200936061 六、發明說明 【發明所屬之技術領域】 本發明係關於一種飮料等液體食品之殺菌方法，較詳 細而言，關於一種使用具有短脈衝寬度之高電場脈衝之液 體食品之殺菌方法。 【先前技術】 〇 以往以來’飲料等液體食品之殺菌，一般係藉由將該 材料加熱，加上一定之熱負荷而進行。然而，對液體食品 材料給予熱負荷之如此的加熱殺菌之中，有難以不損及其 成分而利用食品材料所持有本來的營養成分或風味的問題 。特別是如細菌芽胞般，對熱具有高耐性之微生物之殺菌 而言，爲必須給予液體食品過大的熱負荷才能保證商業的 無菌之現狀。此處所謂商業的無菌，並非完全無菌之狀態 ，係意指在商業上之流通所必要的期間，可防止微生物造 ❹ 成之腐敗變壞之微生物控制狀態。 因此出現了可減低加熱造成之品質劣化般，代替加熱 殺菌之殺菌方法之提案，就如此的殺菌方法之其中一個而 言，藉著對液體食品直接施加高電壓進行殺菌之技術被提 出（例如專利文獻1至3 )。 在特開2000-262261號之中，揭示了藉著施加脈衝寬 度短之高電場脈衝，抑制殺菌對象液之溫度上昇，不引起 加熱造成之對象液之品質劣化般之芽胞形成細菌之殺菌方 法。然而，在此文獻所揭示之實施例中之芽胞形成細菌之 -5- 200936061 殺菌效果，還不能充分說是可達成商業的無菌狀態程度。 在特開2006-2 38827號及日本專利第2964037號之中 ，揭示了藉由交流高電場之施加，使液體食品於未滿1秒 之內溫度上昇，使高溫保持之時間成爲短時間，進行細菌 芽胞之殺菌之方法。然而，藉由此方法，爲了得到充分之 殺菌效果，有必要爲與以往的加熱殺菌同程度，或其以上 之殺菌溫度，難以說是可使液體食品之殺菌溫度降低，充 分地減低加熱造成之品質劣化。 [專利文獻1]特開2000-262261號公報 [專利文獻2]特開2006-238827號公報 [專利文獻3]專利第2964037號公報 【發明內容】 [發明所欲解決之課題] 本發明目的爲提供一種方法，以相較以往的加熱殺菌 更低殺菌溫度、或短的殺菌時間，有效果地將微生物’特 別是細菌芽胞殺菌，達成不引起流通上腐敗或變壞之問題 般之商業的無菌。另外目的爲藉由此方法，充分地減低液 體食品之加熱造成之品質劣化。 [用於解決課題之方法] 本發明人等，爲了解決上述課題進行專心檢討之結果 ，首先發現施加脈衝寬度短之高電場脈衝，將液體食品材 料加熱至既定之殺菌溫度，結果可將上述之細菌芽胞在相 -6- 200936061 較以往之加熱殺菌法更低殺菌溫度有效果地殺菌。 亦即本發明，發現藉由將脈衝寬度定爲未滿200ns及 脈衝上升時間定爲50ns以下，且電場強度定爲10〜 2 0 Ok V/cm之高電場脈衝施加於液體食品材料，使該液體 食品材料加熱到至少1 00 °C之既定溫度，可達成充分之殺 菌效果，而使本發明達到完成。 本發明，係包含以下之形態》 〇 1· 一種液體食品材料之殺菌方法’包含藉由將脈衝寬 度定爲未滿200ns及脈衝上升時間定爲50ns以下，且電 場強度定爲10〜2 0Okv/cm之高電場脈衝施加於液體食品 材料，使前述液體食品材料加熱到至少100 °C之既定溫度 2. 如前述1所記載之殺菌方法’其中前述既定溫度係 100 至 160°c， 3. 如前述1或2所記載之殺菌方法，其中前述高電場 〇 脈衝係重複頻率100HZ以上之脈衝， 4. 如前述1至3之任一者所記載之殺菌方法，其中前 述液體食品材料之導電率係30〜100 OmS/m, 5. 如前述1至4之任一者所記載之殺菌方法，其中加 熱至前述既定溫度所需要的時間爲10秒以下， 6. 如前述1至5之任一者所記載之殺菌方法，其中包 含加熱至前述既定溫度之後，於3 00秒以內冷卻， 7. 如前述1至6之任一者所記載之殺菌方法，其特徵 爲連續地進行， -7- 200936061 8. —種液體食品材料，係藉由前述1至7之任—者所 記載之方法進行殺菌處理， 9. 一種飲料，係藉由前述1至7之任〜者所記載之方 法進行殺菌處理， 1〇_—種殺菌裝置，係具有液體食品材料之收容部之 施加手段，並包含可對導入至該收容部之液體食品材料， 施加寬度未滿200ns、上升時間爲50ns以下，而且電場強 度爲10〜200kV/cm之高電場脈衝之施加手段，而係用於 藉由對導入至該收容部之液體食品材料施加寬度未滿 200ns、上升時間爲50ns以下，而且電場強度爲1〇〜 2 00kV/cm之高電場脈衝，將該材料加熱到至少1〇(rc而將 該材料殺菌。 〔發明之效果〕 藉由本發明，發現可以在相較於以往之加熱殺菌法較 低殺菌溫度，或短的殺菌時間，實施與加熱殺菌法同等殺 菌效果之殺菌。具體而言藉由本發明之方法，可將細菌芽 胞等耐熱性強的微生物，以相較於以往的加熱殺菌法5〜 1 0 °C低殺菌溫度，或一半程度短的殺菌時間，進行與加熱 殺菌法同等殺菌效果之殺菌。另外，在本發明之方法之中 ，由於可在較以往方法低殺菌溫度進行殺菌，因此可抑制 伴隨殺菌時之加熱之液體食品材料之風味或營養成分之劣 化。進一步，在本發明之方法中，與以往的加熱殺菌相比 ，可更快速地殺菌。 -8- 200936061 【實施方式】 本發明之液體食品材料之殺菌方法，係包含將脈衝寬 度定爲未滿200ns及脈衝上升時間定爲50ns以下，且將 電場強度定爲10〜200kV/cm之高電場脈衝施加於液體食 品材料，將此液體食品材料之溫度加熱到至少1 0 0 °C之既 定溫度者。 〇 本發明之殺菌方法，可藉由將脈衝寬度定爲未滿 2〇〇ns，例如1〇〇〜15 0ns、脈衝上升時間定爲50ns以下， 例如10〜50ns、而且電場強度定爲1〇〜200kV/cm、例如 30〜150kV/cm、或60〜120kV/cm之高電場脈衝以100Hz 以上之重複頻率，例如100〜10000Hz、或100〜400Hz之 重複頻率施加於液體食品材料，將其液體食品材料之溫度 加熱到至少l〇〇°C之既定溫度，例如100〜160°C、或100 〜140 °C而進行。爲了將液體食品材料加熱到既定溫度， ❹ 可將高電場脈衝之施加定爲多次，例如100〜500次。 藉由施加，加熱至前述既定溫度所必要的時間，係以 例如定爲5秒以下，或1 0秒以下者爲佳。 另外’在本發明之殺菌方法中，可因應必要在加熱至 前述既定溫產之後，將液體食品材料冷卻（至例如1〇〜30 °C )。冷卻可在加熱至前述既定溫度之後，於例如3 00秒 以內，或於30秒以內’具體而言於1至300秒以內、或 於5至3 0秒以內進行。 脈衝寬度、脈衝上升時間及電場強度，以及施加之次 -9- 200936061 數，係可基於液體食品材料受到加熱之既定溫度而設定。 然後，冷卻至此既定溫度之時間，係以可保證液體食品材 料之商業的無菌之方式設定，液體食品材料之種類或流通 之期間，可因應於流通溫度而變動者。此爲亦即由於液體 食品材料之種類不同，流通時有引起腐敗或變壞之危險性 之微生物之種類而有所不同，再者由於流通之期間或流通 溫度不同，該等微生物之增殖之危險性而相異之故。另外 ，可保證商業的無菌，係意指可達成其液體食品材料在流 通上不會發生腐敗或變壞等問題般的殺菌效果，亦即可達 成至少1位數以上之殺菌效果。在本發明之殺菌方法中， 藉由施加先前規定之脈衝，使液體食品材料加熱到至少 1 00 °c，可達成例如1位數以上之殺菌效果。 就本發明所可適用之液體食品材料而言，只要是殺菌 處理爲必要的液體食品，則並無特別限制。例如清涼飲料 或酒精飮料、具體而言可列舉果汁•果實飲料、茶飮料、 咖啡飮料、果汁氣酒等。本發明之方法特別是適用於具有 30〜1 000mS/m、或90〜700mS/m之導電率之液體食品材 料之殺菌。 本發明之殺菌方法，可藉由例如使用包含連接於可產 生高電場脈衝之電源之一對之對向之平面電極及由適當的 絕緣材料所構成之具有既定容積之液體收容部之施加部之 裝置’於該收谷部加入液體食品材料之後，將前述所規定 之脈衝透過該施加部之電極，由電源施加於該材料，加熱 到至少100°c之既定溫度而進行。 200936061 就電源而言，只要是可產生前述所規定之高電場脈衝 之電源，則並無特別限制。可使用例如組合半導體開關與 磁氣脈衝壓縮回路之產生可重複的高電場脈衝之電源。 另外’本發明之殺菌方法，作爲其中一個形態可使用 具有例如圖1之構成之裝置而實施。圖1中、A係液體食 品材料收容部、B係泵、C係溫度調節手段、D係施加部 、E係冷卻手段、F係閥、G係電源裝置、Η係溫度測定 6 手段，然後1係壓力計。Α至F之各部係藉由配管被連結 ，液體食品材料係被由A往F之方向送液。 使用如圖1般之裝置之情況下，藉由將泵液體食品材 料連續地送液至施加部，然後，於施加部藉由連續地施加 ’可連續地進行液體食品材料之殺菌。 首先’藉由送液用之泵，成爲殺菌對象之液體食品材 料由液體食品材料收容部供給至高電場脈衝之施加部。就 泵而言’可由可定量輸送的莫諾栗（moyno pump )、連結 © 式隔膜栗（diaphragm pump )或旋轉栗等適宜選擇而使用 °另外’亦可將收容部藉由適當的手段加熱或冷卻，藉此 ’可將液體食品材料事先定爲所希望之溫度。 .另外’裝置全體之大小、裝置各部間配管之大小及至 液體食品材料之施加部之供給量等，係基於單位時間所必 要之殺菌處理量，然後，在盡可能藉由前述既定之施加使 液體食品材料加熱至既定溫度之情況下，可適宜地選擇。 藉由溫度調節手段，可將供給至高電場脈衝之施加部 之液體食品材料，因應必要冷卻或加熱。例如收容部中之 -11 - 200936061 液體食品材料之溫度爲室溫（約20°C )之情況下，可藉由 溫度調節手段，使其溫度下降至1〜10 °c、或上升至40〜 60 °C之後，供給至高電場脈衝之施加部。就用於冷卻或加 熱之溫度調節手段而言，可使用例如使用板式熱交換器、 管式熱交換器等進行與冷水、溫水、蒸氣等之熱交換之方 法。在沒有必要調節液體食品材料之溫度之情況下，可省 略溫度調節手段。 接下來，對於供給至施加部之液體食品材料，進行高 電場脈衝之施加。 施加部，可由例如一對之電極與間隔物構成。具體而 言’藉由在連接至電源之以不銹鋼、鈦及鉑等形成之一對 平面電極間’配置以具有絕緣性之機械強度高之樹脂（例 如四氟化乙烯樹脂（PTFE )及超級工程樹脂等）所製作之 間隔物形成流路，然後，藉由將除了流路部分之此電極以 絕緣材料覆蓋可構成施加部。於施加部分別設置有用於使 液體食品材料流入及流出之口。 爲了測定高電場脈衝之剛由施加部流出之後之液體食 品材料之溫度’於施加部流出口之近傍設置有溫度測定手 段。就溫度測定手段而言，可使用高頻率之脈衝及高電壓 下可進行溫度測量之螢光式光纖一溫度計等。 在本發明中’液體食品材料被加熱之殺菌溫度（既定 溫度）’係意指在剛由施加部流出之後所測定之溫度。亦 即’液體食品材料於施加部內，被加熱至在剛由施加部流 出之後所測定之溫度。 -12- 200936061 接下來，由施加部流出之液體食品材料，係可藉由冷 卻手段冷卻。由於由施加部流出之液體食品材料受到加熱 ，因此若長久保持其加熱狀態，則認爲會引起風味或營養 成分之劣化。因此，在有必要避免如此的劣化之情況下， 係以冷卻爲佳。就冷卻手段而言，可使用例如板式熱交換 器及管式熱交換器等之由冷水等進行之熱交換而進行。然 後，藉由如此的手段，可冷卻至例如ίο〜3〇°c。至如此的 U 冷卻之時間，係可在加熱至前述既定溫度之後（亦即由施 加部流出後）、前述所規定之時間以內進行。沒有必要將： 由施加部流出之液體食品材料冷卻之情況下，可省略冷卻| 手段。 本發明之殺菌方法中，將液體食品材料藉由冷卻手段 冷卻之情況下，由殺菌效果之觀點看來，可將由施加部流 出之液體食品材料於既定時間經過後冷卻。 亦即，由施加部流出之液體食品材料，係以例如1至 〇 300秒後、或5至30秒後、或10至20秒後到達冷卻手段 之方式，可調整由施加部至冷卻手段之距離及/或液體食 品材料之送液速度。藉由將由施加部流出之被加熱至既定 溫度之液體食品材料，經過既定時間之後冷卻，藉由高電 場脈衝之施加使所得到之加熱狀態成爲維持在某程度，而 可得到更高殺菌效果。在本發明中，由於藉由高電場脈衝 之施加而被加熱，因此即使在將液體食品材料冷卻之既定 時間，係相較於以往的加熱殺菌法更短之情況下，或較既 定溫度低之情況下，亦可達成高殺菌效果。 -13- 200936061 如此一來經過處理之液體食品材料可透過閥回收，或 可送液至次加工步驟。 在圖1之裝置之中，溫度調節手段係設置於施加部之 前’而本發明之殺菌方法，另外亦可使用將溫度調節手段 設置於施加部之後之裝置而實施。如此的裝置之情況下， 液體食品材料係例如B— D— C— E— F之順序送液，藉 由溫度調節手段’可將由施加部流出之液體食品材料調節 至任意之溫度。 在圖1之裝置之中，由泵至閥係以配管連結而成爲密 閉系統。 因此，藉由將閥定爲具有保壓性能之閥（保壓閥）， 選擇適當的泵，可定爲使此密閉系統受到加壓之狀態。另 外因應必要，爲了確認此密閉系統內之加壓狀態可設置壓 力計。如此的密閉系統之加壓，具體而言0.3MPa以上， 可加壓至例如0.5至l.OMPa。 [實施例] 於以下列舉實施例，對本發明作較具體地說明，而本 發明並非受到該等限定者。 以下之實施例及比較例2、3，係使用除去C(溫度調 節手段）之具有A至I之構成之圖1之裝置而實施。裝置 之詳細係如以下所述。 .液體試樣之收容而言’使用玻璃製之燒杯或不銹鋼製 之容器。就將液體試樣定量地送液之泵而言，係使用隔膜 -14 - 200936061 式之數位定量泵、或莫諾泵。產生高電場脈衝之電源，爲 了將高電壓以高重複頻率施加，係使用過飽和磁氣開關壓 縮型之電源而構成。最大重複頻率爲400Hz。連接至此電 源，將高電壓脈衝施加於液體試樣之施加部，係組合一對 之對向之不銹鋼製之平面電極與聚醚楓製之絕緣間隔物而 構成流路。流路中之液體食品材料之收容量爲約lmL，電 極間隔爲4mm。在施加部流出口之液體試樣之溫度之測定 φ ，係以螢光式探針溫度計進行。試樣溶液之冷卻裝置方面 ，使用不銹鋼製之管式熱交換器，將以低溫恆溫槽冷卻至 以下之乙二醇，作爲冷媒循環供給至熱交換器進行冷 卻。使用手動式針閥之保壓閥作爲閥。另外，爲了測定裝 置內之壓力設置壓力計。 比較例1及4〜8，係將上述裝置之電源及施加部取代 成加熱媒體爲蒸氣之熱交換器而進行殺菌。 〇 [實施例1] (1 )懸浮液試樣之調製 於超純水加入檸檬酸三鈉二水合物，製作於20 °C之導 電率爲12 0mS/m之電解質溶液之後，於此溶液加入細菌芽 胞（枯草桿菌：Bacillus subtilis )，調製含有細菌芽胞之 懸浮液試樣（芽胞濃度：6.5xl06CFU/mL)。 (2)殺菌 使用先前記載之裝置，處理於（1)所調製之懸浮液 -15- 200936061 試樣。 (a)將約20 °C之懸浮液試樣，藉由泵連續地通液至 施加部（通液速度：UmL/分）。然後，於施加部將脈衝 寬度定爲l〇〇ns、脈衝上升時間定爲50ns、而且電場強度 定爲98kV/cm之高電場脈衝以重複頻率100Hz施加500次 。剛流出施加部之後之懸浮液試樣之溫度，亦即既定溫度 爲1 0 5 °C，由施加部流入口至流出口之液通過時間爲5秒 鐘。 其後，將懸浮液試樣藉由冷卻裝置冷卻之後，透過保 壓閥回收懸浮液試樣。回收時之懸浮液試樣之溫度爲15 t 。由施加部流出口至冷卻裝置之距離爲22.5cm，由施加部 流出口到達冷卻裝置所需要的時間爲約1 5秒。該等處理 係以位於冷卻裝置正後之保壓閥，將密閉系統之壓力操作 至0.6MPa而實施。 (i)除了將電場強度定爲103kV/cm以外，係以跑[( a)相同之方式進行懸浮液試樣之處理。另外，剛流出施 加部之後之懸浮液試樣之溫度爲1 1 0 °C。 (u)除了將電場強度定爲i〇8kV/cm以外，係以與（ a )相同之方式進行懸浮液試樣之處理。另外，剛流出施 加部之後之懸浮液試樣之溫度爲115 °C。 [比較例1] (1 )懸浮液試樣之調製 與實施例1相同之方式調製懸浮液試樣（芽胞濃度 -16- 200936061 1 ·2χ 1 07CFU/mL )。 (2 )殺菌 將於（1 )所調製之懸浮液試樣，藉由泵連續地通液 至以蒸氣作爲熱媒體之熱交換器（通液速度：1 lmL/分） 進行加熱處理。熱交換器之通過時間，亦即加熱至既定溫 度之時間爲5秒。 © (a)調節流入熱交換器之蒸氣量，以使在熱交換器 流出口之懸浮液試樣之溫度成爲105 t之方式加熱之後， 經1 5秒鐘之溫度保持時間快速地藉由冷卻裝置冷卻，回 收懸浮液試樣。該等處理係以位於冷卻裝置正後之保壓閥 ，將密閉系統之壓力操作至0.6MPa而實施。 (i)調節流入熱交換器之蒸氣量，以使在熱交換器 流出口之懸浮液試樣之溫度成爲1 1 0 °C之方式加熱之後， 以與（a )相同之方式進行懸浮液試樣之加熱處理。 O (U)調節流入熱交換器之蒸氣量，以使在熱交換器 流出口之懸浮液試樣之溫度成爲1 1 5 °C之方式加熱之後， 以與（a)相同之方式進行懸浮液試樣之加熱處理。 [比較例2 ] (1 )懸浮液試樣之調製 於超純水加入細囷牙胞（枯草桿囷：Bacillus subtilis )，調製含有細菌芽胞之懸浮液試樣（芽胞濃度： 8.1xl06CFU/mL)。本懸浮液在2(TC之導電率爲0.1mS/m -17- 200936061 以下。 (2)殺菌 使用與實施例1相同裝置，處理於（1)所調製之懸 浮液試樣。 將約20°C之懸浮液試樣，藉由泵連續地通液至施加部 (通液速度：1 lmL/分）。然後，於施加部，脈衝寬度定 爲100ns、脈衝上升時間定爲50ns，而且電場強度定爲 100kV/Cm之高電場脈衝，以重複頻率100Hz施加500次 。由於剛流出施加部之後之懸浮液試樣之溫度，並未由約 2 〇°C變化，因此可不進行以冷卻裝置進行之冷卻而回收懸 浮液試樣。該等處理，以位於冷卻裝置正後之保壓閥，將 密閉系統之壓力操作至0.6MPa而實施。 [實施例2] (1 )懸浮液試樣之調製 將細菌芽胞（Alicyclobacillus acidoterrestris)加入 在20°C之導電率爲360mS/m，並且ρΗ3·7之檸檬酸三鈉二 水合物-鹽酸緩衝液，調製懸浮液試樣（芽胞濃度：2 ·4χ 10 CFU/mL)。 (2)殺菌 使用與實施例1相同裝置，處理於（1)所調製之懸 浮液試樣。 -18- 200936061 (a)將約20°C之懸浮液試樣，藉由泵連續地通液至 施加部（通液速度：llmL/分）。然後，於施加部，將脈 衝寬度定爲l〇〇ns、脈衝上升時間定爲50ns、而且電場強 度定爲64kV/cm之高電場脈衝以重複頻率100Hz施加500 次。剛流出施加部之後之懸浮液試樣之溫度係1 00 °C，由 施加部流入口至流出口之液通過時間，亦即加熱到既定溫 度之時間爲5秒鐘。其後，將懸浮液試樣藉由冷卻裝置冷 φ 卻之後，回收懸浮液試樣、回收時之懸浮液試樣之溫度爲 15°C。由施加部流出口至冷卻裝置之距離爲22.5cm，由施 加部流出口到達冷卻裝置所需要的時間爲約1 5秒。該等 處理係以位於冷卻裝置正後之保壓閥，將密閉系統之壓力 操作至0.6MPa而實施。 (i)除了電場強度定爲6 5kV/cm以外，係以與（a) 相同之方式進行懸浮液試樣之處理。另外，剛流出施加部 之後之懸浮液試樣之溫度爲102°C。 ❹ (u)除了將電場強度定爲6 6kV/cm以外，係以與（a )相同之方式進行懸浮液試樣之處理。另外，剛流出施加 .部之後之懸浮液試樣之溫度爲1 05°C。 [比較例3] 除了將電場強度定爲61 kV/cm以外，係以與實施例2 之（a )相同之方式，進行實施例2之於（丨）所調製之懸 浮液試樣之處理。另外，剛流出施加部之後之懸浮液試樣 之溫度爲9 4 °C。 -19- 200936061 [比較例4] (1 )懸浮液試樣之調製 以與實施例2相同之方式調製懸浮液試樣（芽胞濃度 ：2.2x1 06CFU/mL )。 (2)殺菌 將於（1 )調整之懸浮液試樣，藉由泵連續地通液至 以蒸氣作爲熱媒體之熱交換器（通液速度1 ImL/分）進行 加熱處理。熱交換器之通過時間，亦即加熱至既定溫度之 時間爲5秒鐘。 (a)調節流入熱交換器之蒸氣量，以在熱交換器流 出口之懸浮液試樣之溫度成爲95 t之方式加熱之後，經 1 5秒鐘之溫度保持時間快速地藉由冷卻裝置冷卻，回收懸 浮液試樣。該等處理係以位於冷卻裝置正後之保壓閥，將 密閉系統之壓力操作至〇.6MPa而實施。 (i)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲1 02 °C之方式加熱之後，以與 (a)相同之方式進行懸浮液試樣之加熱處理。 (u)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲1 〇 5 °C之方式加熱之後，係以 與（a )相同之方式進行懸浮液試樣之加熱處理。 (e)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲1 1 2 °C之方式加熱之後，係以 與（a )相同之方式進行懸浮液試樣之加熱處理。 -20- 200936061 [實施例3] (1)懸浮液試樣之調製 將細菌芽胞（凝結芽孢桿菌：Bacillus coagulans)加 入於20°C導電率成爲90mS/m、pH爲6.9之磷酸二氫鉀一 磷酸氫二鈉緩衝液，調製懸浮液試樣。（芽胞濃度： 2.2x1 04CFU/mL ) ❹ (2 )殺菌 使用與實施例1相同裝置，處理於（1 )所調製之懸 浮液試樣。 (a)將約2(TC之懸浮液試樣，藉由泵連續地通液至 施加部（通液速度：llmL/分）。然後，於施加部，將脈 衝寬度定爲100ns、脈衝上升時間定爲50ns、而且電場強 度定爲97.5kV/cm之高電場脈衝，以重複頻率100Hz施加 φ 500次。剛流出施加部之後之懸浮液試樣之溫度爲121°C ，由施加部流入口至流出口之液通過時間，亦即加熱至既 定溫度之時間爲5秒鐘。其後，將懸浮液試樣藉由冷卻裝 置冷卻之後，回收懸浮液試樣。回收時之懸浮液試樣之溫 度爲15°C。由施加部流出口至冷卻裝置之距離爲22.5cm ，由施加部流出口到達冷卻裝置所需要的時間爲約1 5秒 〇 該等處理，係以位於冷卻裝置正後之保壓閥，將密閉 系統之壓力操作至〇.6MPa而實施。 -21 - 200936061 (i)除了將電場強度定爲l〇2.5kV/cm以外，係以與 (a)相同之方式進行懸浮液試樣之處理。另外，剛流出 施加部之後之懸浮液試樣之溫度爲128°C。 [比較例5] (1 )懸浮液試樣之調製 與實施例3相同之方式調製懸浮液試樣（芽胞濃度： 1 .5x1 06CFU/mL )。 (2 )殺菌 將於（1 )所調製之懸浮液試樣，藉由栗連續地通液 至以蒸氣作爲熱媒體之熱交換器（通液速度：llmL/分） 進行加熱處理。熱交換器之通過時間、亦即加熱至既定溫 度之時間爲5秒鐘。 (a)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲121 °C之方式加熱之後，經15 秒鐘之溫度保持時間快速地藉由冷卻裝置冷卻，回收懸浮 液試樣。該等處理係以位於冷卻裝置正後之保壓閥，將密 閉系統之壓力操作至0.6MPa而實施。 (i)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲1 27 °C之方式加熱之後，以與 (a )相同之方式進行懸浮液試樣之加熱處理。 (u)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲1 3 5 °C之方式加熱之後，以與 -22- 200936061 (a)相同之方式進行懸浮液試樣之加熱處理。 [評估] 對於在實施例1、比較例1及比較例2、實施例2、比 較例3及比較例4，以及、實施例3及比較例5回收之各 懸浮液試樣，由以下之方式求得殺菌處理後之細菌芽胞之 生存數。 © 採取1 mL回收之各懸浮液試樣，懸浮於9mL之無菌 水，稀釋1 0倍。 進一步採取此稀釋液ImL，懸浮於9mL之無菌水，稀 釋1〇〇倍。將此作業重複合計5次，每次100 μί採取各稀 釋液，塗抹至標準洋菜培養基，於35 °C進行24〜72小時 培養之後，藉著計算生長之菌落數，求得原本的懸浮液試 樣每ImL生存之菌數。另外，培養時間係因應於所使用之 微生物之種類而設定。 ® 將結果表示於表1、表2及表3。表中，殺菌效果， 係對於在各個實施例、比較例而言，將處理前之菌數定爲 No、處理後之菌數定爲N時，L〇g1() ( N〇/N )所求得之値 ，此値爲1.0之情況係意指殺菌效果爲1位數、2.0之情 況下係意指殺菌效果爲2位數。另外，殺菌溫度係對於實 施例1、實施例2、比較例2、比較例3及實施例3而言’ 爲剛流出施加部之後之懸浮液試樣之溫度’對於比較例1 、比較例4及比較例5而言爲剛流出熱交換器之後之懸浮 液試樣之溫度。 -23- 200936061 [表i] 處理前之菌數 (CFU/mL) 處理後之菌數 (CFU/mL) 殺菌溫度 (°C) 殺菌效果 實施例1⑻ 6.5χ106 2.2χ105 105 1.5 實施例l(i) 6.5χ106 6.3 xlO2 110 4.0 實施例l(u) 6.5χ106 6.5Χ101 115 5.0 比較例1⑷ 1.2χ107 7.8χ106 105 0.2 比較例l(i) 1.2χ107 2.5χ106 110 0.7 比較例1⑻ 1.2χ107 3.3χ1〇3 115 3.6 比較例2 8.1xl06 6.8χ106 20 0.1 由實施例1 ( a )，判斷出本發明之方法對於該實施例 所使用之細菌芽胞，在至少105 t以上產生充分之殺菌效 果。 另外，藉由殺菌溫度爲ll〇°C之實施例l(i)之殺菌 效果4.0與比較例l(i)之殺菌效果0·7之比較，判斷出 本發明之方法，在相同殺菌溫度，相較於由以往的熱交換 進行之加熱殺菌，對該實施例所使用之細菌芽胞之殺菌效 果高。此傾向在實施例1 ( a )與比較例1 ( a )之比較、 實施例1 ( u )與比較例1 ( u )之比較亦相同。進一步將 殺菌效果爲4.0與3.6，比較的同等之實施例1 ( i)與比 較例1 ( u )比較，則判斷出本方法與由以往的熱交換進行 之加熱殺菌相比，於至少5 °C低殺菌溫度可得到同等以上 之殺菌效果。 另一方面，施加l〇〇kV/cm之高電場脈衝之比較例2 之殺菌效果爲〇. 1。由於比較例2係懸浮液材料之導電率 -24- 200936061 爲低達0.1 mS/m，因此並未產生高電場脈衝施加造成之溫 度上昇。相對於此，在得到了充分之殺菌效果之實施例1 (a)〜（u)之中，藉由施加與比較例2幾乎同等電場強 度（98〜108kV/cm)之高電場脈衝，懸浮液材料係上昇至 1 05 °C以上之既定溫度，可得到最大5.0之殺菌效果。由 實施例1與比較例2之比較，判斷出爲了得到充分之殺菌 效果，有必要藉由本發明之方法之方式施加高電場脈衝， 〇 使液體試樣之溫度上昇至既定溫度。 [表2] 處理前之菌數 (CFU/mL) 處理後之菌數 (CFU/mL) 殺菌溫度 (°C) 殺菌效果 實施例2⑻ 2.4x10s 1.4x103 100 2.2 實施例2(i) 2.4x105 2.4x1ο1 102 4.0 實施例2⑼ 2·4χ105 8.2x10'1 105 5.5 比較例3 2.4χ105 7.2χ104 94 0.5 比較例4⑻ 2.2χ106 2.2χ106 95 0.0 比較例4(i) 2.2χ106 3.7χ105 102 0.8 比較例4(u) 2.2χ106 5.6χ104 105 1.6 比較例4(e) 2.2χ106 1.3χ102 112 4.2 由實施例2 ( a )與比較例3之比較，判斷出本發明之 方法，係對該實施例所使用之細菌芽胞，於至少loot以 上產生充分之殺菌效果。 另外，由在殺菌溫度爲1 021相同實施例2 ( i )之殺 菌效果4.0與比較例4 ( i )之殺菌效果0.8之比較，判斷 出本發明之方法，係在相同殺菌溫度，相較於由以往的熱 -25- 200936061 交換進行之加熱殺菌，對該實施例所使用之細菌芽胞之殺 菌效果高。此傾向，係在實施例2(u)與比較例4(u) 之比較亦相同。進一步，由殺菌效果爲4.0與4.2幾乎同 等之實施例2 ( i )與比較例4 ( e )之比較，判斷出本方 法，與由以往的熱交換進行之加熱殺菌相比，於1 〇°C低殺 菌溫度可得到同等殺菌效果。 [表3] 處理前之菌數 (CFU/mL) 處理後之菌數 (CFU/mL) 殺菌溫度 (°C) 殺菌效果 實施例3⑻ 2.2χ104 1.2x103 121 1.3 實施例3(i) 2.2χ104 4.5 χΙΟ·1 128 4.7 比較例5⑷ 1.5xl06 3.1x10s 121 0.7 比較例5(i) 1.5xl06 2.5x10s 127 0.8 比較例5⑼ 1.5χ106 5.4χ102 135 3.4BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of sterilizing a liquid food such as a dip material, and more particularly to a method of sterilizing a liquid food using a high electric field pulse having a short pulse width. [Prior Art] In the past, sterilization of liquid foods such as beverages was generally carried out by heating the material and adding a certain heat load. However, in such heat sterilization for imparting a heat load to a liquid food material, there is a problem that it is difficult to use the original nutrient or flavor of the food material without damaging its components. In particular, as in the case of bacterial spores, in the sterilization of microorganisms with high tolerance to heat, it is necessary to give an excessive heat load to the liquid food to ensure the commercial sterility. The so-called commercial sterility here is not a state of complete sterility, and means a state of microbial control which prevents the spoilage of microorganisms from becoming corrupted during the period necessary for commercial circulation. Therefore, there has been proposed a sterilization method which can reduce the quality deterioration caused by heating instead of heat sterilization. In one of such sterilization methods, a technique of directly applying a high voltage to a liquid food for sterilization is proposed (for example, a patent) Documents 1 to 3). Japanese Patent Publication No. 2000-262261 discloses a method for sterilizing spore-forming bacteria by suppressing the temperature rise of the sterilizing target liquid by applying a high electric field pulse having a short pulse width, and causing deterioration of the quality of the target liquid caused by heating. However, the bactericidal effect of the spore forming bacteria in the examples disclosed in this document is not fully sufficient to achieve commercial sterility. In Japanese Patent No. 2006-2 38827 and Japanese Patent No. 2964037, it is disclosed that the application of a high electric field causes the temperature of the liquid food to rise within less than one second, and the time during which the high temperature is maintained becomes a short time. The method of sterilizing bacterial spores. However, in order to obtain a sufficient bactericidal effect by this method, it is necessary to reduce the sterilizing temperature of the liquid food to the same degree as the conventional heat sterilization or the sterilizing temperature of the above, and to sufficiently reduce the heating. Quality is degraded. [Patent Document 1] JP-A-2006-238827 (Patent Document 3) Patent No. 2964037 (Summary of the Invention) [Problems to be Solved by the Invention] The object of the present invention is Providing a method for sterilizing microorganisms, particularly bacterial spores, to achieve commercial sterility without causing spoilage or deterioration in circulation, in comparison with conventional heat sterilization, lower sterilization temperature, or shorter sterilization time. . Another object is to sufficiently reduce the quality deterioration caused by the heating of the liquid food by this method. [Means for Solving the Problem] As a result of intensive review to solve the above problems, the inventors of the present invention first discovered that a high electric field pulse having a short pulse width is applied, and the liquid food material is heated to a predetermined sterilization temperature, and as a result, the above-mentioned Bacterial spores are effectively sterilized at a lower sterilizing temperature than the previous heat sterilization method in the phase -6-200936061. In other words, the present invention finds that a high electric field pulse having a pulse width of less than 200 ns and a pulse rise time of 50 ns or less and an electric field strength of 10 to 2 0 Ok V/cm is applied to the liquid food material. The liquid food material is heated to a predetermined temperature of at least 100 ° C to achieve a sufficient bactericidal effect, and the present invention is completed. The present invention includes the following aspects: 〇1· A method for sterilizing a liquid food material' includes setting the pulse width to less than 200 ns and the pulse rise time to 50 ns or less, and the electric field strength is set to 10 to 2 0 kk/ The high electric field pulse of cm is applied to the liquid food material, and the liquid food material is heated to a predetermined temperature of at least 100 ° C. 2. The sterilization method described in the above 1 wherein the aforementioned predetermined temperature is 100 to 160 ° C, 3. The sterilizing method according to the above-mentioned item 1 or 2, wherein the high electric field 〇 pulse is a pulse having a repetition frequency of 100 Hz or more, and the sterilizing method according to any one of the above 1 to 3, wherein the conductivity of the liquid food material is 5. The sterilization method according to any one of the above 1 to 4, wherein the time required for heating to the predetermined temperature is 10 seconds or less, 6. Any one of the above 1 to 5 The sterilization method according to any one of the above 1 to 6, wherein the sterilizing method is carried out after heating to the predetermined temperature, wherein the sterilizing method is continuously performed, -7-2009360 61. The liquid food material is sterilized by the method described in any one of the above 1 to 7, 9. A beverage is sterilized by the method described in any one of the above 1 to 7. The sterilizing device is a method for applying a sterilizing device for a liquid food material, and includes a liquid food material introduced into the accommodating portion, having a width of less than 200 ns and a rising time of 50 ns or less, and an electric field. A method of applying a high electric field pulse having a strength of 10 to 200 kV/cm, and applying a width of less than 200 ns to a liquid food material introduced into the accommodating portion, a rise time of 50 ns or less, and an electric field intensity of 1 〇 〜 a high electric field pulse of 00 kV/cm, the material is heated to at least 1 Torr (rc to sterilize the material. [Effect of the invention] By the present invention, it has been found that the sterilization temperature can be lowered compared to the conventional heat sterilization method. In the short sterilization time, sterilization with the same bactericidal effect as the heat sterilization method is carried out. Specifically, by the method of the present invention, microorganisms having high heat resistance such as bacterial spores can be compared with the conventional one. The heat sterilization method 5 to 10 ° C low sterilization temperature, or half of the sterilization time, the sterilization with the same sterilization effect as the heat sterilization method. In addition, in the method of the present invention, it can be lower than the conventional method Since the sterilization temperature is sterilized, deterioration of the flavor or nutrient component of the liquid food material accompanying heating during sterilization can be suppressed. Further, in the method of the present invention, sterilization can be performed more quickly than conventional heat sterilization. - 200936061 [Embodiment] The method for sterilizing a liquid food material according to the present invention includes a high electric field pulse having a pulse width of less than 200 ns and a pulse rise time of 50 ns or less and an electric field strength of 10 to 200 kV/cm. Applied to a liquid food material, the temperature of the liquid food material is heated to a predetermined temperature of at least 100 °C. The sterilization method of the present invention can be set to a pulse width of less than 2 ns, for example, 1 〇〇 to 15 0 ns, a pulse rise time of 50 ns or less, for example, 10 to 50 ns, and an electric field strength of 1 〇. A high electric field pulse of ~200 kV/cm, for example 30 to 150 kV/cm, or 60 to 120 kV/cm is applied to the liquid food material at a repetition frequency of 100 Hz or more, for example, 100 to 10000 Hz, or a repetition frequency of 100 to 400 Hz, and the liquid is applied thereto. The temperature of the food material is heated to a predetermined temperature of at least 10 ° C, for example, 100 to 160 ° C, or 100 to 140 ° C. In order to heat the liquid food material to a predetermined temperature, 施加 can apply the application of the high electric field pulse multiple times, for example, 100 to 500 times. The time necessary for heating to the predetermined temperature by application is preferably, for example, 5 seconds or less, or 10 seconds or less. Further, in the sterilization method of the present invention, the liquid food material may be cooled (to, for example, 1 to 30 ° C) after heating to the predetermined temperature. The cooling may be carried out after heating to the aforementioned predetermined temperature, for example, within 300 seconds, or within 30 seconds, specifically within 1 to 300 seconds, or within 5 to 30 seconds. The pulse width, pulse rise time and electric field strength, as well as the number of applications -9-200936061, can be set based on the temperature at which the liquid food material is heated. Then, the time to cool to the predetermined temperature is set in such a manner as to ensure commercial sterility of the liquid food material, and the type or circulation of the liquid food material may vary depending on the circulation temperature. This is because the type of liquid food material is different, the type of microorganisms that cause the risk of spoilage or deterioration during circulation differs, and the risk of proliferation of such microorganisms due to the difference in circulation or circulation temperature. Sexually different. In addition, the commercial sterility can be ensured, which means that the bactericidal effect of the liquid food material can be prevented from being spoiled or deteriorated in circulation, and the bactericidal effect of at least one digit can be achieved. In the sterilization method of the present invention, the sterilizing effect of, for example, one digit or more can be achieved by applying a previously specified pulse to heat the liquid food material to at least 100 °C. The liquid food material to which the present invention is applicable is not particularly limited as long as it is a liquid food necessary for sterilization treatment. For example, a refreshing drink or an alcoholic beverage, specifically, a fruit juice, a fruit drink, a tea drink, a coffee drink, a juice, and the like can be cited. The method of the present invention is particularly suitable for the sterilization of liquid food materials having a conductivity of 30 to 1 000 mS/m or 90 to 700 mS/m. The sterilizing method of the present invention can be applied, for example, by using a planar electrode including a pair of power sources connected to a power source capable of generating a high electric field pulse, and a liquid accommodating portion having a predetermined volume composed of a suitable insulating material. After the device "adds the liquid food material to the valley portion, the pulse specified above is transmitted through the electrode of the application portion, and the material is applied to the material by a power source, and heated to a predetermined temperature of at least 100 ° C. 200936061 In terms of power supply, there is no particular limitation as long as it is a power source capable of generating the aforementioned high electric field pulse. A power source that produces a repeatable high electric field pulse, such as a combined semiconductor switch and a magnetic pulse compression loop, can be used. Further, the sterilization method of the present invention can be carried out as one of the devices having the configuration shown in Fig. 1, for example. In Fig. 1, the A liquid food material storage unit, the B system pump, the C system temperature adjustment means, the D system application unit, the E system cooling means, the F system valve, the G system power supply device, and the lanthanide temperature measurement 6 means, and then 1 Pressure gauge. Each part of the ΑF is connected by a pipe, and the liquid food material is supplied from the direction of A to F. In the case of using the apparatus as shown in Fig. 1, by continuously feeding the pump liquid food material to the application portion, sterilization of the liquid food material can be continuously performed by continuously applying 'in the application portion'. First, the liquid food material to be sterilized by the pump for liquid supply is supplied from the liquid food material accommodating portion to the application portion of the high electric field pulse. As far as the pump is concerned, it can be used by a moyno pump that can be quantitatively transported, a diaphragm pump or a rotary pump, or the like. Alternatively, the housing can be heated by an appropriate means or Cooling, whereby the liquid food material can be pre-determined to the desired temperature. Further, the size of the entire apparatus, the size of the piping between the various parts of the apparatus, and the supply amount to the application portion of the liquid food material are based on the amount of sterilization required per unit time, and then, as far as possible, the liquid is applied by the predetermined application. When the food material is heated to a predetermined temperature, it can be suitably selected. The liquid food material supplied to the application portion of the high electric field pulse can be cooled or heated as necessary by the temperature adjustment means. For example, in the case of the -11 - 200936061 liquid food material, the temperature of the liquid food material is room temperature (about 20 ° C), and the temperature can be lowered to 1 to 10 ° C or to 40 ° by temperature adjustment means. After 60 ° C, it is supplied to the application portion of the high electric field pulse. As the temperature adjusting means for cooling or heating, a method of performing heat exchange with cold water, warm water, steam or the like using, for example, a plate heat exchanger, a tube heat exchanger or the like can be used. Temperature adjustment means can be omitted without the need to adjust the temperature of the liquid food material. Next, the application of a high electric field pulse is performed on the liquid food material supplied to the application portion. The applying portion may be composed of, for example, a pair of electrodes and a spacer. Specifically, 'the resin having high mechanical strength (for example, tetrafluoroethylene resin (PTFE) and super engineering) is disposed between the planar electrodes by one of stainless steel, titanium, and platinum formed in connection with a power source. The spacer formed by the resin or the like forms a flow path, and then the application portion is formed by covering the electrode except the flow path portion with an insulating material. Ports for allowing the liquid food material to flow in and out are provided in the application portion, respectively. In order to measure the temperature of the liquid food material immediately after the high electric field pulse has flowed out from the application portion, a temperature measuring means is provided near the flow outlet of the application portion. As the temperature measuring means, a high-frequency pulse and a fluorescent optical fiber-temperature thermometer capable of temperature measurement at a high voltage can be used. In the present invention, the sterilization temperature (established temperature) at which the liquid food material is heated means the temperature measured immediately after flowing out from the application portion. That is, the liquid food material is heated in the application portion to a temperature measured immediately after flowing out of the application portion. -12- 200936061 Next, the liquid food material flowing out from the application portion can be cooled by means of cooling. Since the liquid food material flowing out from the application portion is heated, it is considered to cause deterioration of flavor or nutrient components if it is kept in a heated state for a long time. Therefore, in the case where it is necessary to avoid such deterioration, it is preferable to cool. The cooling means can be carried out by heat exchange using cold water or the like, for example, a plate type heat exchanger or a tube type heat exchanger. Then, by such means, it can be cooled to, for example, ίο~3〇°c. The time until the U cooling is performed can be carried out after heating to the predetermined temperature (i.e., after flowing out of the application portion) within the time specified above. It is not necessary to: in the case where the liquid food material flowing out from the application portion is cooled, the cooling|discharging means can be omitted. In the sterilization method of the present invention, when the liquid food material is cooled by the cooling means, the liquid food material discharged from the application portion can be cooled after a predetermined period of time from the viewpoint of the sterilization effect. That is, the liquid food material flowing out from the application portion can be adjusted from the application portion to the cooling means by, for example, 1 to 〇300 seconds, or 5 to 30 seconds later, or 10 to 20 seconds later, to reach the cooling means. The feed rate of the distance and/or liquid food material. The liquid food material which is heated to a predetermined temperature by the application portion is cooled after a predetermined period of time, and the obtained heating state is maintained at a certain level by application of a high electric field pulse, whereby a higher sterilization effect can be obtained. In the present invention, since it is heated by the application of a high electric field pulse, even when the liquid food material is cooled for a predetermined period of time, it is shorter than the conventional heat sterilization method, or is lower than a predetermined temperature. In this case, a high bactericidal effect can also be achieved. -13- 200936061 The liquid food material thus treated can be recovered through a valve or can be sent to a secondary processing step. In the apparatus of Fig. 1, the temperature adjusting means is provided before the applying portion, and the sterilization method of the present invention may be carried out by using a device in which the temperature adjusting means is provided after the applying portion. In the case of such a device, the liquid food material is supplied in the order of, for example, B-D-C-E-F, and the liquid food material flowing out from the application portion can be adjusted to an arbitrary temperature by the temperature adjusting means'. In the apparatus of Fig. 1, the pump-to-valve is connected by a pipe to form a closed system. Therefore, by setting the valve as a valve having a pressure holding property (pressure maintaining valve), an appropriate pump can be selected to pressurize the closed system. In addition, the pressure gauge can be set to confirm the pressure in the closed system. The pressurization of such a closed system, specifically, 0.3 MPa or more, may be pressurized to, for example, 0.5 to 1.0 MPa. [Examples] The present invention will be specifically described by the following examples, but the present invention is not limited thereto. The following examples and comparative examples 2 and 3 were carried out using the apparatus of Fig. 1 having a configuration of A to I excluding C (temperature adjusting means). The details of the device are as follows. For the storage of liquid samples, use a glass beaker or a container made of stainless steel. For a pump that supplies a liquid sample quantitatively, a diaphragm type -14 - 200936061 type dosing pump or a Mono pump is used. A power source that generates a high electric field pulse is constructed by applying a high-voltage to a high repetition rate and using a supersaturated magnetic gas switch-compressed power source. The maximum repetition rate is 400 Hz. To this power source, a high voltage pulse is applied to the application portion of the liquid sample, and a pair of opposed stainless steel planar electrodes and an insulating spacer made of polyether maple are combined to form a flow path. The liquid food material in the flow path has a capacity of about 1 mL and an electrode spacing of 4 mm. The measurement of the temperature of the liquid sample at the outlet of the application section is performed by a fluorescent probe thermometer. In the case of the cooling device for the sample solution, a stainless steel tubular heat exchanger is used, and ethylene glycol cooled to a temperature below the low temperature bath is circulated as a refrigerant to the heat exchanger for cooling. Use the pressure valve of the manual needle valve as the valve. In addition, a pressure gauge is provided for measuring the pressure in the apparatus. In Comparative Examples 1 and 4 to 8, the power source and the application portion of the above apparatus were replaced with a heat exchanger in which the heating medium was a vapor, and the mixture was sterilized. 〇 [Example 1] (1) Preparation of a suspension sample was added to ultrapure water to add trisodium citrate dihydrate to prepare an electrolyte solution having a conductivity of 120 mS/m at 20 ° C, and then added to the solution. Bacterial spores (Bacillus subtilis), a suspension sample containing bacterial spores (spore concentration: 6.5 x 106 CFU/mL) was prepared. (2) Sterilization The sample prepared in (1) was treated with the previously described apparatus -15-200936061. (a) A sample of the suspension of about 20 ° C was continuously passed through a pump to the application portion (liquid flow rate: UmL / min). Then, a high electric field pulse having a pulse width of 10 ns, a pulse rise time of 50 ns, and an electric field strength of 98 kV/cm was applied at the application portion 500 times at a repetition frequency of 100 Hz. The temperature of the suspension sample immediately after flowing out of the application portion, i.e., the predetermined temperature was 105 ° C, and the liquid passage time from the application portion to the outlet was 5 seconds. Thereafter, after the suspension sample was cooled by a cooling device, the suspension sample was recovered through a pressure maintaining valve. The temperature of the suspension sample at the time of recovery was 15 t. The distance from the application portion outlet to the cooling device was 22.5 cm, and the time required for the application portion to reach the cooling device was about 15 seconds. These treatments were carried out by operating the pressure of the closed system to 0.6 MPa with a pressure maintaining valve located immediately behind the cooling device. (i) In addition to the electric field strength of 103 kV/cm, the suspension sample was treated in the same manner as [(a). Further, the temperature of the suspension sample immediately after flowing out of the application portion was 110 °C. (u) The treatment of the suspension sample was carried out in the same manner as (a) except that the electric field strength was set to i 〇 8 kV/cm. Further, the temperature of the suspension sample immediately after flowing out of the application portion was 115 °C. [Comparative Example 1] (1) Preparation of suspension sample A suspension sample (spore concentration -16 - 200936061 1 · 2 χ 1 07 CFU/mL) was prepared in the same manner as in Example 1. (2) Sterilization The suspension sample prepared in (1) was continuously heated by a pump to a heat exchanger (vapor flow rate: 1 lmL/min) using steam as a heat medium for heat treatment. The passage time of the heat exchanger, i.e., the time to heat to a predetermined temperature, is 5 seconds. © (a) Adjust the amount of vapor flowing into the heat exchanger so that the temperature of the suspension sample at the heat exchanger outflow port is heated to 105 t, and then rapidly cooled by the temperature retention time of 15 seconds. The device is cooled and the suspension sample is recovered. These treatments were carried out by operating the pressure of the closed system to 0.6 MPa with a pressure maintaining valve located immediately behind the cooling device. (i) adjusting the amount of vapor flowing into the heat exchanger so that the temperature of the suspension sample at the heat exchanger outflow port is heated to 110 ° C, and then performing the suspension test in the same manner as (a) Heat treatment. O (U) adjusts the amount of vapor flowing into the heat exchanger so that the temperature of the suspension sample at the heat exchanger outflow port is heated to 1 15 ° C, and then the suspension is carried out in the same manner as (a) Heat treatment of the sample. [Comparative Example 2] (1) Preparation of a suspension sample was added to an ultrafine water cell (Bacillus subtilis) to prepare a suspension sample containing bacterial spores (spore concentration: 8.1 x 106 CFU/mL) . The suspension is at 2 (the conductivity of TC is 0.1 mS/m -17-200936061 or less. (2) Sterilization Using the same apparatus as in Example 1, the suspension sample prepared in (1) is treated. About 20° The suspension sample of C was continuously passed through the pump to the application portion (through rate: 1 lmL/min). Then, at the application portion, the pulse width was set to 100 ns, the pulse rise time was set to 50 ns, and the electric field strength was set. A high electric field pulse of 100 kV/cm is applied 500 times at a repetition frequency of 100 Hz. Since the temperature of the suspension sample immediately after flowing out of the application portion is not changed by about 2 〇 ° C, it may not be performed by a cooling device. The suspension sample was recovered by cooling, and the treatment was carried out by operating the pressure of the closed system to 0.6 MPa with a pressure maintaining valve located immediately behind the cooling device. [Example 2] (1) Modulation of the suspension sample The bacterial spore (Alicyclobacillus acidoterrestris) was added with a conductivity of 360 mS/m at 20 ° C, and a trisodium citrate dihydrate-hydrochloric acid buffer of ρΗ3·7 to prepare a suspension sample (spore concentration: 2 · 4 χ 10 CFU) /mL) (2) Sterilization is the same as in Example 1. The sample of the suspension prepared in (1) is processed. -18- 200936061 (a) A sample of the suspension of about 20 ° C is continuously passed through the pump to the application portion (through rate: llmL / Then, at the application portion, a high electric field pulse having a pulse width of 10 ns, a pulse rise time of 50 ns, and an electric field intensity of 64 kV/cm was applied 500 times at a repetition frequency of 100 Hz. The temperature of the suspension sample thereafter is 100 ° C, and the liquid passage time from the inlet of the application portion to the outlet port, that is, the time for heating to a predetermined temperature is 5 seconds. Thereafter, the suspension sample is used. After the cooling device is cooled φ, the suspension sample is recovered, and the temperature of the suspension sample at the time of recovery is 15 ° C. The distance from the application portion outlet to the cooling device is 22.5 cm, and the outlet portion of the application portion reaches the cooling device. The required time is about 15 seconds. The treatment is carried out by operating the pressure of the closed system to 0.6 MPa with a pressure maintaining valve located immediately behind the cooling device. (i) Except that the electric field strength is set at 65 kV/cm, Treatment of suspension samples in the same manner as (a) Further, the temperature of the suspension sample immediately after flowing out of the application portion was 102 ° C. ❹ (u) A suspension sample was prepared in the same manner as (a) except that the electric field intensity was set to 6 6 kV/cm. In addition, the temperature of the suspension sample immediately after flowing out of the application portion was 10 ° C. [Comparative Example 3] In addition to the electric field intensity of 61 kV/cm, it was compared with (a) of Example 2. In the same manner, the treatment of the suspension sample prepared in (Example) of Example 2 was carried out. Further, the temperature of the suspension sample immediately after flowing out of the application portion was 94 °C. -19-200936061 [Comparative Example 4] (1) Preparation of suspension sample A suspension sample (spore concentration: 2.2 x 1 06 CFU/mL) was prepared in the same manner as in Example 2. (2) Sterilization The suspension sample to be adjusted in (1) was continuously heated by a pump to a heat exchanger (vapor passing rate of 1 ImL/min) using steam as a heat medium. The passage time of the heat exchanger, i.e., the time to heat to a predetermined temperature, is 5 seconds. (a) adjusting the amount of vapor flowing into the heat exchanger to be rapidly cooled by the cooling device after a temperature of 15 seconds after the temperature of the suspension sample at the heat exchanger outlet is heated to 95 t. , recover the suspension sample. These treatments were carried out by operating the pressure of the closed system to 〇6 MPa with a pressure maintaining valve located immediately behind the cooling device. (i) adjusting the amount of vapor flowing into the heat exchanger, and heating the suspension sample in the same manner as (a) after the temperature of the suspension sample at the heat exchanger outflow port is heated to 1200 °C. deal with. (u) adjusting the amount of vapor flowing into the heat exchanger, and after the temperature of the suspension sample at the heat exchanger outlet is heated to 1 〇 5 ° C, the suspension sample is carried out in the same manner as (a) Heat treatment. (e) adjusting the amount of vapor flowing into the heat exchanger, and heating the sample at a temperature at which the temperature of the suspension sample at the heat exchanger outlet is 1 1 2 ° C, and then performing the suspension sample in the same manner as (a) Heat treatment. -20- 200936061 [Example 3] (1) Preparation of suspension sample Bacterial spores (Bacillus coagulans) were added to potassium dihydrogen phosphate at 20 ° C with a conductivity of 90 mS/m and a pH of 6.9. Disodium hydrogen phosphate buffer was used to prepare a suspension sample. (Core concentration: 2.2 x 1 04 CFU/mL) ❹ (2) Sterilization Using the same apparatus as in Example 1, the suspended suspension sample prepared in (1) was treated. (a) A suspension sample of about 2 (TC) was continuously passed through a pump to an application portion (liquid flow rate: llmL/min). Then, at the application portion, the pulse width was set to 100 ns, and the pulse rise time was set. A high electric field pulse of 50 ns and an electric field strength of 97.5 kV/cm was applied, and φ 500 times was applied at a repetition frequency of 100 Hz. The temperature of the suspension sample immediately after flowing out of the application portion was 121 ° C, and the inlet portion of the application portion was passed to The liquid passage time of the outflow port, that is, the time of heating to a predetermined temperature is 5 seconds. Thereafter, the suspension sample is cooled by a cooling device, and the suspension sample is recovered. The temperature of the suspension sample at the time of recovery It is 15 ° C. The distance from the application portion to the cooling device is 22.5 cm, and the time required for the cooling device to reach the cooling device from the application portion outlet is about 15 seconds. The treatment is located at the front of the cooling device. The pressure valve is operated by operating the pressure of the closed system to 〇6 MPa. -21 - 200936061 (i) The suspension test is carried out in the same manner as (a) except that the electric field strength is set to l〇2.5 kV/cm. The treatment of the sample. In addition, the suspension just after flowing out of the application part The temperature of the liquid sample was 128 ° C. [Comparative Example 5] (1) Preparation of suspension sample A suspension sample (spore concentration: 1.5×1 06 CFU/mL) was prepared in the same manner as in Example 3. (2) The sterilization sample prepared by the sterilization of (1) is continuously heated to a heat exchanger (vapor flow rate: llmL/min) using steam as a heat medium. The heat exchanger is passed. The time, that is, the time to heat to a predetermined temperature is 5 seconds. (a) The amount of vapor flowing into the heat exchanger is adjusted, and after the temperature of the suspension sample at the heat exchanger outflow port is heated to 121 ° C, The temperature holding time of 15 seconds was quickly cooled by a cooling device to recover a sample of the suspension. The treatment was carried out by operating the pressure of the closed system to 0.6 MPa with a pressure maintaining valve located immediately behind the cooling device. The amount of vapor flowing into the heat exchanger was adjusted, and the temperature of the suspension sample at the heat exchanger outflow port was heated to 1,27 ° C, and then the suspension sample was subjected to heat treatment in the same manner as in (a). (u) adjust the amount of vapor flowing into the heat exchanger, in the hot After the temperature of the suspension sample at the outlet of the vessel is heated to 135 ° C, the suspension sample is subjected to heat treatment in the same manner as in -22-200936061 (a). [Evaluation] For Example 1 In each of the suspension samples recovered in Comparative Example 1 and Comparative Example 2, Example 2, Comparative Example 3, and Comparative Example 4, and Example 3 and Comparative Example 5, bacterial spores after sterilization treatment were obtained in the following manner. Survival number © Take 1 mL of each suspension sample, suspend it in 9 mL of sterile water, and dilute it by 10 times. Take 1 mL of this dilution, suspend it in 9 mL of sterile water, and dilute it 1 time. The operation was repeated 5 times, each dilution was taken at 100 μί, and applied to a standard acacia medium, and cultured at 35 ° C for 24 to 72 hours, and then the original suspension was obtained by counting the number of colonies grown. The number of bacteria per sample of ImL survived. Further, the culture time is set depending on the type of microorganism to be used. ® The results are shown in Table 1, Table 2 and Table 3. In the table, the bactericidal effect is determined by setting the number of bacteria before treatment to No and the number of bacteria after treatment to N in each of the examples and comparative examples, and L〇g1()(N〇/N) After obtaining this, the case where the 値 is 1.0 means that the bactericidal effect is 1 digit, and 2.0 means that the bactericidal effect is 2 digits. Further, the sterilization temperature was "the temperature of the suspension sample just after flowing out of the application portion" for Example 1, Example 2, Comparative Example 2, Comparative Example 3, and Example 3 for Comparative Example 1 and Comparative Example 4 And Comparative Example 5 is the temperature of the suspension sample immediately after flowing out of the heat exchanger. -23- 200936061 [Table i] Number of bacteria before treatment (CFU/mL) Number of bacteria after treatment (CFU/mL) Sterilization temperature (°C) Sterilization effect Example 1 (8) 6.5χ106 2.2χ105 105 1.5 Example l (i 6.5χ106 6.3 xlO2 110 4.0 Example l(u) 6.5χ106 6.5Χ101 115 5.0 Comparative Example 1(4) 1.2χ107 7.8χ106 105 0.2 Comparative Example l(i) 1.2χ107 2.5χ106 110 0.7 Comparative Example 1(8) 1.2χ107 3.3χ1〇3 115 3.6 Comparative Example 2 8.1xl06 6.8χ106 20 0.1 From Example 1 (a), it was judged that the method of the present invention produced a sufficient bactericidal effect on at least 105 t or more for the bacterial spores used in the examples. Further, by comparing the bactericidal effect 4.0 of the example 1 (i) having a sterilization temperature of ll 〇 ° C with the bactericidal effect 0·7 of the comparative example 1 (i), the method of the present invention was judged at the same sterilization temperature, The bactericidal effect of the bacterial spores used in the examples is high compared to the heat sterilization by the conventional heat exchange. This tendency is also the same in comparison between Example 1 (a) and Comparative Example 1 (a), and Example 1 (u) and Comparative Example 1 (u). Further, the bactericidal effect was 4.0 and 3.6, and the comparative example 1 (i) was compared with the comparative example 1 (u), and it was judged that the method was at least 5 ° as compared with the heat sterilization by the conventional heat exchange. C low sterilization temperature can achieve the same sterilization effect. On the other hand, the sterilizing effect of Comparative Example 2 in which a high electric field pulse of l〇〇kV/cm was applied was 0.1. Since the conductivity of the suspension material of Comparative Example 2 was -24-200936061 as low as 0.1 mS/m, the temperature rise due to the application of a high electric field pulse was not generated. On the other hand, in Example 1 (a) to (u) in which a sufficient bactericidal effect was obtained, a high electric field pulse of almost the same electric field strength (98 to 108 kV/cm) as that of Comparative Example 2 was applied, and the suspension was suspended. When the material is raised to a predetermined temperature above 10 °C, a bactericidal effect of up to 5.0 can be obtained. From the comparison between Example 1 and Comparative Example 2, it was judged that in order to obtain a sufficient sterilization effect, it is necessary to apply a high electric field pulse by the method of the present invention to raise the temperature of the liquid sample to a predetermined temperature. [Table 2] Number of bacteria before treatment (CFU/mL) Number of bacteria after treatment (CFU/mL) Sterilization temperature (°C) Sterilization effect Example 2 (8) 2.4×10s 1.4×103 100 2.2 Example 2 (i) 2.4×105 2.4 X1ο1 102 4.0 Example 2(9) 2·4χ105 8.2x10'1 105 5.5 Comparative Example 3 2.4χ105 7.2χ104 94 0.5 Comparative Example 4(8) 2.2χ106 2.2χ106 95 0.0 Comparative Example 4(i) 2.2χ106 3.7χ105 102 0.8 Comparative Example 4(u 2.2χ106 5.6χ104 105 1.6 Comparative Example 4(e) 2.2χ106 1.3χ102 112 4.2 From the comparison of Example 2 (a) with Comparative Example 3, the method of the present invention was judged to be the bacterial spore used in the Example. , at least loot above to produce sufficient bactericidal effect. Further, by comparing the bactericidal effect 4.0 of the same Example 2 (i) with the sterilizing effect of the sterilizing temperature of 021 and the bactericidal effect of the comparative example 4 (i) of 0.8, it was judged that the method of the present invention is at the same sterilization temperature as compared with The heat sterilization by the conventional heat-25-200936061 exchange has a high bactericidal effect on the bacterial spores used in the examples. This tendency is also the same in comparison between Example 2 (u) and Comparative Example 4 (u). Further, in comparison with Comparative Example 2 (i) in which the sterilization effect was 4.0 and 4.2, the method was judged to be in comparison with the heat sterilization by the conventional heat exchange at 1 〇 ° C low sterilization temperature can get the same bactericidal effect. [Table 3] Number of bacteria before treatment (CFU/mL) Number of bacteria after treatment (CFU/mL) Sterilization temperature (°C) Sterilization effect Example 3 (8) 2.2χ104 1.2x103 121 1.3 Example 3(i) 2.2χ104 4.5 χΙΟ·1 128 4.7 Comparative Example 5(4) 1.5xl06 3.1x10s 121 0.7 Comparative Example 5(i) 1.5xl06 2.5x10s 127 0.8 Comparative Example 5(9) 1.5χ106 5.4χ102 135 3.4
由實施例3(a)，判斷出本發明之方法，對該實施例 所使用之細菌芽胞，於至少121 °C以上產生充分之殺菌效 果。 另外，殺菌溫度爲在128°C與127°C幾乎相同之實施 例3 ( i )與比較例5 ( i)之殺菌效果分別爲4.7與〇.8， 藉由兩者之比較，判斷出本發明之方法，在相同殺菌溫度 ’相較於由以往的熱交換進行之加熱殺菌，對該實施例所 使用之細菌芽胞之殺菌效果高。進一步，由殺菌效果爲 4.7之實施例3 ( i )與殺菌效果爲3.4之比較例5 ( u )之 比較，判斷出本方法，與由以往的熱交換進行之加熱殺菌 -26- 200936061 相比，於至少7°C以上低殺菌溫度可得到同等以上之殺菌 效果。 [實施例4] (1 )懸浮液試樣之調製 將細菌芽胞（嗜熱桿菌：Geobacillus stearothermophilus)加入使用 KH2P〇4 水溶液與 Na2HP〇4 φ 水溶液而製作成在20°C之導電率爲400mS/m、pH爲6.5 之磷酸緩衝液，調製含有細菌芽胞之懸浮液試樣（芽胞濃 度：3.6xl05CFU/mL )。 (2 )殺菌 使用與實施例1相同之裝置，如以下之順序處理於（ 1)所調製之懸浮液試樣。 (a)將約20°C之懸浮液試樣，藉由泵連續地通液至 〇 施加部（通液速度：llmL/分）。然後，於施加部之通液 中，將脈衝寬度定爲100ns、脈衝上升時間定爲50ns，並 且16.1〜18.2kV/cm之範圍之電場強度之高電場脈衝以重 複頻率100Hz施加500次。剛流出施加部之後之懸浮液試 樣之溫度’亦即既定溫度爲125 °C，由施加部流入口至流 出口之液通過時間爲5秒鐘。 其後’將懸浮液試樣藉由冷卻裝置冷卻之後、透過保 壓閥回收懸浮液試樣。回收時之懸浮液試樣之溫度爲15°C 。在本實施例中’以由施加部流出口到達冷卻裝置之時間 -27- 200936061 爲約14秒而實施。該等處理係以位於冷卻裝置正後之保 壓閥，將密閉系統之壓力操作至〇.6MPa而實施。 (i)除了將電場強度定爲16.4〜18.41^/(：111之範圍以 外，以與（a )相同之方式進行懸浮液試樣之處理。另外 ，剛流出施加部之後之懸浮液試樣之溫度爲1 3 0 °C。 (u)除了將電場強度定爲16.7〜18.81^/(；111之範圍 以外，以與（a )相同之方式進行懸浮液試樣之處理。另 外，剛流出施加部之後之懸浮液試樣之溫度爲1 3 5 °C。 [比較例6 ] (1 )懸浮液試樣之調製 與實施例4相同之方式調製懸浮液試樣（芽胞濃度 17.3xl05CFU/mL )。 (2)殺菌 將於（1 )所調製之懸浮液試樣，藉由栗連續地通液 至以蒸氣作爲熱媒體之熱交換器（通液速度：1 lmL/分） 進行加熱處理》熱交換器之通過時間，亦即加熱至既定溫 度之時間爲5秒鐘。 (a)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲1 25 t之方式進行加熱。其後 ，將懸浮液試樣藉由冷卻裝置冷卻，透過保壓閥回收懸浮 液試樣。回收時之懸浮液試樣之溫度爲15 °C。在本比較例 中，由熱交換器流出口到達冷卻裝置所需要之時間爲約14 -28- 200936061 秒。該等處理係以位於冷卻裝置正後之保壓閥，將密閉系 統之壓力操作至0.6MPa而實施。 (i)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲130 °C之方式加熱之後，以與 (a)相同之方式進行懸浮液試樣之加熱處理。 (u)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲135 °C之方式加熱之後，以與 φ ( a )相同之方式進行懸浮液試樣之加熱處理。 [實施例5] (1)懸浮液試樣之調製 將細菌牙胞（Moorella thermoacetica)加入使用 KH2P〇4水溶液與Na2HP04水溶液製作成在20°C之導電率 爲400mS/m、pH爲6.5之磷酸緩衝液，調製含有細菌芽胞 之懸浮液試樣（芽胞濃度9.8xl03CFU/mL)。 Φ (2 )殺菌 使用與實施例1相同之裝置，如以下之順序處理於（ 1)所調製之懸浮液試樣。 (a)將約20°C之懸浮液試樣，藉由泵連續地通液至 施加部（通液速度：llmL/分）。然後，於施加部之通液 中，將脈衝寬度定爲100ns、脈衝上升時間定爲50ns'而 且電場強度定爲15.2〜16.7kV/cm之範圍之高電場脈衝以 重複頻率100Hz施加5 00次。剛流出施加部之後之懸浮液 -29- 200936061 試樣之溫度、亦即既定溫度爲132 °C，由施加部流入口至 流出口之液通過時間爲5秒鐘。 其後’將懸浮液試樣藉由冷卻裝置冷卻之後，透過保 壓閥回收懸浮液試樣。回收時之懸浮液試樣之溫度爲1 5 °C 。在本實施例中，以距離相異之三種配管長構成由施加部 流出口至冷卻裝置而實施，由施加部流出口到達冷卻裝置 所需要的時間分別爲約1 1、1 4、及1 7秒。該等處理係以 位於冷卻裝置正後之保壓閥，將密閉系統之壓力操作至 0.6MPa而實施。 (i)除了將電場強度定爲15.6〜16.91^/(；111之範圍以 外’以與（a)相同之方式進行懸浮液試樣之處理。另外 ，剛流出施加部之後之懸浮液試樣之溫度爲136°C。 (u)除了將電場強度定爲15.5〜17.2kV/cm之範圍 以外，以與（a)相同之方式進行懸浮液試樣之處理。另 外，剛流出施加部之後之懸浮液試樣之溫度爲140 °C。 [比較例7] (1 )懸浮液試樣之調製 以與實施例5相同之方式調製懸浮液試樣（芽胞濃度 5.〇x 104CFU/mL )。 (2 )殺菌 將於（1)所調製之懸浮液試樣，藉由栗連續地通液 至以蒸氣作爲熱媒體之熱交換器（通液速度：llmL/分） 200936061 進行加熱處理。熱交換器之通過時間、亦即加熱至既定溫 度之時間爲5秒鐘。 (a)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲1 32 t之方式加熱。其後，將 懸浮液試樣藉由冷卻裝置冷卻之後，透過保壓閥回收懸浮 液試樣。回收時之懸浮液試樣之溫度爲1 5 °C。在本比較例 中，以距離相異之三種配管長構成由熱交換器流出口至冷 © 卻裝置而實施，由熱交換器流出口到達冷卻裝置所需要的 時間分別爲約1 1、1 4、及1 7秒。該等處理係以位於冷卻 裝置正後之保壓閥，將密閉系統之壓力操作至0.6MPa而 實施。 (i)調節流入熱交換器之蒸氣量，在熱交換器流出 口之懸浮液試樣之溫度成爲1 3 6 °C之方式加熱之後，以與 (a )相同之方式進行懸浮液試樣之加熱處理。 (u)調節流入熱交換器之蒸氣量，在熱交換器流出 ❹ 口之懸浮液試樣之溫度成爲140 °C之方式加熱之後，以與 (a)相同之方式進行懸浮液試樣之加熱處理。 [評估] 對於在實施例4、比較例6回收之各懸浮液試樣而言 ’殺菌處理後之細菌芽胞之生存數係由以下之方式求得。 採取lmL回收之各懸浮液試樣，懸浮於9mL之無菌水稀 釋10倍，進一步採取此稀釋液ImL，懸浮於9mL之無菌 水稀釋100倍。每次ΙΟΟμί採取懸浮液及所得到之10倍 -31 - 200936061 、100倍稀釋液，塗抹至標準洋菜培養基，於55°c進行72 小時培養之後，計算生長之菌落數，求得原本的懸浮液試 樣每lmL生存之菌數。 於實施例5、比較例7回收之各懸浮液試樣之殺菌處 理後之細菌芽胞之生存數，係由以下之方式求得。採取 lmL回收之各懸浮液試樣，懸浮於9mL之無菌水稀釋10 倍。任一者採取此稀釋液lmL，懸浮於9mL之無菌水稀釋 1〇〇倍。每次ΙΟΟμί採取懸浮液及所得到之10倍、100倍 稀釋液至空的培養皿。預先調製好之溫度約60°c之滅菌過 的改良TGC洋菜培養基，將約20mL分注至上述之培養皿 ，將先前採取之菌懸浮液與培養基充分混合。 確認洋菜培養基冷卻凝固之後，厭氣下於55 °C進行1 週培養，藉著計算生長之菌落數，求得原本的懸浮液試樣 每lmL生存之菌數。 將實施例4與比較例6之結果表示於表4，實施例5 與比較例7之結果表示於表5，以及圖2〜圖3。表中之殺 菌溫度，對於實施例4及實施例5而言爲剛流出施加部之 後之懸浮液試樣之溫度，對於比較例6及比較例7而言爲 剛流出熱交換器之後之懸浮液試樣之溫度。另外，表5中 ，殺菌時間，係由施加部流出口或熱交換器流出口到達冷 卻裝置所需要的時間。 圖2〜圖3係將對殺菌時間之處理後之菌數（對數値 )之變化對每個殺菌溫度作圖者。圖形中之直線，係由每 個殺菌溫度之作圖藉由最小平方法求得之迴歸直線，對每 -32- 200936061 條直線記載迴歸式。此處，迴歸式之傾斜度係表示對殺菌 時間之處理後菌數之衰減速度。 [表4] 處理前之菌數 (CFU/mL) 處理後之菌數 (CFU/mL) 殺菌溫度 (°C) 殺菌效果 實施例4⑻ 3.6x10s 3.2χ104 125 1.0 實施例4(i) 3.6χ105 1.8xl03 130 2.3 實施例4⑻ 3.6χ105 2-OxlO1 135 4.3 比較例6⑻ 7.3 x10s 1.7x10s 125 0.6 比較例6(i) 7.3xl05 4.5 xlO4 130 1.2 比較例6⑼ 7.3 x10s 4.1xl02 135 3.2 由實施例4 ( a )，判斷出本發明之方法對該實施例所 使用之細菌芽胞，在至少125 °C以上產生充分之殺菌效果 〇 另外，藉由實施例4(i)之殺菌效果2.3與比較例6 (i )之殺菌效果1.2之比較’判斷出本發明之方法，係在 相同殺菌溫度’相較於由以往的熱交換進行之加熱殺菌， 對該實施例所使用之細菌芽胞之殺菌效果高。此傾向係在 實施例4(a)與比較例6(a)之比較、實施例4(u)與 比較例6 ( u )之比較亦相同。進一步若比較殺菌效果爲 1.0與1.2比較的同等之實施例4(a)與比較例6(i)， 判斷出本方法與由以往的熱交換進行之加熱殺菌相比，即 使約5 °C低殺菌溫度’可得到同等程度之殺菌效果。 -33- 200936061 [表5] 處理前之菌數 (CFU/mL) 處理後之菌數 (CFU/mL) 殺菌溫度 (°C) 殺菌時間 (秒） 殺菌效果 實施例5⑻ 9.8χ103 3·2χ103 132 11 0.5 實施例5⑻ 9.8χ103 3·4χ103 132 14 0.5 實施例5⑻ 9.8χ103 1.2χ103 132 17 0.9 比較例7⑻ 5.0χ104 2.1χ104 132 11 0.4 比較例7⑻ 5.0x104 2.0χ104 132 14 0.4 比較例7⑻ 5.〇χ104 2.1χ104 132 17 0.4 實施例5(i) 9.8χ103 1.4χ103 136 11 0.8 實施例5(i) 9.8χ103 1.3χ103 136 14 0.9 實施例5(i) 9·8χ103 5.4x102 136 17 1.1 比較例7(i) 5.〇χ104 Ι.ΙχΙΟ4 136 11 0.6 比較例7(i) 5.0χ104 9-ΙχΙΟ3 136 14 0.7 比較例7(i) 5.〇χ104 9.3 χΙΟ3 136 17 0.7 實施例5⑻ 9.8χ103 1.3 χΙΟ2 140 11 1.9 實施例5⑻ 9.8χ103 Ι.ΙχΙΟ2 140 14 1.9 實施例5⑻ 9·8χ103 0 140 17 3.9 比較例7⑻ 5.〇χ104 1.2χ103 140 11 1.6 比較例7⑻ 5.〇χ104 8.4χ102 140 14 1.8 比較例7⑻ 5.〇χ104 7.8 χΙΟ2 140 17 1.8 由實施例5 ( i )，判斷出本發明之方法，係對於該實 施例所使用之細菌芽胞而言，至少殺菌溫度136 °C、殺菌 時間1 7秒以上產生充分之殺菌效果。 另外，藉由實施例5之殺菌效果與比較例7之殺菌效 果之比較，判斷出本發明之方法，係在相同殺菌溫度、殺 菌時間，相較於由以往的熱交換進行之加熱殺菌，對於該 實施例所使用之細菌芽胞之殺菌效果高。 如由比較例7判斷般，在由以往的熱交換進行之方法 -34- 200936061 中，即使將殺菌時間（亦即由熱交換器流出口到達冷卻裝 置所需要的時間）由11秒延長至17秒，殺菌效果也幾乎 不增加。相對於此，判斷出在利用高電場脈衝之本發明之 方法中，藉由延長殺菌時間（由施加部流出口到達冷卻裝 置所需要的時間），殺菌效果大幅增加。例如在實施例5 (u )中，藉由將殺菌時間定爲1 1秒至1 7秒，殺菌效果 爲高達1.9至3.9。依據圖2與圖3之迴歸直線之傾斜度 〇 之比較，亦可說是相同之情形。迴歸直線之傾斜度，係表 示對殺菌時間之處理後菌數之衰減速度，而由圖中之迴歸 式看來，可知在本發明之殺菌方法中，與以往的加熱殺菌 相比，此衰減速度係變爲約2倍大。亦即，藉由本發明之 方法，在相同殺菌溫度之殺菌時間，係可縮短至一半程度 。由以上看來，本發明之方法可對細菌芽胞造成以往加熱 殺菌法所沒有的耐熱性之降低。認爲此即由於高電場脈衝 施加對細菌芽胞加上熱以外之物理的刺激所引起之現象。 ❹ [實施例6] (1 )橘子榨汁液之製作 將市售之和歌山產有田橘子搾汁，將以目之間隔 45μιη、3 3 0網目之測試用篩過濾所得到之濾液，製成香味 評估用之橘子搾汁液。此橘子搾汁液在25 之pH爲3 _76 、導電率爲294mS/m。另外，糖度爲13.1。 (2 )殺菌 -35- 200936061 使用與實施例1相同裝置’以高電場脈衝處理於（1 )所製作之橘子搾汁液。殺菌處理，係與實施例2 (i )相 同之殺菌效果爲目標在殺菌溫度102 °c進行。在此殺菌溫 度條件之細菌芽胞（Alicyclobacillus acidoterrestris)之 殺菌效果係由實施例2 ( i )爲4.0。 將約1 (TC之橘子搾汁液，藉由栗連續地通液至施加部 (通液速度：1 lmL/分）。然後，於施加部將脈衝寬度定 爲100ns、脈衝上升時間定爲50ns、而且電場強度定爲 80kV/cm之高電場脈衝以重複頻率100Hz施加500次。剛 流出施加部之後之橘子搾汁液之溫度爲1 〇2 °C，由施加部 流入口至流出口之液通過時間，亦即加熱至既定溫度之時 間爲5秒鐘。其後，將橘子搾汁液藉由冷卻裝置冷卻之後 ，回收橘子搾汁液。回收時之橘子搾汁液之溫度爲1 5 °C。 由施加部流出口至冷卻裝置之距離爲22.5 cm ’由施加部流 出口到達冷卻裝置所需要的時間爲約1 5秒鐘。該等處理 係以位於冷卻裝置正後之保壓閥，將密閉系統之壓力操作 至0.6MPa而實施。 [比較例8] 使用與於實施例6所製作者相同之橘子搾汁液’進行 以熱交換器進行之加熱殺菌處理。殺菌處理係與比較例4 (e)相同之殺菌效果爲目標在殺菌溫度H2°C進行。在此 殺菌溫度條件之細菌芽胞 （Ali cyclobacillus acidoterrestris )之殺菌效果，係由比較例4 ( e )爲4.2 ’ 200936061 成爲與實施例2 (i)幾乎同等殺菌效果之殺菌條件。 將約10°c之橘子搾汁液，藉由泵以蒸氣作爲熱媒體之 熱交換器連續地通液至（通液速度：i lmL/分）進行加熱 處理。調節流入熱交換器之蒸氣量，熱交換器流出口之橘 子搾汁液之殺菌溫度成爲1 1 2乞之方式加熱之後，經1 5秒 鐘之溫度保持時間快速地藉由冷卻裝置冷卻至約15 t，回 收橘子搾汁液。橘子搾汁液通過以蒸氣作爲熱媒體之熱交 φ 換器之時間，亦即橘子搾汁液被加熱至既定溫度之時間爲 5秒鐘。另外，該等連續處理，係藉由位於冷卻裝置正後 之保壓閥’將密閉系統之壓力操作至〇.6MPa而實施。 [評估] (1 )感官測試 以殺菌處理前之橘子搾汁液作爲對照，對於實施例6 及比較例8之施加殺菌處理之橘子搾汁液之香味，關於由 φ 加熱殺菌造成之風味劣化進行感官測試。感官測試，係藉 由5名經過訓練之測試員（A〜E )，對於1 :外觀之色度 、2:新鮮度、3:劣化臭味少量程度、4:酸味、5:甜味 五個評估項目而進行。將結果表示於表6。表中，〇係與 對照之橘子搾汁液風味爲同等、△係與對照之橘子搾汁液 相比風味梢差、X係表示與對照之橘子搾汁液相比風味明 顯地劣化。 -37- 200936061From the embodiment 3 (a), the method of the present invention was judged, and the bacterial spores used in the examples produced sufficient bactericidal effects at least 121 °C. In addition, the bactericidal effects of Example 3 (i) and Comparative Example 5 (i), which were almost the same at 128 ° C and 127 ° C, were 4.7 and 〇8, respectively, and the comparison between the two was judged. According to the method of the invention, the bactericidal effect of the bacterial buds used in the examples is high at the same sterilizing temperature as compared with the heat sterilization by the conventional heat exchange. Further, in comparison with Comparative Example 5 (i) in which the bactericidal effect was 4.7 and the comparative example 5 (u) having a bactericidal effect of 3.4, the present method was judged to be compared with the heat sterilization by the conventional heat exchange -26-200936061. A sterilization effect equal to or higher than that at a low sterilization temperature of at least 7 ° C can be obtained. [Example 4] (1) Preparation of suspension sample A bacterial spore (Geobacillus stearothermophilus) was added to an aqueous solution of KH2P〇4 and Na2HP〇4 φ to prepare a conductivity of 400 mS at 20 °C. m. Phosphoric acid buffer having a pH of 6.5, and preparing a suspension sample containing bacterial spores (spore concentration: 3.6 x 105 CFU/mL). (2) Sterilization Using the same apparatus as in Example 1, the suspension sample prepared in (1) was treated in the following order. (a) A suspension sample of about 20 ° C was continuously passed through a pump to a 施加 application portion (liquid flow rate: llmL / min). Then, in the liquid passing through the application portion, the pulse width was set to 100 ns, the pulse rise time was set to 50 ns, and the electric field intensity of the electric field intensity in the range of 16.1 to 18.2 kV/cm was applied 500 times at a repetition frequency of 100 Hz. The temperature of the suspension sample immediately after flowing out of the application portion was also a predetermined temperature of 125 ° C, and the liquid passage time from the application portion to the flow outlet was 5 seconds. Thereafter, after the suspension sample was cooled by a cooling device, the suspension sample was recovered through a pressure maintaining valve. The temperature of the suspension sample at the time of recovery was 15 °C. In the present embodiment, 'the time -27-200936061 from the application portion outlet to the cooling device is carried out for about 14 seconds. These treatments were carried out by operating the pressure of the closed system to 〇6 MPa with a pressure maintaining valve located immediately behind the cooling device. (i) The treatment of the suspension sample is carried out in the same manner as (a) except that the electric field intensity is set to be in the range of 16.4 to 18.41 Å / (: 111. Further, the suspension sample immediately after flowing out of the application portion The temperature was 130 ° C. (u) The suspension sample was treated in the same manner as (a) except that the electric field strength was set to be in the range of 16.7 to 18.81 Å/(; 111. The temperature of the suspension sample after the portion was 135 ° C. [Comparative Example 6] (1) Preparation of suspension sample A suspension sample (spore concentration: 17.3 x 105 CFU/mL) was prepared in the same manner as in Example 4. (2) Sterilization The suspension sample prepared in (1) is continuously heated to a heat exchanger using steam as a heat medium (through rate: 1 lmL/min) for heat treatment. The passage time of the exchanger, that is, the time to be heated to a predetermined temperature is 5 seconds. (a) The amount of steam flowing into the heat exchanger is adjusted, and the temperature of the suspension sample at the outlet of the heat exchanger becomes 1 25 t. Heating is performed. Thereafter, the suspension sample is cooled by a cooling device and recovered through a pressure maintaining valve. Suspension sample. The temperature of the suspension sample at the time of recovery was 15 ° C. In this comparative example, the time required for the heat exchanger outflow to reach the cooling device was about 14 -28 - 200936061 sec. The pressure of the closed system is operated to 0.6 MPa by a pressure maintaining valve located immediately behind the cooling device. (i) The amount of steam flowing into the heat exchanger is adjusted, and the temperature of the suspension sample at the heat exchanger outflow port becomes After heating at 130 ° C, the suspension sample is subjected to heat treatment in the same manner as (a). (u) The amount of vapor flowing into the heat exchanger is adjusted, and the temperature of the suspension sample at the heat exchanger outflow port is adjusted. After heating at 135 ° C, the suspension sample was heat treated in the same manner as φ ( a ). [Example 5] (1) Preparation of suspension sample The bacterial cell (Moorella thermoacetica) was added. A phosphate buffer solution having a conductivity of 400 mS/m and a pH of 6.5 at 20 ° C was prepared using an aqueous solution of KH 2 P 4 and a solution of Na 2 HP 04 to prepare a suspension sample containing bacterial spores (spore concentration: 9.8×10 3 CFU/mL). 2) sterilization use and example 1 In the same apparatus, the suspension sample prepared in (1) is processed in the following order: (a) A suspension sample of about 20 ° C is continuously passed through the pump to the application portion by the pump (liquid flow rate: llmL/min.) Then, in the liquid passing through the application portion, the pulse width is set to 100 ns, the pulse rise time is set to 50 ns', and the electric field strength is set to a high electric field pulse in the range of 15.2 to 16.7 kV/cm at a repetition frequency of 100 Hz. Apply 5 00 times. Suspension immediately after flowing out of the application section -29- 200936061 The temperature of the sample, that is, the predetermined temperature was 132 ° C, and the liquid passage time from the inlet of the application portion to the outlet port was 5 seconds. Thereafter, after the suspension sample was cooled by a cooling device, the suspension sample was recovered through a pressure maintaining valve. The temperature of the suspension sample at the time of recovery was 15 °C. In the present embodiment, the three types of pipe lengths differing from each other are configured by the application portion outlet to the cooling device, and the time required for the application portion outlet to reach the cooling device is about 1 1 , 1 4 , and 1 7 , respectively. second. These treatments were carried out by operating the pressure of the closed system to 0.6 MPa with a pressure maintaining valve located immediately behind the cooling device. (i) The treatment of the suspension sample is carried out in the same manner as (a) except that the electric field strength is set to be in the range of 15.6 to 16.91^/(; 111. Further, the suspension sample immediately after flowing out of the application portion The temperature was 136 ° C. (u) The suspension sample was treated in the same manner as (a) except that the electric field intensity was set to be in the range of 15.5 to 17.2 kV/cm. The temperature of the liquid sample was 140 ° C. [Comparative Example 7] (1) Preparation of suspension sample A suspension sample (spore concentration 5. 〇 x 104 CFU/mL) was prepared in the same manner as in Example 5. 2) Sterilize the suspension sample prepared in (1) by heat-passing the liquid to the heat exchanger (vapor flow rate: llmL/min) 200936061 with steam as the heat medium. The passage time, that is, the time to be heated to a predetermined temperature was 5 seconds. (a) The amount of vapor flowing into the heat exchanger was adjusted, and the temperature of the suspension sample at the outlet of the heat exchanger was heated to 1,32 t. Thereafter, after the suspension sample is cooled by the cooling device, it is returned through the pressure maintaining valve. Suspension sample. The temperature of the suspension sample at the time of recovery is 15 ° C. In this comparative example, the three kinds of pipe lengths which are different in distance are formed by the heat exchanger outlet to the cooling device. The time required for the heat exchanger outlet to reach the cooling device is about 1 1 , 14 , and 17 seconds, respectively. The treatment is performed by a pressure maintaining valve located directly behind the cooling device to operate the pressure of the closed system to 0.6 MPa. (i) adjusting the amount of vapor flowing into the heat exchanger, and heating in the same manner as (a) after the temperature of the suspension sample at the heat exchanger outlet is heated to 136 °C. Heat treatment of the sample (u) The amount of vapor flowing into the heat exchanger is adjusted, and after the temperature of the suspension sample of the heat exchanger outlet port is heated to 140 ° C, the same manner as in (a) is carried out. Heat treatment of the suspension sample. [Evaluation] For each of the suspension samples recovered in Example 4 and Comparative Example 6, the survival number of the bacterial spores after the sterilization treatment was determined by the following method. Each suspension sample was suspended in 9 mL Dilute the broth water 10 times, further take 1 mL of this dilution, and dilute it in 9 mL of sterile water to dilute 100 times. Take a suspension of each ΙΟΟμί and obtain 10 times-31 - 200936061, 100 times dilution, and apply to standard agar. The culture medium was cultured at 55 ° C for 72 hours, and the number of colonies grown was counted, and the number of bacteria per 1 mL of the original suspension sample was determined. The sterilization of each suspension sample recovered in Example 5 and Comparative Example 7 The survival number of the bacterial spores after the treatment was determined by the following method: 1 mL of each suspension sample recovered was suspended in 10 mL of sterile water and diluted 10 times. Either 1 mL of this dilution was taken and suspended in 9 mL of sterile water for 1 〇〇. Take a suspension and the resulting 10-fold, 100-fold dilution to the empty Petri dish each time ΙΟΟμί. The sterilized modified TGC agar medium having a temperature of about 60 ° C was preliminarily prepared, and about 20 mL was dispensed into the above-mentioned culture dish, and the previously taken bacterial suspension was thoroughly mixed with the medium. After confirming the cooling and solidification of the acacia medium, the culture was carried out for one week at 55 ° C under anaerobic conditions, and the number of colonies of the original suspension sample per l mL was determined by calculating the number of colonies grown. The results of Example 4 and Comparative Example 6 are shown in Table 4, and the results of Example 5 and Comparative Example 7 are shown in Table 5 and Figs. 2 to 3. The sterilization temperature in the table is the temperature of the suspension sample immediately after flowing out of the application portion for Example 4 and Example 5, and the suspension immediately after flowing out of the heat exchanger for Comparative Example 6 and Comparative Example 7. The temperature of the sample. Further, in Table 5, the sterilization time is the time required to reach the cooling device by the application portion outlet or the heat exchanger outlet. Fig. 2 to Fig. 3 are graphs showing changes in the number of bacteria (logarithm 値) after the treatment of the sterilization time for each sterilization temperature. The straight line in the graph is the regression line obtained by the least square method from the plot of each sterilization temperature, and the regression equation is recorded for each -32-200936061 line. Here, the slope of the regression equation indicates the decay rate of the number of bacteria after the treatment of the sterilization time. [Table 4] Number of bacteria before treatment (CFU/mL) Number of bacteria after treatment (CFU/mL) Sterilization temperature (°C) Sterilization effect Example 4 (8) 3.6×10s 3.2χ104 125 1.0 Example 4(i) 3.6χ105 1.8 Xl03 130 2.3 Example 4(8) 3.6χ105 2-OxlO1 135 4.3 Comparative Example 6(8) 7.3 x10s 1.7x10s 125 0.6 Comparative Example 6(i) 7.3xl05 4.5 xlO4 130 1.2 Comparative Example 6(9) 7.3 x10s 4.1xl02 135 3.2 By Example 4 (a) It is judged that the method of the present invention produces a sufficient bactericidal effect on the bacterial spores used in the embodiment at least at least 125 ° C. In addition, the bactericidal effect 2.3 and the comparative example 6 (i) of Example 4 (i) Comparison of the bactericidal effect 1.2 'It is judged that the method of the present invention is based on the same sterilizing temperature' as compared with the heat sterilization by the conventional heat exchange, and the bactericidal effect of the bacterial spores used in the examples is high. This tendency is also the same in comparison between Example 4 (a) and Comparative Example 6 (a), and Example 4 (u) and Comparative Example 6 (u). Further, when the comparative example 4 (a) and the comparative example 6 (i) in which the bactericidal effect was 1.0 and 1.2 were compared, it was judged that the method was lower than about 5 ° C compared with the heat sterilization by the conventional heat exchange. The sterilization temperature 'can obtain the same degree of sterilization effect. -33- 200936061 [Table 5] Number of bacteria before treatment (CFU/mL) Number of bacteria after treatment (CFU/mL) Sterilization temperature (°C) Sterilization time (seconds) Sterilization effect Example 5 (8) 9.8χ103 3·2χ103 132 11 0.5 Example 5(8) 9.8χ103 3·4χ103 132 14 0.5 Example 5(8) 9.8χ103 1.2χ103 132 17 0.9 Comparative Example 7(8) 5.0χ104 2.1χ104 132 11 0.4 Comparative Example 7(8) 5.0x104 2.0χ104 132 14 0.4 Comparative Example 7(8) 5.〇χ104 2.1χ104 132 17 0.4 Example 5(i) 9.8χ103 1.4χ103 136 11 0.8 Example 5(i) 9.8χ103 1.3χ103 136 14 0.9 Example 5(i) 9·8χ103 5.4x102 136 17 1.1 Comparative Example 7(i 5.〇χ104 Ι.ΙχΙΟ4 136 11 0.6 Comparative Example 7(i) 5.0χ104 9-ΙχΙΟ3 136 14 0.7 Comparative Example 7(i) 5.〇χ104 9.3 χΙΟ3 136 17 0.7 Example 5(8) 9.8χ103 1.3 χΙΟ2 140 11 1.9 Example 5(8) 9.8χ103 Ι.ΙχΙΟ2 140 14 1.9 Example 5(8) 9·8χ103 0 140 17 3.9 Comparative Example 7(8) 5.〇χ104 1.2χ103 140 11 1.6 Comparative Example 7(8) 5.〇χ104 8.4χ102 140 14 1.8 Comparative Example 7(8) 5. 〇χ104 7.8 χΙΟ2 140 17 1.8 From the embodiment 5 (i), the method of the present invention is judged For the system of Example bacterium used in the spores, the sterilizing temperature of at least 136 ° C, sterilization time is 7 seconds or more to produce sufficient effects of sterilization. Further, by comparing the sterilizing effect of Example 5 with the sterilizing effect of Comparative Example 7, it was judged that the method of the present invention is the same sterilization temperature and sterilization time as compared with the heat sterilization by the conventional heat exchange. The bacterial spores used in this example have a high bactericidal effect. As judged by the comparative example 7, in the method of the conventional heat exchange, -34-200936061, even if the sterilization time (that is, the time required for the heat exchanger outlet to reach the cooling device) is extended from 11 seconds to 17 In seconds, the bactericidal effect hardly increases. On the other hand, it was judged that in the method of the present invention using a high electric field pulse, the sterilization effect is greatly increased by prolonging the sterilization time (the time required for the application portion to flow to the cooling device). For example, in the embodiment 5 (u), the bactericidal effect is as high as 1.9 to 3.9 by setting the sterilization time to 1 1 second to 17 seconds. The comparison between the inclination of the regression line of Fig. 2 and Fig. 3 can be said to be the same. The inclination of the regression line indicates the decay rate of the number of bacteria after the treatment of the sterilization time, and it can be seen from the regression equation in the figure that the attenuation rate is higher than that of the conventional heat sterilization in the sterilization method of the present invention. The system becomes about 2 times larger. That is, by the method of the present invention, the sterilization time at the same sterilization temperature can be shortened to half. From the above, the method of the present invention can cause a decrease in heat resistance which is not required for the conventional heat sterilization method for bacterial spores. This is believed to be caused by the application of a high electric field pulse to the physical stimulation of the bacterial spore plus heat. ❹ [Example 6] (1) Preparation of orange juice extracting juice The commercially available Wakayama rice-fielded mandarin orange juice was extracted, and the filtrate obtained by filtering the sieves at 45 μιηη, 303 mesh was observed to obtain a fragrance evaluation. Use the orange to squeeze the juice. This orange juice has a pH of 3 _76 at 25 and a conductivity of 294 mS/m. In addition, the sugar content was 13.1. (2) Sterilization -35-200936061 Using the same apparatus as in Example 1, the orange juice produced by (1) was treated with a high electric field pulse. The sterilization treatment was carried out in the same manner as in the sterilization effect at 102 ° C in the same sterilization effect as in Example 2 (i). The bactericidal effect of the bacterial spore (Alicyclobacillus acidoterrestris) at this sterilization temperature condition was 4.0 from Example 2 (i). About 1 (the orange of TC is squeezed out of the juice, and the liquid is continuously supplied to the application portion (liquid flow rate: 1 lmL/min). Then, the pulse width is set to 100 ns in the application portion, and the pulse rise time is set to 50 ns. Further, a high electric field pulse having an electric field strength of 80 kV/cm was applied 500 times at a repetition frequency of 100 Hz. The temperature of the orange juice immediately after flowing out of the application portion was 1 〇 2 ° C, and the liquid passage time from the application portion to the outflow port That is, the time for heating to a predetermined temperature is 5 seconds. Thereafter, the orange juice is cooled by a cooling device, and then the orange juice is recovered. The temperature of the orange juice extracted at the time of recovery is 15 ° C. The distance from the outlet to the cooling device is 22.5 cm. The time required to reach the cooling device from the outlet of the application section is about 15 seconds. These treatments are based on the pressure-retaining valve located directly behind the cooling device, and the pressure of the closed system. The operation was carried out to 0.6 MPa. [Comparative Example 8] The heat sterilization treatment by a heat exchanger was carried out using the same orange juice as that produced in Example 6. The sterilization treatment was the same as that of Comparative Example 4 (e). Sterilization effect The sterilization effect was carried out at a sterilization temperature of H2 ° C. The bactericidal effect of the bacterial spore (Ali cyclobacillus acidoterrestris) at the sterilization temperature was 4.2. 200936061 from Comparative Example 4 (e), which was almost the same as the sterilization of Example 2 (i). Sterilization conditions of the effect. The juice of about 10 °c is squeezed out, and the heat is continuously passed through the heat exchanger of the pump using steam as a heat medium to (liquid flow rate: i lmL / min) for heat treatment. The amount of steam in the evaporator, the sterilization temperature of the orange juice in the heat exchanger outlet is heated to 1 2 2, and then the temperature is maintained for 15 seconds by the cooling device to cool to about 15 t. Squeezing the juice. The orange juice is squeezed through the steam as the heat medium for the heat exchanger, that is, the orange juice is heated to a predetermined temperature for 5 seconds. In addition, the continuous treatment is by cooling. The pressure-retaining valve at the rear of the device is operated by operating the pressure of the closed system to 〇6 MPa. [Evaluation] (1) The sensory test is carried out by pressing the juice of the orange before the sterilization treatment as a control. 6 and Comparative Example 8, the scent of the orange juice extracted by the sterilizing treatment, and the sensory test on the flavor deterioration caused by φ heat sterilization. The sensory test was performed by 5 trained testers (A to E), for 1 : appearance color, 2: freshness, 3: deterioration of odor, a small amount, 4: sourness, 5: sweetness, five evaluation items were carried out. The results are shown in Table 6. In the table, the tangerine and the control orange The juice flavor was the same, the △ system was compared with the control orange juice, and the flavor was significantly worse than that of the control orange juice. -37- 200936061
1.外觀之色度 測試員A 測試員B 測試員C 測試員D 測試員E 實施例6 Δ Δ Δ Δ 〇 比較例8 Δ Δ Δ X 〇 2_新鮮度 測試員A 測試員B 測試員C 測試員D 測試員E 實施例6 Δ 〇 〇 Δ 〇〜△ 比較例8 △〜X Δ X X Δ 3.劣化臭味少量程度 測試員A 測試員B 測試員C 測試員D 測試員E 實施例6 Δ 〇 Δ Δ Δ 比較例8 △〜X X X X X 4.酸味 測試員A 測試員B 測試員C 測試員D 測試員E 實施例6 Δ 〇 〇 〇 〇 比較例8 Δ △〜X Δ Δ Δ 5.甜味 測試員A 測試員B 測試員C 測試員D 測試員E 實施例6 〇 △ Δ Δ Δ 比較例8 Δ △〜X X X X -38- 200936061 由上述之結果，判斷出本發明之殺菌方法，與由以往 的熱交換進行之加熱殺菌相比，抑制了風味之劣化，橘子 搾汁液本來的風味會殘留更多。 (2 )成分分析 對於殺菌處理前之橘子搾汁液，以及施加殺菌處理之 實施例6及比較例8之橘子搾汁液，測定維他命c之濃度 。測定係依照一般稱爲靛酚滴定法之方法進行。已知維他 命C，由於加熱處理濃度會減少’作爲加熱造成之品質劣 化之代用特性而進行成分分析。將其結果表示於表7。 [表7] 維他命C (mg/1 00g) 殺菌處理前之橘子搾汁液 34 實施例6 3 3 比較例8 32 由此結果看來’判斷出本發明之殺菌方法與由以往的 熱交換進行之加熱殺菌相比’可抑制殺菌造成之維他命C 濃度之減少。維他命C係橘子搾汁液本來具有之營養成分 之1個，判斷出藉由本發明之方法可抑制其劣化。 【圖式簡單說明】 圖1係表示用於實施本發明之殺菌方法之裝置之一形 態之圖。 -39- 200936061 圖2係表示對實施例5之殺菌時間之生存菌數之變化 之圖形。 圖3係表示對比較例7之殺菌時間之生存菌數之變化 之圖形。 【主要元件符號說明】 A ： 液體食品材料收容部 B :栗 C ： 溫度調節手段 D ： 施加部 E ： 冷卻手段 F : 閥 G ： 電源裝置 Η ： 溫度測定手段 I : 壓力計 〇 -40-1. Appearance Chroma Tester A Tester B Tester C Tester D Tester E Example 6 Δ Δ Δ Δ 〇 Comparative Example 8 Δ Δ Δ X 〇 2_ Freshness Tester A Tester B Tester C Tester D Tester E Example 6 Δ 〇〇Δ 〇 △ △ Comparative Example 8 Δ~X Δ XX Δ 3. Deteriorated odor A small amount Tester A Tester B Tester C Tester D Tester E Example 6 Δ 〇Δ Δ Δ Comparative Example 8 △~XXXXX 4. Sour Tester A Tester B Tester C Tester D Tester E Example 6 Δ 〇〇〇〇 Comparative Example 8 Δ Δ~X Δ Δ Δ 5. Sweet Taste Tester A Tester B Tester C Tester D Tester E Example 6 〇 Δ Δ Δ Δ Comparative Example 8 Δ Δ~XXXX -38- 200936061 From the above results, the sterilization method of the present invention was judged Compared with the heat sterilization by the conventional heat exchange, the deterioration of the flavor is suppressed, and the original flavor of the orange juice is more retained. (2) Component analysis The concentration of vitamin C was measured for the orange juice before the sterilization treatment and the orange juice of Example 6 and Comparative Example 8 to which the sterilization treatment was applied. The measurement is carried out according to a method generally referred to as indophenol titration. It is known that vitamin C is subjected to component analysis because the concentration of heat treatment is reduced as a substitute characteristic of quality deterioration caused by heating. The results are shown in Table 7. [Table 7] Vitamin C (mg/1 00g) Orange juice before sterilizing treatment 34 Example 6 3 3 Comparative Example 8 32 From the results, it was judged that the sterilizing method of the present invention was carried out by conventional heat exchange. Compared with heat sterilization, it can inhibit the decrease of vitamin C concentration caused by sterilization. Vitamin C is one of the nutrients originally possessed by the orange juice, and it is judged that the deterioration can be suppressed by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a view showing the state of one of the devices for carrying out the sterilization method of the present invention. -39- 200936061 Fig. 2 is a graph showing the change in the number of viable cells in the sterilization time of Example 5. Fig. 3 is a graph showing the change in the number of viable cells in the sterilization time of Comparative Example 7. [Description of main component symbols] A : Liquid food material storage unit B : Chestnut C : Temperature adjustment means D : Application part E : Cooling means F : Valve G : Power supply unit Η : Temperature measuring means I : Pressure gauge 〇 -40-