Microsoft PowerPoint - 発表原稿.ppt

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1 シート状培地サニ太くんの評価 - AOAC Performance Tested Methods- 牛山正志 1) 岩崎美穂子 1) 青山茂之 1) Ann M. Schulz 2) Wendy Lepper 2) 森田裕 1) 1) チッソ横浜研究所 2)Sillikar Laboratories Research Center

2 AOAC Method Validation Programs Official Methods of Analysis(OMA) Proprietary Non-Proprietary Performance Tested Methods(PTM) Peer-Verified Methods(PVM)

3 AOAC Performance Tested Methods Validated Performance Criteria Specificity rate Sensitivity rate False Positive and False Negative Rates Percent Agreement to Reference Method Ruggedness Matrix effects Recovery Limited Inter-Laboratory Reproducibility Limit of detection Limit of quantitation Precision Accuracy Quality Assurance Policy

4 AOAC PTM サニ太くん評価 Analytes 一般生菌定量試験 Matrices 全食品 ( 除色素 ) 15(14) グループから少なくとも 1 種ずつ Accuracy(AOAC 法との比較 ) 1 食品最低 3 ロット Precision-Repeatability 4 汚染レベル各 5 試料での再現性 Ruggedness 至適条件から統計的に有意差のない範囲の確定温度時間 Lot to Lot ( 兼 Stability, Shelf Life) 最低 3 ロット Limit of quantitation (Limit of detection) Independent validation Study In-house Validation の確認

5 Foods Group1 Raw meats 生または冷凍豚肉 Group2 Fresh produce キャベツ Group3 Processed meats ソーセージ焼き豚 Group4 Seafoods 冷凍エビ Group5 Dairy アイスクリーム Group6 Egg/Egg products から付き卵厚焼き卵 Group7 Spices オレガノ一味唐辛子 Group8 Dry foods そば粉青のり Group9 Fruits/juices レーズン Group10 Uncooked pasta うどん ( 乾麺 ) Group11 Nuts ピーナッツバター Group12 Confectionary ミルクチョコレート ( チップ ) Group13 Pet food キャットフード ( 乾燥タイプ ) Group14 Food dye Group15 Miscellaneous 冷凍ピザ

6 Accuracy: サニ太くん一般生菌用と AOAC Official Method の比較それぞれ50gの食品をAOAC Method にしたがってホモジナイズから付き卵は AOAC method したがって調製滅菌生理食塩水で10 倍希釈列を作製試料懸濁液希釈懸濁液 1mLを5 枚のサニ太くんおよび滅菌シャーレに加え滅菌シャーレはAOAC Method にしたがって混釈時間培養後計数

7 食品 50g 懸濁液 AOAC Method (450mL バターフィールド緩衝液ホモジナイズ ) ( から付き卵 :940.37) Accuracy 1mL 滅菌生理食塩水で希釈 35 培養希釈懸濁液 (1:100, 1:100, ) 1mL 48 時間後計数

8 AOAC Method とサニ太くん一般生菌用の比較 APC(Log 10 cfu/g) Food lot Lot#1 Lot#2 Lot#3 食品サニ太くん AOAC Method サニ太くん AOAC Method サニ太くん AOAC Method 冷凍または生豚肉キャベツ焼き豚冷凍エビアイスクリームから付き卵オレガノ一味唐辛子そば粉レーズンうどんピーナッツバターミルクチョコレートキャットフード冷凍ピザ

9 AOAC Official Method とサニ太くん一般生菌用の比較 8 7 y = x R 2 = Sanita-kun (logcfu/g) Plate Count Agar (logcfu/g)

10 SDrs of each 3 lots of food sample by Sanita-kun Aerobic Count and plate count agar SDr Food lot Lot#1 Lot#2 Lot#3 Food samples Sanita-kun Plate count agar Sanita-kun Plate count agar Sanita-kun Plate count agar lower dilution higher dilution lower dilution higher dilution lower dilution higher dilution lower dilution higher dilution lower dilution higher dilution lower dilution higher dilution Frozen or Raw Pork Cabbage Roast Pork Frozen Shrimps Ice Cream Shell Egg Oreganos Red Pepper Buckwheat flour Raisins Udon Noodles Peanut Butter Milk Chocolate Dry Cat Food Frozen Pizza Red figure : significantly higher (P<0.01), Blue figure : significantly higher (P<0.05)

11 Food 25g <10, , 約 1000, 約 2500 cfu/g Independent validation study 225mL バターフィールド緩衝液ストマッキング 2 分懸濁液 1mL バターフィールド緩衝液で希釈 35 培養希釈懸濁液 (1:100, 1:100, ) 1mL 48(24) 時間後計数

12 Independent validation study 食品ホットドッグ牛挽肉目標菌数 (cfu/g) <10 <10 (7/8) AOAC Methods(48 時間 ) サニ太くん (24±2 時間 ) サニ太くん (48 時間 ) 8 連の平均 SDr 8 連の平均 SDr 8 連の平均 SDr (cfu/g) (cfu/g) (cfu/g) <10 < <10 <10 <10 <

13 Precision: Repeatability Several lots of the 14 food types obtained at local retail markets. The contamination level of each sample was determined according to the method Several lots of each food type were mixed to prepare four target levels (<10, 10-25, ca 100, ca 250 cfu/ml). Each prepared sample was divided into five subsamples. Each 50g of subsample was homogenized with 450mL of sterile physiological saline and diluted with sterile physiological saline. 1mL of homogenates or dilutions were inoculated to duplicate Sanitakun and incubated at 35 for 48 hours. Red colonies formed on the sheets were counted.

14 食品食品食品食品食品混合 Repeatability 食品 (<10) 食品 (10-25) 食品 ( 約 100) 食品 ( 約 250) cfu 10 n /g 5 分割食品 50g 4 汚染水準 450mL 滅菌生理食塩水 35 培養ストマッカー袋ストマック 2 分懸濁液 48 時間後計数 1mL 滅菌生理食塩水で希釈希釈懸濁液 (1:100, 1:100, ) 1mL

15 Repeatability test results Food Pork Cabbage Frankfurter Shrimp Ice cream Fried egg (Atuyakitamago) Red pepper Target level (cfu/ml) Average count SD SDr Food Target level (cfu/ml) Buckwheat Average count SD SDr < < flour Ca Ca Ca Ca < Raisins < Ca Ca 100 No sample Ca Ca 250 No sample < Udon noodles < Ca Ca Ca Ca < Peanut butter < No sample Ca Ca 100 No sample Ca Ca 250 No sample < Milk < chocolate Ca 100 No sample chips Ca 100 No sample Ca 250 No sample Ca 250 No sample < Dry cat food < Ca Ca Ca Ca < Frozen pizza < Ca Ca Ca Ca all foods Target level Average SDr (cfu/ml) count < Ca Ca

16 Ruggedness Testing -The effect of incubation temperature and time- Each 50g of sample was added to stomacher bag. 450 ml of sterile physiological saline was added to the stomacher bag and stomached for 2 min. Sample dilutions (1:100, 1:1000, 1:10000, ) were prepared to add sterile physiological saline. 1 ml of sample homogenate and dilutions were applied on five replicates Sanita-kun sheets and incubated at 30, 32.5, 35, 37 for hours. Colonies counted at 24, 27-30, 41, and/or 48 hours after incubation.

17 食品 50g Ruggedness Testing 450mL 滅菌生理食塩水ストマッカー袋ストマック 2 分 30, 32.5, 35, 37 培養懸濁液 1mL 滅菌生理食塩水で希釈希釈懸濁液 (1:100, 1:100, ) 1mL 24, 27-30, 41, 44-45, 48 時間後計数

18 Effect of incubation time at 32.5 Food APC(log 10 cfu/g) Incubation time(hr) Raw Pork Cabbage Roast Pork Frozen Shrimp Frozen Shrimp Fried egg Red pepper Red pepper Buckwheat flour Buckwheat flour Noodles (Udon) Dry cat food Frozen pizza Frozen pizza Red figure : Significanly lower than 48hr(P<0.01), Blue figure : Significantly lower than 48hr(P<0.05)

19 Effect of incubation temperature at 48hr incubation Food APC(log 10 cfu/g) Incubation temperature( ) Raw Pork Cabbage Roast Pork Frozen Shrimp Frozen Shrimp Fried egg Red pepper Red pepper Buckwheat flour Buckwheat flour Noodles (Udon) Dry cat food Frozen pizza Frozen pizza Red figure : Significanly lower or higher than 32.5 (P<0.01) Blue figure : Significantly lower or higher than 32.5 (P<0.05)

20 Lot to lot study -Comparison of the three lots of Sanita-kun- The three lots of the Sanita-kun sheets were 9909TC(manufactured in Sep 1999), 00918TC(Sep 2000) and TC(Nov 2000). Eleven foods were prepared homogenates according to Method Sample dilutions ( 1:100, 1:1000, 1:1000, ) were prepared to add sterile physiological saline. 1mL of sample homogenate and dilutions were applied on the duplicate Sanita-kun sheets and incubated at 35 for 48 hours. Colonies on each Sanita-kun sheets were counted after the incubation.

21 Lot to lot study -Comparison of the three lots of Sanita-kun- 99/9 00/9 00/ TC 00918TC TC 1mL Food s homogenate or dilutions Incubated at 35 for 48 hours and counted

22 サニ太くんロット間比較 Food APC(log 10 cfu/g) SDr of APC 9909TC 00918TC TC Average 生肉ソーセージ冷凍エビアイスクリーム厚焼き卵一味唐辛子オレガノそば粉うどんピーナッツバターキャットフード

23 Limit of quantitation 食品平均 (10 連 ) SD SDr 豚肉キャベツソーセージエビアイスクリームから付き卵一味唐辛子そば粉うどんピーナッツバターミルクチョコレートチップキャットフード冷凍ピザ

24 まとめサニ太くん一般生菌用をAOACPTMプログラムにより評価し Performance Tested Status( 認証 ) に達したほとんどの食品でサニ太くんと標準寒天で統計的に同等な菌数が認められ回帰式はy=0.986x 相関係数は0.99であった Independent Lab. Validationにおいても同様な結果が得られサニ太くんとAOAC Methodsが同等であることが示された多くの食品で30~35 44~48 時間で生育数に差は認められなかったサニ太くん3ロット間の差は認められず少なくとも 14ヶ月間の保存性が確認された

320 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.1, -,* -,/ (,**1) 8 * * * * E#ect of Superheated Steam Treatment on the Preservation an

320 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.1, -,* -,/ (,**1) 8 * ** *** * ** *** E#ect of Superheated Steam Treatment on the Preservation and Sensory Characteristics of Buckwheat Noodles Kazuhiro