TW200421979A - Novel lactic acid bacteria and bacteriocin thereof, and method for processing fish and legume foodstuffs using the same and the products obtained - Google Patents
Novel lactic acid bacteria and bacteriocin thereof, and method for processing fish and legume foodstuffs using the same and the products obtained Download PDFInfo
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- TW200421979A TW200421979A TW092109120A TW92109120A TW200421979A TW 200421979 A TW200421979 A TW 200421979A TW 092109120 A TW092109120 A TW 092109120A TW 92109120 A TW92109120 A TW 92109120A TW 200421979 A TW200421979 A TW 200421979A
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- Prior art keywords
- lactic acid
- item
- acid bacteria
- patent application
- fish
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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Description
200421979 ⑴ 玖、發明說明 【發明所屬之技術領域】 本發明提供一種新穎之乳酸菌及其細菌素、利用該類 乳酸菌之魚肉食品加工方法與產品以及豆類食品加工方法 與產品。更具體而言,本發明係提供一種篩選自肉中的新 穎性乳酸菌,可使用於利用魚類或豆類作爲原料之發酵加 工食品加工方法,且提供由該方法所製得之可抑制雜菌生 長、風味特佳、經濟價値更高之加工食品。本發明更提供 一種由該新穎性乳酸菌所產生的細菌素,該細菌素可有效 地抑制其他雜菌的生長,進而確保保存期間食品的品質。 【先前技術】 傳統食品已漸漸不能滿足消費者的需求,由市面上健 康食品、精緻食品的充斥,不難瞭解食品的多元化、安全 性與機能性是現今食品業開發的重點。 乳酸菌是一群可發酵醣類產生乳酸的微生物,常在蔬 菜、榖類、乳品及肉品等發酵產品中被當成發酵菌酉I ( starter )使用,可以增進食品之營養價値(Act〇n a/., 1977)、抑制腸內病原菌生長(Bacus and Brown, 1981)、 提高乳糖之利用性(Siddons and Coates, 1 985)、具有抗 癌(Oda ef a/., 1983)與降低膽固醇(Mann and Spoerry, 1 974)功能等特性。發酵過程中乳酸菌因利用醣類進行糖 解作用,而提供產品特殊風味;亦能分泌乳酸、醋酸等有 機酸分子,使製品之p Η値下降,抑制微生物的生長,延 -6 - (2) (2)200421979 長產品的儲藏期限;此外有些乳酸菌尙能產生過氧化氫、 雙乙醯、細菌素等產物以抑制腐敗菌或病原菌之生長 (Gibbs, 1 987; Klaenham mer, 1 988; Daeschel. 1 989; S c h i 11 i n g e r a n d Lucke , 1 989)。 乳酸菌能產生抑制病原菌生長之物質(森地,1 997), 主要的物質爲細菌素(Bacteriocins)、diacetyl、H2O2和 次級代謝產物。細菌素是一種含蛋白質之大分子,具有抑 制微生物生長作用(Scbillinger and Holzapfel,1 996; Roller and Lusengo,1997)。能產生細菌素之乳酸菌有 Lactobacillus fermentum (Deklerk and Smit, 1 967)、 Lactobacillus plantarum (Sedewitz et al., 1 983)、
Lactobacills helveticu s (Joerger and Klaenhammer, 1 986) 、 Lactob acill u acidophilus (Muriana and
Klaenhammer, 1 987) 、 Lactob a ci 11 u plantarum (West and Warner, 1 988) 和 Ped iococcu s pentosaceus (Daeschel and Klaenhammer, 1 985)。
Pediococcus ac/of//aci/c/所產生的細菌素能抑制鮮 肉、發酵香腸、發酵甘藍菜、碎牛肉和乳酪中 Listeria monocytogenes ^^^(Nielsen et al., 1 990; Choi and
Beuchat, 1 994; Motlagh et al., 1 9 9 2; Parente et al., 1 996; Vignolo et a/., 1 996; Cutter and Siragusa, 1 996) ,增加冷藏之保存期限。//aci/s ATCC 1 1454 , Pediococcus pentosaceus ATC C 43200 和 Pediococcus pentosaceus ATCC 43201 所產生的細菌素 (3) (3) 200421979 添加於含 3〜4%食鹽之冷藏調理食品中,能抑制 C/ostr 丨 cUum botu/丨·ηυ/ν Μ子的萌芽與生長(Okereke and Montville,1991) 〇 Lactococcus lactis 和 Pediococcus peniosaceoys所產生的細菌素能抑制革蘭氏陽性之病原 菌,$〇 Bacillus cereus 、 Clostridium perfnngenes、 Staphylococcus aureus 和 L/sie厂/a monocyiogenes ;此 外,對於某些革蘭氏陰性病原菌,如 Aeromonas hydro phi! a A Η 2 、 Escherichia coli 0 1 5 7 : H 7 、 Vibrio cholerae 8 51 禾 D V. parahaemolyticus A865957 (Spelhaug and Harlander, 1 98 9; Helander et al., 1 997) 亦有抑制作用。Laciococcus /aci/s subsp. /aci/s 所產生 的 nisin 已被列爲 GRAS (generally recognized as safe) (FDA, 1 992),使用於冷藏乳酪以抑制 Clostndium ύοία//Α?ί/Α77孢子之萌芽生長(園田等,1996卜 乳酸菌除產生細菌素外,亦產生丁二酮和Η202。丁 二酮(2,3-butanedione)爲乳酸菌中間代謝產物丙酮酸 (pryuvate)所合成之終產物(Kandler,1 983; Monnet ei a/·, 1 99 4)。Jay (1982)硏究丁二酮在 200 pg/ml 能抑制 革蘭氏陽性菌。丁二酮也是GRAS,但具強烈風味,揮發 性高,最好限量使用。H202爲乳酸菌在生長時,分別由 丙酮酸、乳糖酶和NADH經由丙酮酸氧化酶,L-乳糖酶和 NADH 氧化酶與 〇2 作用產生(Kandler,1 983; Sedewitz et a/·,1983),能抑制有害微生物的生長(森地,1997)。 H2〇2能與其他成分形成良好抑菌物質,如以乳糖過氧化 -8- (4) (4)200421979 酶(lactoperoxidase)作用於硫氰酸鹽(thiocyanate) ,形成中間氧化產物以抑制微生物,此過程爲”乳糖過氧 化乳糖過氧化酶抗菌系統 (lactoperoxidase antibacterial system ) ”,能增加食品之保存期限 (Harnulv et al.} 1982)。 由Pec//ococc〃s菌屬生產之細菌素種類及特性,包括 由 Ped · acidilactici H 生 產之 Pediocin AcH、 Ped · acidilactici PA1.0 生 產 之 Pediocin PA-1 、 Ped. pentosaceu s FBB61 生 產 Pediocin A。此外 Ped. cere vesiae FBB63 禾口 Ped · acidilactici PC 生產之,細菌素 目前仍未命名。
Bhunia等人在1 987年自發酵香腸中篩選出能分泌細 菌素 Pediocin AcH 之菌株 Ped. ac/d//aci/c/ strain Η ,其 分子量約2,700Da (SDS-PAGE)。經結果證實此細菌素可 抑帋ij Lactobacilli 、 Leuconostocs 、 Staphylococcus aureus、Clostridium perfringens ' Pseudomonas putida 和 Usier/a monocyiogfenes等微生物的生長。具有對蛋 白分解酵素敏感、對熱安定。Pediocin AcH因作用於細 胞膜上,造成細胞膜內鉀離子等之流失,導致細胞分解 (Bhunia et al., 1991 卜 【發明內容】
有鑑於上述課題,本發明人等詳細硏究結果發現,使 用由肉品中所舖選分離的Pecf/ococci/s penfosaceus YJL (5) (5)200421979 和 YJS菌株等特殊乳酸菌可改善鯖魚等魚類及豆類之加 工適性,且由探討乳酸菌發酵過程中所產生之產物對魚肉 品質(質地、風味、色澤等)及豆類之影響、及乳酸菌所 產生之蛋白酶對魚肉質地及豆類之影響,可增加魚類及豆 類之加工範圍,更提升其利用性。又,進一步利用該新穎 乳酸菌於在不同條件下進行魚肉或豆類發酵時,則因所產 生之產物及所生產之蛋白酶作用程度的不同,最終產品之 質地、色、香、味影響亦不同。因此,本發明提供一種可 以利用發酵條件來控制並生產不同新產品之新食品加工技 由上可知,本發明提供如下之發明。 1. 一 種寄存號碼爲 BCRC910210 之 pentosaceus YJL 〇 2. 一種寄存號碼爲 BCRC910211 之 Pediococcus pentosaceus YJS 〇 3. —種魚肉加工方法,其爲使用如第1項或第2項 之乳酸菌、或如第1項或第2項之混合乳酸菌、或其他乳 酸菌之混合乳酸菌進行魚肉發酵者;其加工方法爲:魚肉 加0.3〜2.0%食鹽及0_5〜3倍水後均質之,經100〜115 t殺菌15〜30分鐘後冷卻至25〜40°C ,調整基質水分含 量(由不稀釋〜五倍稀釋)及添加1.0〜6.0%之糖後接種乳 酸菌,在25〜40°C發酵6〜30小時後可任意添加適當量 之調味劑及香辛料或可包裝之。 4. 如第3項之加工方法,其中其他乳酸菌的混合乳 -10- (6) (6)200421979 酸菌爲 1 種以上選自 Lacio6ac///usp/anfara/77 CCRC1 0069, Lactococcus lactis subsp. lactis CCRC 123 15, Lactobacillus helveticus CCRC 1 4092 者。 5·如第3項之魚肉加工方法,其中魚肉原料爲至少 一種選自於紅色肉、白色肉魚、其混合魚肉、冷凍魚漿等 - 者。 -
6.如第3項之魚肉加工方法,其中糖原料爲至少一 種選自於蔗糖、葡萄糖、甜菜等。 H 7 .如第3項之魚肉加工方法.,其中魚肉基質可爲不 稀釋〜五倍稀釋之魚肉漿。 8 ·如第3項之魚肉加工方法,其中調味劑爲至少一 種選自於一般之水果、加工之水果醬、芝麻、花生等。 9.如第3項之魚肉加工方法,其中香辛料爲至少一 種選自於一般之薑、蒜、味淋、酒、五香粉等。 1 0·如第3項之魚肉加工方法,其中發酵後基質之 pH 在 3.8 〜5.5。 1 1 . 一種魚肉加工食品,其特徵爲使用如第1項或第 2項之乳酸菌、或如第1項或第2項之混合乳酸菌、或其 他乳酸菌之混合乳酸菌進行魚肉原料發酵所得者。 1 2 ·如第1 1項之加工方法,其中其他乳酸菌的混合 · 乳酸菌爲 1 種以上選自 Lactobacillus plantarum - CCRC1 0069, Lactococcus lactis subsp. lactis CCRC 12315, Lacio&ac///i;s CCRC 1 4092 者。 1 3. —種魚肉加工食品,其爲利用如第3項之魚肉加 -11 - (7) (7)200421979 工方法進行魚肉原料發酵所得者。 14. 一種魚肉加工食品,其爲利用如第3項之魚肉加 工方法進行魚肉原料發酵後在1 〇 〇〜1 1 5 ° C加熱殺菌、成 型、部分乾燥所得之類似起司產品者。 1 5 ·如第1 1〜1 4項中任一項之魚肉加工食品,其中魚 肉原料爲至少一種選自紅色肉、白色肉魚、其混合魚肉或 冷凍魚漿等。 16·—種豆類加工方法,其爲使用如第1項或第2項 之乳酸菌、或如第1項或第2項之混合乳酸菌、或其他乳 酸菌之混合乳酸菌進行豆類發酵者;其加工方法爲:經浸 泡豆類加水後均質、過濾後在經1 00〜1 1 5°C加熱殺菌1 5 〜30分鐘後冷卻至25〜40 °C,調整基質水分含量(50 %〜 98%)及添加1.0〜6.0%之糖後接種乳酸菌,在25〜40。(: 發酵6〜30小時後任意添加適當量之調味劑或可包裝之。 1 7·如第1 6項之加工方法,其中其他乳酸菌的混合 乳酸菌爲1種以上選自p/aniariv/T? CCRC1 0069, Lactococcus lactis subsp. lactis CCRC 12315, Lactobacillus helveticus CCRC 1 4092 者。 18·如第16項之豆類加工方法,其中豆類原料爲至少 一種選自黃豆或黑豆。 19. 如第16項之豆類加工方法,其中糖原料爲至少一 種選自蔗糖、葡萄糖、甜菜等。 20. 如第16項之豆類加工方法,其中豆類發酵基質之 水分含量爲50%〜98%。 -12- (8) (8)200421979 2 1 .如弟1 6項之丑類加工方法,其中調味劑爲至少一^ 種選自一般之水果、加工之水果醬等。 22·如第16項之豆類加工方法,其中發酵後豆類基質 之pH在4.5〜6.0。 23. —種豆類加工食品,其爲使用如第1項或第2項 之乳酸菌、或如第1項或第2項之混合乳酸菌、或其他乳 酸菌之混合乳酸菌進行豆類原料發酵所得者。 24. 如第23項之加工方法,其中其他乳酸菌的混合 乳酸菌爲 1 種以上選自 Lactobacillus plantarum CCRC1 0069, Lactococcus lactis subsp. lactis CCRC 12 315, Lactobacillus helveticus CCRC 14092 者。 25. —種豆類加工食品,其爲利用如第14項之豆類加 工方法進行豆類原料發酵所得者。 26. 如第23項〜第25項之豆類加工食品,其中豆類 爲至少一種選自於黃豆、黑豆、或其混合物者。 27. —種豆類加工食品,其爲利用如第3項之豆類加 工方法進行豆類原料發酵後在90〜11 5°C加熱殺菌、成型 、部分乾燥所得之類似起司產品者。 28. —種細菌素,其特徵爲來自如第1項之乳酸菌。 29. 如第28項之細菌素,其爲分子量爲20〜30 kDa 的抑菌性物質。 3 0.—種細菌素,其特徵爲來自如第2項之的乳酸菌 31 .如第30項之細菌素,其爲分子量爲20〜30 kDa (9) (9)200421979 的抑菌性物質。 以下茲更詳細地說明本發明。本發明係提供新穎之乳 酸菌及其細菌素、利用該類乳酸菌之魚肉食品加工方法與 產品以及丑類食品加工方法與產品。 本發明所使用的具有新穎性之乳酸菌係由肉品中所篩 選分離出白勺 Pediococcus pentosaceus YJL YJS 菌株 。Ped/ococct/s菌屬爲乳酸四鏈球菌,不具運動性、不產 孢子且爲觸媒陰性之革蘭氏陽性菌。 本發明提供一種魚肉加工方法,其特徵爲使用 Pediococcus pentosaceus YJL 和 YJS 菌株或 Pediococcus pentosaceus YJL m YJS 菌株的混合学L 酸菌 或其他?L 酸菌 $口 Lactobacillus plantarum CCRC1 0069, Lactococcus lactis subsp. lactis CCRC 12315, Lactobacillus helveticus CCRC 1 4092 等進行紅色肉或 白色肉魚或其混合魚肉或冷凍魚漿等魚肉發酵,其加工方 法爲將作爲魚肉原料的魚肉基質之魚肉漿加入0.3〜2.0% 食鹽及0.3〜1.5倍水後均質之,經1 〇 〇〜1 1 5 °C殺菌1 5〜 30分鐘後冷卻至25〜40°C ,調整基質水分含量(由不稀釋 〜五倍稀釋)及添加1.0〜6.0 %之至少一種選自蔗糖、葡 萄糖、甜菜等糖後接種乳酸菌,在25〜40 °C發酵6〜30 小時後,使該發酵基質的最後pH在3.8〜5.0,可任意添 加適當量之至少一種選自水果、加工之水果醬、芝麻、花 生等調味劑及香辛料並視情況做任意包裝。 本發明又提供一種魚肉加工食品,其爲使用如 -14- (10) (10)200421979
Pediococcus pentosaceus YJL BCRC91 0210 和 YJS BCRC91021 1 菌株之乳酸菌或如 Pediococcus peniosaceus yjL和YJS菌株之混合乳酸菌或其他乳酸菌 如 Lactobacillus plantarum CCRC10069, Lactococcus I act is subsp. lactis CCRC 12315, Lactobacillus helveticus C C R C 1 4092等進行至少一種選自紅色肉或白 色肉魚或其混合魚肉或冷凍魚漿之魚肉原料發酵所得者。
本發明又提供一種魚肉加工食品,其爲使用如 Pediococcus pentosaceus YJL BCRC910210 禾口 YJS BCRC91021 1 菌株之乳酸菌或如 Pediococcus pentosaceus YJL "S菌株之混合乳酸菌或其他乳酸菌 如 Lactobacillus plentdrum CCRC1 0069, Ldctococcus lactis subsp. lactis CCRC 12315, Lactobacillus
Zie/vef/ci/s CCRC 1 4092等進行至少一種選自紅色肉或白 色肉魚或其混合魚肉或冷凍魚漿之魚肉原料,經發酵、加 熱殺菌、成型、部分乾燥後所得之類似起司產品者。
本發明又提供一種豆類加工方法,其特徵爲如 Pediococcus pentosaceus YJL BCRC910210 禾口 YJS BCRC910211 菌株之乳酸菌或如 pecy/〇c〇cct/s peniosaceus YJL和YJS菌株之混合乳酸菌或其他乳酸菌 如 Lactobacillus plantdrum CCRC1 0069, Lacfococct/s lactis subsp. lactis CCRC 12315, Lactobacillus /7e/\zei/c£is CCRC 1 4092等進行豆類發酵者;其加工方法 爲:經浸泡至少一種選自黃豆或黑豆之豆類加水後均質、 -15- (11) (11)200421979 過濾之,在100〜11 加熱殺菌15〜30分鐘後冷卻至 25〜40°C,調整基質水分含量(50%〜98%)及添加1 ·0 〜6.0 %之至少一種選自蔗糖、葡萄糖、甜菜之糖後接種 乳酸菌,在25〜40°C發酵6〜30小時後可任意添加適當 量之調味劑並視情況做包裝。 本發明又提供一種豆類加工食品,其爲使用如 Pediococcus pentosaceus YJL BCRC910210 和 Y JS BCRC91021 1 菌株之乳酸菌或如 Pediococcus pentosaceus YJL iD "S菌株之混合乳酸菌或其他乳酸菌 如 Lactobacillus pla ntaru m CCRC1 0069, Lactococcus I act is subsp. I act is CCRC 12 315, Lactobacillus helveticus CC RC 1 4092等進行至少一種選自黃豆或黑豆 或其混合物者豆類原料發酵所得者。 本發明又提供一種豆類加工食品,其爲使用如 Pediococcus pentosaceus YJL BCRC910210 和 YJS BCRC910211 菌株之乳酸菌或如 Pediococcus pentosaceus Y儿和YJS菌株之混合乳酸菌或其他乳酸菌 如 Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315, Lactobacillus CCRC 1 4092等進行至少一種選自黃豆或黑豆 或其混合物者豆類原料,經發酵、加熱殺菌、成型、部分 乾燥後所得之類似起司產品者。 本發明又提供一種分子量爲20〜30 kDa的抑菌性物 質之細菌素,其爲來自如Ped/ococci/s peniosacei/s /儿 -16- (12) (12)200421979 菌株之乳酸菌。 本發明又提供一種分子量爲2 0〜3 0 k D a的抑菌性物 質之細菌素,其特徵爲來自如Pecf/ococct/s peniosace^ys yjs菌株之的乳酸菌。 【實施方式】 以下茲例舉實施例,以更具體地說明本發明,惟本發 明並未限定於此等。 主要儀器 低溫振盪培養箱·· Orbital shaking incubator (HOTECH 718, Hotech Instruments Co., Taiwan). 低溫高速離心機:Automatic high speed refrigerated centrifuge (SCR 20B , Hitachi, Japan). 色差儀:Model TC-1 800MK-II, Tokyo Denshoku Co., Japan. pH 測定儀·· pH Meter (HM-30S, TOA Electronic Co., Japan). 恒溫恆濕培養箱·· T C -1 2 0 H D , T u n g t e c i n s t r u m e n t s C., LTD. 碎肉乳化機·· UM-12,Stephan, Germany. 凍乾機:Model FD-20-84, Fts ststems, INC.,U.S.A. (13)200421979 胺基酸測定儀:A m i η 〇 A c i d A n a I y ζ θ r ( H i t a c h i L - 8500,Japan). 真空減壓濃縮機:Rotavapor (Biichi RE111, Buchi,
Switzerland). 迷你電泳·· Electrophoresis Cell (Mi ni-PROTEAN II, Bio-Rad, U. S. A.).
電源供應器:Power Supply (Model 200/2.0, Bio-Rad, U. S. A.). 止泡均質機·· Waring Blender (subjoined with a baffle, Japan). 冷凍櫃:-30〇C 及- 80〇C 爲 Bio-Freezer (Model 8442, Forma Scientific, U. S. A.). 分光光度計:Hitachi U-2001, Hitachi, Japan. 尾部血壓量測器·· SoHron BP 98-A, Japan. 實施例1 _
Pediococcus pentosaceus YJL BCRC910210 ^
Pediococcus pentosaceus YJS BCRC910211 S: ^ 定結果 由表一結果得知在MRSA選擇性培養基上呈現乳酸菌 · 乳白色典型菌落之Pediococcus pentosaceus yjL菌株爲 ^ 不具運動性、觸媒陰性之革蘭氏陽性四連球菌,可利用葡 萄糖發酵產酸且不產氣;此外,Pecf/ococMs pentosaceus /JS菌株亦爲觸媒陰性、不具運動性之革蘭 -18- (14) (14)200421979 氏陽性四連球菌,可利用葡萄糖發酵產酸且不產氣。由以 上結果根據1988年Simpson與1991年Pilone學者所提 菌種分類表(圖一)即可將此二株乳酸菌歸屬於 Ped/ococct/s 菌屬。 進一步測試此二菌株之生長溫度範圍、耐鹽度和P Η 範圍等。由表一結果顯示:MPL菌株可於4〜45°C、pH 4.0〜7.0間生長,10%鹽濃度以上不能生長◊而 Pediococcus pentosaceus WS 菌株可於 15 〜45〇C 下生 長,但不能於4°C下生長且可於pH 4.0〜7.0間生長,此 外,亦可在1〇.〇 %鹽濃度下生長。在醣類發酵測試方面( $口 表二),Pediococcus pentosaceus YJL Pediococcus pentosaceus YJS二株乳酸菌可發酵核糖(ribose )、但 是不能利用D-阿拉伯糖、L-木糖等醣類進行發酵。此特 性與1978年Back學者提出pec//ococci/s菌屬之菌種分 類表,可知此二菌株均屬於PecZ/ococci/s peniosaceas。 200421979 5) s「Aco^a)oeco07c:(l>da; 画龄 + + + + + + + + + + + + + sco ±H&B^^srA38s5§aa:^Ί「Λ3δδο;§αα;,—« _J「>snQ)oessc:(l)da. + + + + + + + + + + + + + + + + _龄 66·ε (Η<ε) Hd 班« (Η<ε) ooz (Η<ε) om (Η<ε)°.ιο (Η<ε) 0.寸 (Η<ε) sCNi (Με) 00 ^fessloBN£盤«^ (Η<ε) ο·ζ hcl (Η<ε)99 hq. (Η<ε) o.s hq. (Η<ε) 0·寸 hq. (Hd lemulcsMlMOJQxds盤瞰胡鋇醛 (H<z)o。寸 Rz) oosl (tKco) POCSJ (Η<ε) asCN RCN) ροε RCN) Ρ9ε (H<3) CX0 寸 3(h<3)ps々 (£MOJ0) (A6oloJZdJOUJJelnll0o) ilass (ΛΙΗΙ)δ賴 OS2 aseliseo)^·^® (cmls E29)釦获也鼴 # «吆*舔蝤^14-长«„丨,,:8 。稍ftgisys^*瑯驭奪 _ -20- (16) (16)200421979 API 50CHL系統鑑定 將 Pediococcus pentosaceus YJL WS 菌株與購 自食工所之標準菌 Pediococcus pentosaceus CCRC 1 4024三株乳酸菌進行API 50C H L system鑑定測試,結 果列於表三。由表中可知標準菌 Pediococcus pentosaceus CCRC 1 4024可利用核糖、半乳糖、D-葡萄 糖、D-果糖、D-甘露糖、N-乙醯胺基葡糖、苦杏仁苷、 arbutine、esculin、纖維二糖酶、麥芽糖、melibiose、 蔗糖、海藻糖、salicin。但不能利用β-gentiobiose、D-阿拉伯糖、木糖及果糖等。測試結果經系統硏判爲 Pediococcus pentosaceus ,判別率達 9 9.8% 〇 因 β-gentiobiose爲負反應與系統値相反造成誤差。
Pediococcus pentosaceus 菌株可手[]用核糖、半 乳糖、D-葡萄糖、D-果糖、D-甘露糖、N-乙醯胺基葡糖 、苦杏仁苷、arbutine、escul in、纖維二糖酶、麥芽糖、 melibiose、蔗糖、海藻糖。但對於 salicin、β-gentiobiose、D-阿拉伯糖、木糖及果糖等均不能利用。 上述結果經系統硏判爲 Ped/ococct/s pentosaceus ,因 salicin、β-gentiobiose爲負反應與系統値有不同測試結 果,使 Peaf/ococcas penfosacet/s 菌株之判別率爲 9 9.6% 〇
Pediococcus pentosaceus yJS 此菌株可手[j 用核糖、 半乳糖、D-葡萄糖、D-果糖、D-甘露糖、N-乙醯胺基葡 糖、苦杏仁苷、arbutine、esculin、salicin、纖維二糖酶 -21 - (17) (17)200421979 、麥芽糖、melibiose及蔗糖,但不能利用β-gentiobiose 、D -阿拉伯糖、木糖、s a I i c i η和果糖等。有二種測試結 果 salicin、p_gentiobiose爲負反應與系統値不同,使測 試結果之判別率爲99.3%。綜合以上傳統鑑定方法與API 50CHL乳酸菌系統鑑定法比對,可確定此二株乳酸菌爲 Pediococcus. pentosaceus。
-22- 200421979 8
Ico「l>g3la)ioggo/glq)la·!0. CO66 l-l「l>-l«onlq)loggo~clq)IQ..lQ. + + + + 臂SO 寸IT-loltt:lu>ol«olal(l)loggojctq)dIQ. CO66 码纗璀味圃泡Τέ ΊΗυοΙΛldv:?s*-9>.s825§Q.CL^lrA3a:Jeso7sCLQ:,u 揪 (a》gpAqol-selo)llafal
(%) als/JSOqlso 知 csd ·0. 海长-塌B-i vsolqoluiso-Ql φρ·3)οιι UBUI .Ο'Λ I09s.<3 4).·2υ>οοΛ 16-G->.£a)2-i3 il>x (®SOI2i)海«i? (olos)胜海硪-3 (auplles )«n琛« 00 海掸 a)(AOUEeqa: 海味M狴鎇Ig.z 9SOI2 ^o^ccns (S21es )海妝麻 (asolAX-l)海长j (so-eos-1)海祙彐Jl e>soonu.--J (|022<-1〕海珥13囱--| (es23el)墀成 (soulq2v -Ί)海 Jnul (υ®600ΛΙ0)®ιι (Ι290ΛΙ9)坦扣 5euoonlo (asolueleo )海^升 ullnoIAui loi?UJ (asolAX-a )雔长-a 15olna souejnld socin-ey-a (asouues-a )雔®in-a d>SOXA_l-Q(assnlo-a)海ItaKla)woonLL-Q (as20ELL-a )墀睞—a (lojlqsv.a ) u^^lIIl-a (9so=qeJ<-a)海溢醮担-a 9solqol«o ®u!jnqj< (θ.ΕΙΒΡΒΛαι v )«nu 你«a (uopl )ffi^楸 (luopv)胜诨啪領虽 91Βυοαπισ>-οϊα>ο-ιο v4Ecoon l?OJS-CN 旺 fVR-K-长谳: -23 - (19) (19)200421979 生化測試 P· pentosaceus Y儿及 P. pentosaceus WS 均對精 胺酸具水解能力,但無法利用尿素、t e t r a z ο I i u m r e d及 丙酮酸等。此二株菌之菌體型態於TEM下觀察結果顯示 此二菌株均爲無鞭毛之四連球菌。 抗生素敏感性試驗 抗生素敏感性試驗結果如表三所示,由表中得知P. pentosaceus YJL fi Ceftazidime (30 meg) > Moxalactam (30 meg)、 Nalidoxic acid (30 meg)、具耐抗性 (resistant);對 Gentamicin (10 meg)微耐抗性 (intermediate resistant);對安比西林 (10 meg)、 Cefotaxime (30 meg)、Cefuroxime (30 meg)、盤尼西林 (10 meg)微敏感性 (moderately susceptible);對 Clidamycin (2 meg)、紅黴素(15 meg)、Imipenem (10 meg)、Netilmicin (30 meg)、四環素 (30 meg)、 Ticarcillin (75 meg)、Vancomycin (30 meg)具敏感性 (susceptible) ° P. pentosaceus YJS Gentamicin (10 meg) Moxalactam (30 meg)、Nalidoxic acid (30 meg)、 Vancomycin (30 meg)具耐抗性;對四環素(30 meg) 微耐抗;對安比西林(10 meg)、Cefotaxime (30 meg)、 Ceftazidime (30 meg)具微敏感性 (moderately susceptible);而對 Cefuroxime (30 meg)、Clidamycin -24- (20) (20)200421979 (2 meg)、紅黴素(15 meg)、Imipenem (i〇 meg)、 Netilmicin (30 meg)、盤尼西林(10 meg)、Ticarci I lin (75 meg)具敏感性。 根據上述試驗結果可知此二株乳酸菌在生理生化特性 上有差異,因此將之分命名爲 Pediococcus pentosaceus YJL (P. pentosaceus YJL)和 Pediococcus pentosaceus YJS [P. pentosaceus YJS) 〇
-25- (21)200421979 ^M#i'®®«^^^s「>-s8sssdQ:^—lr>-3sss§aCL,H« s「Aco3CDo(Dco5c(l)dCL-J「A(o^(l>oa3ssc:a>dCL®s ώιΛΙ el eQ: (6301)««
Q: S Iss id S S S S S1AI SIAI SIAI
S S S S2 S a: ys Iss SIAI Q: SIAI SIAI CVJT-OCNI 61, SCN InT-61 eCN 9广 loT— 8l· COCNJ COCN COCN4 oco CNJsr·· CNJCNJ—sl· CNJCNJ-sl· 6cnj-c\icnj ^rvcoLzv? CVJsl·
ZSCN 6TST- U-OT-· oovgT- ^rvcol, oov?
6 寸T- n 61 CNJT-ετ-· 寸T- CNJT-COV-寸l· 寸τ— 寸Τ-寸一 aT-CNI oco loz oco oco oco oco CH CH Lol· ocsi ocoοε oco 0T-· υΌΛΕΟουωΛ u!lloJS!l (u!oA3ei=91)*酹Ξ (umoca}d) #®s/^ υοΙΕΙΙΙΘΝ pocoolx!p!l03N lucolocolcoxol/M ΕωυθφΕΙ uoIEissCD (uoAEOJ£AJLlJ)«uy υιοΛ£ΒΡΕ·ιο ΘΕ!Χ22Θ0 ΘΕΙΡΝΒ^ΘΟ ΘΕ!ΧΒ15θο (uldl) #ss 。?£1^«£11展运:0| #i鲍鏹毗0):担龌癱蕤coIAIiHi*蕤 Ίώ运 -26- (22) (22)200421979 實施例2 手[J 用 Lactobacillus plantarum CCRC 1 0069 ' Lactococcus lactis subsp. lactis CCRC 12315 、
Lactobacillus helveticus CCRC 1 4092、Pediococcus pentosaceus YJL BCRC910210 及 P. pentosaceus YJS BCRC91021 1的魚類加工方法 冷凍鯖魚於室溫下經流水解凍,去除內臟及頭後,利 用採肉機採肉。以2% NaCI溶液等體積均質鹽溶,再作 三倍稀釋之魚肉基質。經1〇〇°C殺菌20分鐘後冷卻至40 °C,最後添加4%蔗糖、1 %葡萄糖及乳酸菌以滅菌玻 離棒均勻混合後置於37°C發酵48小時。在此基質中乳酸 菌接種的濃度約爲1〇5 CFU/g。實驗中檢測之項目包括 P Η値、乳酸菌數及好氣性菌數。此外,亦檢測原物料及 加工製程中可能存在主要微生物菌相之變化,包括 Pseudomonas、Staphylococcus 禾口 Enterobacteriaceae 等菌群,藉以瞭解乳酸菌之抑菌能力。最後添加適當量之 桑椹及糖後作官能品評。 結果:37 °C發酵48小時後,五種乳酸菌發酵製品之 pH値自6.2-6.3降低至4.5-4.7,未添加乳酸菌組則pH 値上升至7·4-7·6;且VBN値於發酵24小時後由8.2-8.6上升至50.2-51.4 mg/100 g,甚至於發酵48小時後 亦提高至70.1-71.3 mg/100 g。然而添加乳酸菌組則僅由 8.1-8.6些微上升至21.0-24.8 mg/100 g。該項結果顯示 利用乳酸菌發酵可抑制VBN之生成,同時由於細菌素之 -27- (23) (23)200421979 生成而能可有效抑制腐敗菌或病原菌如 Pseiyciomonas、 Staphylococcus 及 Enterobacteriaceae 等之生長(表 四).五種乳酸菌發酵製品之 Hunter L (indicator of transparency)、b (indicator of yellow/blue)和白度 (whiteness)均高於未添加乳酸菌組 (L: from 47.61-4 9.98 increased to 59.03-65.06; b: from 7.14-8.64 to 9.35-1 1.68; whiteness: from 46.8-49.3% increased to 57.9-6 3.5%,p<0.05)。五種乳酸菌發酵產品之口感、風 味及整體接受性均受好評。由表五發現發酵24小時產品 接受性優於48小時組,但大體上不同乳酸菌之接種其差 異不大。
-28- 200421979 \—/ (24 舰鑛 ®coCDcooyoPA7CDcoCLpucocoA70oooo/>ft/dct3ico CDeaoeA/aoeqo/acUJ-画氍忒_祕由忉瑯骠盤v『/v+s黜粼晒饀Ίέ±ρζε袒黟逻疵驄忉誕艎兴逛—Kl , am
Pseudo. 4.10±0.15e 8.04±0.26b 9.04±0.21 a 3.22±0.11 b 4.00±0.26 a 4.20±0.30a 3.31±0.22b 3.52±0.19b 4.53±0.20a 3·31±0.21 b 3.32±0.19 b 4.33±0.20a 3.3±0.14b 3·49±0·20 b 4.33±0.17a 3.3±0.14b 3.09±0.21b 4.33±0.19a -c Q. S (〇 2.97±0.13c 6.63±0.23b 7.54±0.15a 2.77±0.21 c 3·38±0·22 b 4.13±0.22a 3·10±0·17 b 3.20±0.15b 4.43±0.18a 3.10±0.17b 3·20±0·15 b 4.43±0.19a 2.80±0.25b 3.00±0.16b 4.21±0.20a 2.80±0.25b 3.00±0.16b 4.21±0.20a E LL 〇 O) ja CO ω 方 CO GQ Entero. 2.78±0.13b 9.04±0.27a 9.08±0.18a 3.15±0.15c 3·66±0·21 b 4·10±0·18 a 3.08±0.19e 3.38±0.13bc 4.40±0.23b 3_18±0.19b 3·38±0·13 b 4.50±0.24a 3·19±0·11 b 3.95±0.21 a 4.30±0.15a 3.19±0·11 e 3.95±0.20 b 4.30±0.14a CD < 」 3.34±0.15c 6.35±0.32b 7.24±0.17a 6.40±0.16b 9.46±0.19a 9.49±0.17a 6.34±0_21 c 8.65±0.17a 8·85±0.21 a 6.34±0.21 b 8.65±0.17a 8.85±0.22a 6.45±0.12b 9.26±0.12a 9.18±0_20a _〇C〇(C CM 寸 T- 广 T- CN • · 參 ? ? s CD 00 ^ CM T- cd σί σί o 0. < 4.20±0.31e 7.78±0.36b 8.17±0.33a 6.23±0.18b 9.38±0_33a 9.27±0.24a 6·28±0·21 c 8.20±0.32b 9_32±0.32a 6.28±0.20c 8.20±0.31 b 9.32±0.35a 6.43±0.19b 9.36±0.20a 9.20±0.27a 6.43±0.19b 9.36±0.21 a 9.20±0.31 a 發酵時間 X: o寸00 σ CSJ 寸 起始物t CO z < CO 〇 Q LU (soovd) ysi
•S「Au)23(Domsoc9d snooooopeCLUJlJrA srwoesoced(οηοοοοο!ΡΘί1.·οιζ60<ιο}ίοο(οοο!ω>φχ:(η3ι!οωοουωΊ lo一 glcocsiloQroos/oe/ dsqns s/pe/ Ss080pe7 :g :690CHoa:oo55ece/d sn//oeqfoc3el :<:鬆装超呂腾雔:SN -29 - 200421979 \—/ 5 (2 账遽ifeng雜iIn*N®i顆盤七<4-0黜餾脑趦忒卜3^〇〇相黟裰疵驄^||#爷逛1^,:5« 5 1111 1111 CO CO CO (0 O 00 T-卜 • · * * T- 〇 v- 〇 +1+1+1+1 O) 〇〇 X- 00 CD (〇 CO CO CD ¢0 CO T- ο p ^ 6 d V; +1 +1 +i +J r- σ> (N p N: CD ^ 6.8±1.2a 7.1±1.1 a 7.3±0.8a 7.0±1.3a 7.0±1.3a 7.1±1.2a 7.4±1.1 a 7.0±1.1a 7.0±1.1 a 7.1±1.2a 7.211.0 a 7.1±1.2a •恆溫培養時間(小時) 寸 CN It Jill 'II 1 7.6±1.2a 7.6±1.1a 8.2±1.1a 7.9±0.8a 7.7±1.2a 7.9±1.0a 8.3±1.2a 7.7±1.2a CQ CO CO (Q Ο) T- ?! +i ϊ +« 〇> 05 CO CNi 卜卜· 〇〇· 〇6 CO CO CO c〇 h厂q厂 O X- T- Y; +l +l +l + N. σ> ττ cv4 卜’卜· 〇〇· 〇〇 7.9±0.9a 8.0±1.1a 8.2±1.0a 8.0±1.1a 〇 4·1±1·1 a** 4.2±1_2a 3.3±0.5a 4.0±0.5a 4.2±1.1b 3.8±1.2b 3.3±1·1 b 3.9±1.0b 4.2±1.2b 4.0±1.1 b 3.4±0.9b 4.0±1.2b ^0*0*0。 卜 〇 (D O ?i +i ?i ^ (〇 σ> h- cr> CO CO co co 4.0±1.0b 4.0±0.8b 3.7±0.6b 4.0±1.1b ja -° λ O 〇〇 CD V Ϊ ?! ?! Ϊ 卜· O⑦厂 CO ^ CO·寸 官能評估 5> o | fei 给备艾制 卜ϋ f鹚 n5 eg co W 卜ϋ f瑯 ^ ^ C is J3备艾制 卜ϋ $锄 笔张霸鉚 π° δ eg H c ^ ζ嵌魔》 nggg 卜E f瑯 ^ ^ sly Μ 雲震麵潮 S 卜E f瑯 ^ w w is 寫雲震翻 起始物4 O) 2 < GQ o D Lli .?.ovs ^.1¾Ϊ as+lMlnidz^龌{w鹅 _嫛00脈傾}«仵漱··,ttgs < f % -30- (26) (26)200421979 實施例 3 禾[j 用 Lactobacillus plantarum CCRC1 0069 ' Lactococcus lactis subsp. lactis CCRC 12315 、
Lactobacillus helveticus CCRC 1 4092 、 Pediococcus pentosaceus YJL BCRC910210 及 P. pentosaceus YJS BCRC910211的魚肉起司及優格力口工方法 冷凍金線鰱魚漿於5 °C下解凍一夜,再以1 . 〇 % N a C I 溶液等體積均質,經100〜11 5°C殺菌15分鐘後冷卻至 30°C,添加4%蔗糖及乳酸菌以滅菌玻離棒均勻混合後置 於37°C發酵24小時。在此基質中乳酸菌接種的濃度約爲 105CFU/g。最後添加適當量之芝麻或花生粉後於100〜 115 °C殺菌15分鐘,成型(如圖三及四所示)爲方形並 在適度乾燥後進行官能品評。 結果:37°C發酵24小時後,五種乳酸菌發酵製品之 pH値均降低至4.6-4.8,五種乳酸菌發酵產品之口感、風 味及整體接受性均受好評。 實施例 4 罕[J 用 Pediococcus pentosaceus YJL 及 Pediococcus peniosacet/s YJS的豆類力口工方法 經浸泡之黃豆加水後均質、過濾之,以100〜11 5°C 加熱殺菌20分鐘後冷卻至30 °C ,調整基質水分含量爲 60%,最後添加4%蔗糖與1%葡萄糖及乳酸菌以滅菌玻離 棒均勻混合後置於37 °C發酵24小時。在此基質中乳酸菌 -31 - (27) (27)200421979 接種的濃度約爲1 05 C F U / g。並檢測ρ Η値、乳酸菌數、 好氣性菌數、主要微生物菌相〜Pseacfomonas、 Staphylococcus 和 Er?ier〇jbacienaceae 等菌群之變化。 最後添加適當量之草莓及糖後作成布丁(如圖五),並作 官能品評。 結果:37 °C發酵24小時後,乳酸菌發酵製品之pH 値自6.0-6 ·2降低至4.7-4.9,未添加乳酸菌組則pH値上 升至 7.5-7.7 ; Pseudomonas 、 Staphylococcus 及
EnieT〇bacier/aceae等之生長均有效被抑制(表六)。五 種乳酸菌發酵製品之Hunter L、b和白度(whiteness)均 高於未添加乳酸菌組(ρ < 〇 . 〇 5)。五種乳酸菌發酵產品之 口感、風味及整體接受性均受好評(表七)。 -32- 200421979
I
^000 ωωοοΕΟΈΦωη^ pue 500000/Λι/ο.5οοφδο.2-/θίο52^£-画趦^-晒祕^^^盤\「/04-71蝕粼晒趦^^3。卜£袒黟|111,<撇 Pseudo. 4.10±0.15c 8.10±0.26b 3.3±0.14b 3.29±0·20 b 3.20±0.14b 3.09±0.20b N 」 ε LL 〇 O) 15 u. ① CO ω iS co 2.97±0.13c 6.73±0.23b 2.80±0.25b 3.10±0.16b 2.90±0.25b 3.05±0·11 b Entero. 2.78±0.13b 9.14±0.27a 3·19±0·11 b 3·55±0·21 a 3.09±0.11 e 3·45±0·21 b LAB 3.34±0.15c 6.33±0.33b 6.45士0.12 b 9.29±0.12a 6.35±0.12b 9.28±0.14a APC 4.20±0.31e 7.79±0.37b 6.43±0.19b 9.37±0.20a 6.23±0.19b 9·32±0·21 a 發酵時間(小時)_ 〇艺 〇艺 °艺 起始物+ CO z < 0Q •(90d>d)Mllf爱 lisi ^ ^ sf ^00.^00 ^ ^ icofx S8S0 名 0〇f SO38O/P0C/ ώ 一 7ΓΛ saoso;5d Sn880/P9c/ :v 一蓉^snill〕SN · -33- 200421979 9 (2
_煺痣ngijl)l{nn^您盤七S+-H 触粼画鍫 Ίέκ-ρζε 袒麟Ini, ¥ 漱 4.0±1.3a“ 4.1±1.5a 4.0±0.8a 7_6±1.2a 7.6±1.1a 7.9±0.8a 7.7±1.2a 7.9±1.0a 7.7±1.2a 〇 4·1±1 ·1 a“ 4.2±1.2a 4.0±0.5a 4·3±1.1 a** 4.0±1.2a 4.0±0.7a 4·2±1·1 a** 4.2±1.4a 4.1±0.6a 官能評估 5 ? ^ l·- E w w 廳 彆#潮 Ππ 瘦 ω ? 1¾ 给$职 H E t$ ng 5 2 ^ 卜 LL 逝 一 w 廳 昭S 起始物 CO z < CQ .?.ovsis ω -34- (30) (30)200421979 實施例 5 來自 Ped i oco cc u s pe n to s ace u s Y J L 及 Ped i 〇 co cc u s peniosaceas YJS的細菌素之萃取及定性結果 1.粗細菌素的置備(Isolation of bacteriocins) 乳酸菌接種至MRS培養基,置於37°C培養48小時 後,以5,000 X g,離心30 min後,上層液經0_ 45 μηη膜 過濾(No. 4654, Gelman),以去除菌體。抑菌活性測定則 以L. monocytogenes CCRC 1 4845做爲指示菌株,所得 濾液則進行抑菌活性測定確認其爲具有抑菌活性之粗細菌 素液。 2.氯仿的萃取(Chloroform extraction)
Wi ^ Μ 0.1% Pediococcus pentosaceus YJL and Pediococcus pentosaceus 400 mL MRSig 養基,置於37 °C培養18小時。所得菌液以9,500 g離心 15 min (4°C ),上層液再以0.45 μη膜過濾,所得濾液混 合200 mL氯仿劇烈攪拌20 min後進行10,400 g (4°C) 離心20 min。離心後則分爲四相,其中溶劑與水層之界 面層 (solvent-aqueous interface layer)及沈澱層 (precipitates)具有最大之抑菌活性,利用 5_ 1 0 m L buffer (0.1 M Tris_HCI, pH 7.0)懸浮溶解。其餘二相包 括水層(aqueous phase)與溶劑層(solvent phase) 均不具有活性。懸浮後之緩衝液再以真空濃縮機(40°C ) -35- (31) (31)200421979 (RotavaporR114, BCJCHI)去除氯仿,最後濃縮至約2-3 m L ( B u r i a n e k and Yousef, 2000),分 S(l 命名爲 Pediocin YJL 和 Pediocin YJS。 3. SDS-PAGE (Sodium dodecyI sulfate polyacrylamide gel electrophoresis) 爲確定細菌素之純度並訂定其分子量,將細菌素液溶 解在樣品緩衝液 dissociating buffer (62.5 mM Tris-HCI buffer, pH 6.8, containing 3 % SDS and 0.002% bromophenol blue)並隔水煮沸5min。再以 8-15%丙嫌 醯胺進行SDS-PAGE電泳分析 (Laemmli, 1 970)。泳動 完成後之膠片以 15% TCA進行固定,Coomassie brilliant blue G-250染色,最後以 25% 甲醇脫色,並 將膠片夾於玻璃紙中陰乾即可。 4. 蛋白質濃度(Protein concentration) 蛋白質定量依Bradford (1 976)的方法進行,並以牛 血淸白蛋白 (bovine serum albumin)爲標準品。 -36- (32) 200421979 液加至1 5 ml適當的培養基(451 )中,充分振盪,再 將含菌液的培養基倒入培養皿中,靜置3 0 m i η後移入冰 箱(4 °C )中放置1小時。之後,以直徑8 mm的金屬環在 培養基表面挖洞,再取細菌素溶液30 μΙ,滴於洞內,並 於指示菌株最適生長溫度下培養24小時後,觀察是否有 抑制環並記錄其大小。
6.生化特性(Biochemical Properties) 6.1 酵素敏感性(Sensitivity of bacteriocin to the proteolytic enzymes )
將細菌素液以1.0 N H CI或1.0 N N a Ο H分別調至 pH 4_0、5·0、6.0、7·0、8.0後,各別加入所測試酵素 2 0.0 mg/ml (pepsin [from Porcine Stomach Mucosa, Sigma] 、 α-chymotrypsin [from Bovine Pancreas, Sigma】、pronase [from Streptomyces griseus, Sigma] 、bromelain [from Pineapple stem, Sigma],37 °C 作用 2小時後,於80°C下加熱15分鐘,使蛋白質分解酵素失 活,再置於冰浴槽中使其溫度迅速冷卻至室溫,測其抑菌 活性(Piddock, 1 990)。 6.2 熱安定性(Thermostability of purified bactenocins) 首先將抑菌物質分別以1.0 N HCI或NaOH調整其 pH 爲 4.0、5.0、6.0、7.0、8.0,並於 80 °C、100 °C 下 15min、30min、45min、60min 及 121 °C、1 5 m i n 之條件 -37- (33) (33)200421979 進行熱處理後迅速置於冰浴槽中使其溫度冷卻至室溫,再 測其抑菌活性 (Piddock, 1 990)。 6.3 抑菌範圍試驗(Bacteriocin spectrum of activity) 細菌素液Pentocin YJL和Pentocin YJS對於食品中 一 常見病原菌及腐敗菌之抑菌範圍測試如表八所示。 -
-38- 200421979 \J/ (34 雲 050 0^00 050 oso 050 050 050 0^00 ναϋ-pvaLL 050 oso oyoo 050 050 050 oso oyoo 050 u!Bsl.oJd qo5o
Co
P9CVJ/0CON0/0S6+ VN pzco/ VN ρζε / ρζε / vn oozco/ VS1 pzco/ VN P9CNI/ VN pzco/LLIAmsi pzco/LLIAmsl pzco/LIJAmsi pzco/ VN pzco/0CONO/OS0+ VN ρζε / VN ρζε / vn ρζε / asH ρζε / vn pzco/ VN pzco/ VN pzco/ VN pzco/ VN oozco/ VN Θ2θιοιιοομ€1> cocoecoloolvoaJ'cscCDs 乂p(uii(d.9jcs In丨 sQ(o//CDoe4ssnoooo5ofa)ico 0α>^κτιοο±ν.!2ρ!Ε」θραΘ snoOOOOIAJCdJSs coCNJ6lnCNJ00l-<(onQ>.yne snooooo/xi/dsco ιΛτ-·εετ"001<(ο·ζ:ωβ/π/\ίοΓ7α>7α;α. coCVJLOcolool<coc:(Dosa/on//(oec:oayopna)sCL In寸00寸1.0叱00<0〇>£7〇)50//006700/〇5/.7〇)、<0/7 l/\ncoQ)cQ)o)o^fooc:oa/ e*c:Q>is/7 ι2(οθυθσ)ο}Λοοί/οεmfjefcorj SLcoT-oolv soi/fxo e/a)/sqfa/>/ coocou 001V//00aJ/l/ocai/osUJ 63ονιΙΛοο1<//οοω5ο/ν:(ϋι/ο<οιυ 8^tocolool<co(Dc:Q)c3)owa)e-/Q)ioCDqo7(l>icUJ o>loCNJAAool\/snoc960Jods UJnlplJlsolo ooZNA>ooI\/snaJ(Dosnllocom sCSIOAooIVSIluqns snlloeGQ loCNJCNIoA ooI<cs!l!lclns snlloeg o>loOAAool\/sccalnoJ!osnllocoCQ 的 ιι(Όοθ(ο』(Λ(ουοαιο」θ< 0IOZ8 005(0//(000¾ sa)ca)o)//eo/\/ ..0^00 •M 寸c\l:SE#0#(瑯 -39- (35) (35)200421979 7.結果 7.1 P · pentosaceus Y J L and P · pentosaceus YJS,細菌 素之純化 根據超過濾膜濃縮所得細菌素液進行SDS-PAGE電 泳分析及經蛋白酶作用後均無抑菌活性,顯見Pentocins YJL和YJS其分子量爲27與25 kDa且該細菌素爲一蛋 白質 ° 此外,Pentocin YJL 和 Pentocin YJS 分另[]在 pH 4·0-8·0與pH4.0-6·0時,8(rc加熱30min仍有80%殘 存活性。Pentocin YJS 甚至 pH 4.0 and 5.0 於 100 °c 加 熱3 0 m i η後亦分別有4 1 %及 3 7 %殘存活性。p e n t 〇 c i η YJL在pH 4.0下,經100°C加熱30min後仍有34%殘存活 性。該現象顯示二細菌素可廣泛應用在作爲多種加工製程 上之天然保鮮劑。 7.2抑菌範圍
Pentocin YJL和Pentocin YJS對於食品中常見病原 菌及腐敗菌之抑菌範圍測試結果列於表九。由表中可觀察 到.Pentocin YJL 對革蘭氏陰性菌 Shigella、Ε· aerogenes、P. vulgaris 、 S· dysente门’ae、V. cholerae 及革蘭氏陽性菌 B. subtilis、B. cereus、B. circular、 L. monocytogenes、S. ep/cfe厂m/cf/s 等有明顯的抑芾((表現 。細菌素 Pentocin YJS對革蘭氏陰性菌 S/?/ge//a、Κ· oxyioca、\Λ cho/erae 和革蘭氏陽性菌 β. 、β. cereus ' B. circular 及 L· monocyiogf e/?es 等有抑菌表現 -40- (36)200421979 。顯示此二細菌素爲一廣效性細菌素。
-41 - 200421979 \]/ (37 srA.E00c9d 1「Λ uooced + + + + + + + + + + + + + + + + + + + + + + + + + + + 醒蹰觀运瘵螩画SS「A^-I「A suOoced , v« 6LOIzll· Ool±v(nl30cl(DIl5olJods UJn!PUJSoo 8ZZ πίο01νοο^δφοίο^///ο(οω KrLnzolCJos smiqns snlloem Lozzai 03±\/co!l!iqns snlloeg 6100ool<coce/no/oSA7///oeCQ e!J910eqo)c!EJoil9Jods ίηι^ο.ί2ωοφω』snoooooKCDJoo 066寸一οο1ν(ο!ρ!Ε」θρ!όθ snoooooAi/deco coCN6LOCNool-<coA7a/A7CDSA7ooooo//i/dcnJco uo寸 8寸|^〇〇:〇〇(〇〇)口〇〇)5/〇〇1/〇〇;.2-/01〇/7 slα>Θυθσ)ο}Αοουοωωμθ}.ί2η ϋ£Βα:8θυθσ)ο}Λοουοίυωμθ^π +)9 Θ(α」θιοίιοομ€ι!> εοο6ε toolv f.2>ws 00Η< coCNILOsT—ool<coc:a)osa;OA7//scocoayopA7<Dcoc/ δτ-ετ-οοιν δομχο e//0/sqa/>/ εοε uoolv //8 esoce-cusUJ 6CNJCNIU001V//8 esOA/芝 osg 8t70coT--ool<coQ)c:a)o)ayCDe Ja)%oe-Qa/cl)icUJ (ο=(οοθω』ωωυοίυο」θ< oloz8oo±vco//cnoa)^s(Dc:ao)/mo/\/ (丨)0 •Εω 9 <15sss„+..q ;EUU 9 > alfffisl·, ώ -42- (38) (38)200421979 7.3產孢菌及其孢子萌發之抑制 由上述結果發現細菌素 Pentocin YJL和 Pentocin YJS 對 3ac///i;s和C/osir/of/i/m等產孢菌有良好之抑菌 效果,因此進一步探討對 Sac///(/s ATCC 1 0225 、Β· subti/is ATCC 1 0254、S. cereus 等產孢菌之孢子 進行抑制活性試驗。由表十發現此二細菌素均具有明顯抑 制孢子萌發之效果,以細菌素Pentocin YJL而言,抑制 Bacillus subtilis ATCC 1 0225 、 B. subtilis ATCC 1 0254 、S· cere/vs產孢菌之抑菌環面積分別爲120.3、174.3、 236.3 mm2;抑制孢子萌發之抑菌環面積分別爲64.0、 96.3、189.0 mm2。以細菌素 Pentocin YJS 而言,抑制 產孢菌之抑菌環面積分別爲189.0、146.3、236.3 mm2; 抑制孢子萌發之抑菌環面積分別爲85.0、108.0、189.0 mm2。由結果得知細菌素Pentocin YJL和Pentocin YJS 均對S. cereus及其孢子萌發之抑制環面積最大,可達 236.3 > 189.0 mm2。此外,二株菌之細菌素對產孢菌之 抑菌效果較抑制孢子佳。 本發明並不限定於上述實施型態,於不脫離其要旨之 範圍內,可施以諸種改變而加以實施。 -43- 200421979 \J/ 9 (3
srAcoOI 968l·seCN oi coCDn 9S8 9681 睬餃蝱胡屮庳哞觀孃緘^画蹈_諒瘵骸画粜〇)「>-惡1「>-|321^01,+-條 (EE) s$s 觀s 1ΓΛ.εοοιιιθω. 0 681 00.9003ε·96ε·寸 ZT—0·寸9 cooCNIl· i BZZUoolvsesCD 屮蹈 S0m寸lr)CNcaoo±vco///snsCQ sCNICNIcn oolvco///icocon///os -44- (40) (40)200421979 產業上利用性 本發明提供一種新穎性乳酸菌、使用其之豆類、魚肉 食品加工方法、及使用其之丑類、魚肉加工食品。更具體 而言,本發明係提供一種篩選自肉中的新穎性乳酸菌,其 可使用於利用魚類或豆類作爲原料之發酵食品加工方法上 ,且提供由該方法所製得之可抑制雜菌生長、風味特佳、 經濟價値更高之發酵食品。本發明更提供一種由該新穎性 乳酸菌所產生的細菌素,該細菌素可有效地抑制其他雜菌 於食品中的生長,進而確保保存期間食品的品質。 【圖式簡單說明】 圖一表示乳酸四連球菌之鑑定流程。
圖二表示純化 Pentocins YJL and YJS 之 SDS-PAGE (8〜1 5 %聚丙烯胺)。 圖三表示發酵發酵魚肉起司之實例。 圖四表示發酵魚肉優格(yogurt)。 圖五表示發酵黃豆布丁之實例。
Claims (1)
- (1) (1)200421979 拾、申請專利範園 1. 一 種寄存號碼爲 BCRC910210 之 Ped/ococcas pentosaceus 丫JL 〇 2· — 種寄存號碼爲 BCRC910211 之 Peci/ococci/s pentosaceus Y J S。 3. —種魚肉加工方法,其特徵爲使用如申請專利範 圍第彳項或第2項之乳酸菌、或如申請專利範圍第1項或 第2項之混合乳酸菌、或其他乳酸菌之混合乳酸菌進行魚 肉發酵者;其加工方法爲:魚肉加 0.3〜2.0 %食鹽及 〇 . 5〜3倍水後均質之,經1 〇 〇〜1彳5 °c殺菌1 5〜3 0分鐘後 冷卻至25〜4CTC,調整基質水分含量(由不稀釋〜五倍稀 釋)及添加1.0〜6_0%之糖後接種乳酸菌,在25〜4(TC發 酵6〜30小時後可任意添加適當量之調味劑及香辛料或可 包裝之。 4 ·如申請專利範圍第3項之加工方法,其中其他乳 酸菌的混合乳酸菌爲 1種以上選自 Laciobac/7/t/s p I a n ta r u m CCRC1 0069, Lactococcu s I act is subsp. /aci/s CCRC 12315, Lactobacillus helveticus CCRC 14092 者。 5·如申請專利範圍第3項之魚肉加工方法,其中魚 肉原料爲至少一種選自於紅色肉、白色肉魚、其混合魚肉 、冷凍魚漿等者。 6·如申請專利範圍第3項之魚肉加工方法,其中糖 原料爲至少一種選自於蔗糖、葡萄糖、甜菜等。 -46- (2) (2)200421979 7 ·如申請專利範圍第3項之魚肉加工方法其中魚 肉基質可爲不稀釋〜五倍稀釋之魚肉漿。 8.如申請專利範圍第3項之魚肉加工方法其中嗎 味劑爲至少一種選自於一般之水果、加工之水果醬、芝麻 、花生等。 9 ·如申請專利範圍第3項之魚肉加工方法,其中香 辛料爲至少一種選自於一般之薑、蒜、味淋、酒、五香粉 等。 10·如申請專利範圍第3項之魚肉加工方法,其中發 酵後基質之pH在3.8〜5.5。 1 1 · 一種魚肉加工食品,其特徵爲使用如申請專利範 圍第1項或第2項之乳酸菌、或如申請專利範圍第1項或 第2項之混合乳酸菌、或其他乳酸菌之混合乳酸菌進行魚 肉原料發酵所得者。 1 2 ·如申請專利範圍第1彳項之加工方法,其中其他 乳酸菌的混合乳酸菌爲1種以上選自Lacio0ac///iys plantarum CCRC1 0069, Lactococcus lactis subsp. lactis CCRC 12315, Lactobacillus helveticus CCRC 14092 者 。 1 3 · —種魚肉加工食品,其特徵爲利用如申請專利範 圍第3項之魚肉加工方法進行魚肉原料發酵所得者。 1 4· 一種魚肉加工食品,其特徵爲利用如申請專利範 圍第3項之魚肉加工方法進行魚肉原料發酵後在9〇〜 1 15QC加熱殺菌、成型、部分乾燥所得之類似起司產品者 -47- (3) (3)200421979 1 5.如申請專利範圍第1 1〜1 4項中任一項之魚肉加工 食品,其中魚肉原料爲至少一種選自紅色肉、白色肉魚、 其混合魚肉或冷凍魚漿等。 1 6· —種豆類加工方法,其特徵爲使用如申請專利範 圍第1項或第2項之乳酸菌、或如申請專利範圍第彳項或 第2項之混合乳酸菌、或其他乳酸菌之混合乳酸菌進行豆 類發酵者;其加工方法爲:經浸泡豆類加水後均質、過濾 後在經100〜115°C加熱殺菌15〜30分鐘後冷卻至25〜40 °C,調整基質水分含量(50%〜98%)及添加1.0〜6.0%之 糖後接種乳酸菌,在25〜4CTC發酵6〜30小時後任意添 加適當量之調味劑或可包裝之。 1 7.如申請專利範圍第1 6項之加工方法,其中其他 乳酸菌的混合乳酸菌爲1種以上選自Lacio6ac///us plantarum CCRC1 0069, Lactococcus lactis subsp. /acf/s CCRC 12 315, Lactobacillus helveticus CCRC 14092 者。 1 8.如申請專利範圍第1 6項之豆類加工方法,其中豆 類原料爲至少一種選自黃豆或黑豆。 19·如申請專利範圍第16項之豆類加工方法,其中糖 原料爲至少一種選自蔗糖、葡萄糖、甜菜等。 2 0_如申請專利範圍第16項之豆類加工方法,其中豆 類發酵基質之水分含量爲50 %〜98%。 2 1 .如申請專利範圍第1 6項之豆類加工方法,其中調 -48- (4) (4)200421979 味劑爲至少一種選自一般之水果、加工之水果醬等。 2 2 .如申請專利範圍第彳6項之豆類加工方法,其中發 酵後豆類基質之pH在4.5〜6.0。 23· —種豆類加工食品,其特徵爲使用如申請專利範 圍第1項或第2項之乳酸菌、或如申請專利範圍第1項或 第2項之混合乳酸菌、或其他乳酸菌之混合乳酸菌進行豆 類原料發酵所得者。 2 4 ·如申請專利範圍第2 3項之加工方法,其中其他 乳酸菌的混合乳酸菌爲1種以上選自Lacio6ac/7/i/s p I a n ta r u m CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315, Lactobacillus helveticus CCRC 14092 者 。 25. —種豆類加工食品,其特徵爲利用如申請專利範 圍第1 4項之豆類加工方法進行豆類原料發酵所得者。 26. 如申請專利範圍第23項〜第25項中任一項之豆 類加工食品,其中豆類爲至少一種選自於黃豆、黑豆、或 其混合物者。 27. —種豆類加工食品,其特徵爲利用如申請專利範 圍第3項之豆類加工方法進行豆類原料發酵後在90〜115 °C加熱殺菌、成型、部分乾燥所得之類似起司產品者。 28. —種細菌素,其特徵爲來自如申請專利範圍第1 項之乳酸菌。 29. 如申請專利範圍第28項之細菌素,其爲分子量爲 20〜30 kDa的抑菌性物質。 -49- 200421979 (5) 30. —種細菌素,其特徵爲來自如申請專利範圍第2 項之的乳酸菌。 3 1 .如申請專利範圍第30項之細菌素,其爲分子量爲 2 0〜3 0 k D a的抑菌性物質。
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TWI243646B TWI243646B (en) | 2005-11-21 |
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US (1) | US20040208978A1 (zh) |
JP (1) | JP2004313171A (zh) |
TW (1) | TWI243646B (zh) |
Families Citing this family (10)
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JP4243442B2 (ja) * | 2001-08-06 | 2009-03-25 | 日本水産株式会社 | 水産発酵食品の製造方法 |
EP1716258B1 (en) * | 2004-04-15 | 2010-03-31 | Chr. Hansen A/S | Method for reducing the content of pathogenic organisms present in food materials |
EP1676489A3 (en) * | 2004-12-16 | 2006-07-12 | Shonan Pure Co. Ltd. | Method of producing food and food produced by the method |
KR101109746B1 (ko) | 2009-11-13 | 2012-03-13 | 충북대학교 산학협력단 | 균체외 다당을 생성하고 건강기능성 효과를 갖는 신규한 페디오코커스 펜토사세우스 |
TWI433651B (zh) * | 2011-04-28 | 2014-04-11 | Univ Nat Penghu | 新穎乳酸菌株、含彼之組合物及其用途 |
US20140271994A1 (en) * | 2013-03-15 | 2014-09-18 | Richard Baird Smittle | Meat slurry culture |
JP7032619B2 (ja) * | 2016-11-14 | 2022-03-09 | 信州中野商工会議所 | バチルス属細菌の増殖を抑制する抗菌性乳酸菌株、及び、それを用いた食品の製造方法 |
CN110093289B (zh) * | 2019-05-05 | 2020-12-04 | 西南大学 | 一株乳酸片球菌及其应用 |
CN110982745B (zh) * | 2019-12-24 | 2021-06-25 | 扬州大学 | 一种戊糖片球菌z-1、戊糖片球菌细菌素z-1及戊糖片球菌细菌素z-1的生产方法 |
CN113699091B (zh) * | 2021-10-27 | 2022-02-01 | 北京大北农科技集团股份有限公司 | 一种重组枯草芽孢杆菌及其构建方法和应用 |
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2003
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- 2003-08-26 JP JP2003301771A patent/JP2004313171A/ja active Pending
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TWI243646B (en) | 2005-11-21 |
US20040208978A1 (en) | 2004-10-21 |
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