對動物的危害 23
五、組織胺生產菌 25
六、三聚氰胺 27
七、三聚氰胺對動物的危害 38
八、三聚氰胺的分析方法 41
参、進口飼料用魚粉與魚溶漿之一般成份分析、化學及微生物品質探討
一、前言 46
二、材料與方法 49
三、結果與討論 64
肆、高效能液相層析儀檢測飼料用魚粉三聚氰胺含量之方法建立
一、前言 78
二、材料與方法 81
三、結果與討論 90
伍、結論 108
陸、參考文獻 111
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