201242517 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種新穎乳酸菌株，特別是指一種可以降低發光桿菌 (洲伽办州從⑹)生長且可提升海鱺對發光桿菌抵抗力之乳酸菌 株，以及含有該乳酸菌株之組合物及該組合物之用途。 【先前技術】 隨著全球人口增加，人類對於糧食的需求也日與俱增；在捕魚業漁獲 量曰漸減少的情況下，養殖漁業成為重要的肉品供應來源之一。海鱺 办m)是一種熱帶海洋魚類，也是海鱺科(办 唯-的魚種。目為親職境的高騎度以及其快速的生長率，海罐成為 理想的海水箱網養殖魚種。台灣海鱺養殖業始於199〇年代初期；於1997 年人工育種成功後，此-高經濟價值的魚種便廣泛地被養瘦於海水箱網中 (Liao et al., 2004) ° 儘管海鱺產量逐年增加，包含發光桿菌（或稱巴斯德桿菌） subsp·冲浼洳，坤)與弧菌(vibri〇)等細菌性病原 菌已對海嫌魚苗造成嚴重的疫情。其中以發光桿菌ρφ對海鱺造成最嚴重 的威脅’死亡率也最高。發光桿菌坤為革蘭氏陰性菌，會造成感染魚隻 的内臟產生白色的結節’並且經由受感染的吞伽胞散佈傳染。到目前為 止’海嫌養殖業者對抗發光桿菌Ρφ最主要的方法是使用抗生素 ;然而， 有越來越多對抗生素產生抗性的發光桿發現，而且使用抗生素也會有 藥物殘留等食全的_。另—方面，由於賴的免疫記憶力(immune memory)不像多其他的魚種(如：石斑魚)那麼有效，因此對海鱺接種發光 桿菌疫苗的效果並不穩定。 由此可見’上述習用針對海罐抵抗發光桿菌感染的方法仍有諸多缺 201242517 失，實非一良善之設計者，而亟待加以改良。 在許多研究中’乳3线〇伽aeid baeteria，LAB)已被使用作為益生菌 (probiotics)。乳酸菌產生的有機酸與細菌素(bacteri〇dn准夠抑制或直接殺死 許多微生物(ErC〇lani et al.，1976; Mathur et al，2〇〇5)，且乳酸菌抗酸及抗膽 汁的能力可以提高該菌在動物腸胃道的存活率(Iri_ et d，纖）。此外， 前人研究顯示’約有m至的腸道_具有成為益生菌的潛力(Sugitaet al., 2002) » 是以’本案發明人鑑於上述習用針對海鱺抵抗發光桿菌感染的方法所 衍生的各項親’以及乳義作為益生_可能性，乃亟思加贿良創新， 並經多年苦錄麟,㈣究後’終於成功研發完成本件新輒義株 '含 彼之組合物及其用途。 【發明内容】 本說明書中所述之所有技術性及科學術語，除非另外有所定義，皆為 該所屬領域具有通常技藝者可共同瞭解的意義。 本發明之目的即在於提供-種新穎乳酸菌株·乳酸戊糖片球菌 ’該菌株係自飢餓三天後海鱺的腸道篩選而來。 本發明之次一目的係在於提供一種含有新穎乳酸菌株_乳酸戊糖片球菌 之組合物，該組合物具有抑制發光桿菌 而_/ae)等病原菌生長，以及提升海鱺免疫力的效果。 本發明之另一目的係在於提供一種含有新穎乳酸菌株-乳酸戊糖片球菌 (/^•〇咖〇^邮0娜咖)之組合物的應用，該組合物可作為魚飼料、房疫苗 之添加物，以增進魚類抵抗發光桿菌等病原菌之能力。 可達成上述發明目的之新穎乳酸菌株、含彼之組合物及其用途，包括 有.一種新穎乳酸戍糖片球菌株，或該菌株的繼 代培養後代；該菌株係篩選自海鱺腸道樣品，並經過耐酸性、耐膽鹽性篩 201242517 選，以及餵食海鱺後進行攻毒試驗所得到可以幫助海鱺抵抗發光桿菌Pdp 感染的乳酸菌株。 該新顆乳酸戊糖片球菌株(P pertiosacew·?)已寄存於新竹食品工業發展 研究所生物資源保存及研究中心，寄存編號為BCRC910480的菌株，寄存 曰期為2010年7月22曰。 將本發明所篩選到之乳酸戊糖片球菌(户40Π菌株培養懸 浮液與發光桿菌Ρφ —起培養，結果顯示，本發明之新穎乳酸戊糖片球菌 4012菌株可以抑制病原菌發光桿菌Ρφ之生長。 將本發明之乳酸戊糖片球菌(/! pewtoyacews) 4012菌株與市售魚飼料混 合以餵食海鱺，結果顯示本發明乳酸戊糖片球菌(p p⑼沿）4012菌株 可明顯促進海鱺生長率；搭配接種發光桿菌疫苗後，可以增加海鱺對發光 桿菌Pdp感染的抵抗力，其相對存活率顯著高於只有接種疫苗的魚隻以及 對照組。 本發明並進一步提供含有本發明之乳酸戊糖片球菌(户 4012菌株之組合物，該組合物係用於抑制發光桿菌之生長；該組合物 係可製成動物飼料添加物或動物用醫療組合物等形式。 其中該組合物可進一步包含一藥學上可接受之載劑，該載劑包含但不 限於.溶劑(solvent)、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑 (decomposer)、黏結劑(binding agent)、賦形劑(excipient)、安定劑(stabilizing agent)、螯合劑(chelating agent)、稀釋劑(diluent)、膠凝劑(gelling agent)、防 腐劑(preservative)、潤滑劑(lubricant)、界面活性劑(surfactant)、佐劑 (adjuvant)，及其他類似或適用本發明之載劑。 本發明另提供一增強魚類對抗發光桿菌的免疫力之方法，包含：施予 一有效量之乳酸戊糖片球菌CP ⑼tosaceus) 4012菌株、或含彼之組合物， 及一發光桿菌疫苗予一魚類，即可增強其對抗發光桿菌之免疫力；其中該 乳酸戊糖片球菌CR 4012菌株可進一步混合於一飼料或動物用 201242517 醫療組合物中餵食魚類，並搭配該發光桿菌疫苗接種，以提高其對抗發光 桿菌之免疫力。 另’本發明亦提供前述乳酸戍糖片球菌(P /pewtosacew·?) 4012菌株用於 製備保護魚類免於感染發光桿菌的方法或用途。 本發明所述之新穎乳酸戊糖片球菌4012菌株，亦應包 含其繼代培養之後代，或突變株，但仍具有與本發明所述之菌種特性、基 因體(genomic)、或功能性相同者。 術語“有效量”意謂，於個體之用量，該用量可有效增進、改善該個體之 免疫力以對抗發光桿菌，進而預防及保護該個體免於感染發光桿菌及其衍 生之相關疾病。 術語“預防、保護，，意謂，相較於未使用本發明之疫苗者，使用本發明之 疫苗者，將可有效增強其免疫力以對抗發光桿菌，增加其存活率，進而預 防及保護其免於發光桿菌引起的疾病及其衍生之相關疾病。 【實施方式】 本發明係以下面的實施例予以示範闡明，但本發明不受下述實施例所 限制。 實施例一海鱺腸道乳酸菌株之篩選與分離 1·海鱺腸道乳酸菌篩選 收集域三錢的_腸舰品，於概__ 棘基(職a 69966)中，在37°C厭氧環境下培養(De Man心觸）。將分離出的乳酸 菌在體外初步細魏性及_鹽測試，以及_性試驗後，勒三株乳 酸菌株。其中，代號為伽in4G12的乳_株對胃酸與膽鹽的耐受度最高， 因此被挑選作為餵食海鱺的試驗材料。 201242517 2.以16S rDNA定序鑑定海鱺腸道乳酸菌株 以偷臟定序對_選出之乳酸菌進行蘭種鑑定，以下列引子對進 行 PCR 反應(Weisburg et al.，1991)。 正向引子： 5，-agagtttgatcatggctcag-3’ (SEQIDNo.: 1) 反向引子： 5，-aaggaggtgatccagcc-31 (SEQ ID No.: 2) 以 PCR System 2700 (Applied Biosystems 公司）進行 pcR 反應，pcR 產 物以Am 3730 (AppUed Bi〇systems公司）進行定序，接著以Ncm網站上 BLAST功能進行序列比對，根據比對結果，侧菌株屬於乳酸戊糖片球菌 {Pediococcus pentosaceus) ° 實施例二海鱺腸道乳酸菌株抗發光桿菌Ρφ之效果 1.乳酸菌株4012抑制發光桿菌Ρφ的生長 取實施例-分離得到的乳義株4〇12的代謝產物來測試抑制發光桿菌 户Φ的效果。發光桿i Ρφ菌株Ρ40係由國立彩湖大學古鎮釣教授所提供， 於37 C下’以MRS液體培養基培養至吸光值〇〇6。。為2，然後以5,〇〇〇xgr 離心15分鐘後，收集上清液(pH值為將收集的上清液加入Bffi液體培 養基中(Becton，Dickinson and Company) ’ BHI培養基與上清液的混合比例 為4··1，並紀錄發光桿菌户办在此一混合培養基内的生長曲線。負對照組係 以ΒΗΙ液體培養基混合MRS液體培養基(pH值為6_2)培養發光桿菌户φ，201242517 VI. Description of the Invention: [Technical Field] The present invention relates to a novel lactic acid strain, in particular to a method for reducing the growth of luminescent bacillus (from the genus (6)) and improving the resistance of sea lice to luminescent bacteria. A lactic acid strain, and a composition comprising the lactic acid strain and use of the composition. [Prior Art] With the increase of the global population, the demand for food has also increased day by day; in the case of a gradual decline in fishing catches, aquaculture has become an important source of meat supply. Haitang Office m) is a tropical marine fish, also a species of sea bream (a unique species). The high degree of riding in the pro-employment and its rapid growth rate, sea cans become the ideal sea-bowl cultured species. Taiwan's seabream farming began in the early 1990s; after artificial breeding in 1997, this high-value fish species was widely thinned in the seawater tank network (Liao et al., 2004) ° The production of sea bream has increased year by year, including bacterial pathogens such as luminescent bacillus (or P. pastoris), subsp., Kun, and vibri〇, which have caused serious epidemics to sea squid. Among them, the luminescent bacterium ρφ poses the most serious threat to sea otters, and the mortality rate is also the highest. Photobacterium bacillus is a Gram-negative bacterium that causes the viscera of infected fish to produce white nodules' and is transmitted through infected swallow gamma cells. So far, the most important method for anti-bacteria 对抗 Ρ φ is to use antibiotics; however, more and more luminescent rods that are resistant to antibiotics have been found, and antibiotics can also have drug residues and other foods. . On the other hand, since the immune memory of Lai is not as effective as many other species (such as grouper), the effect of inoculation of the sputum vaccine on sea lice is not stable. It can be seen that the above-mentioned methods for the anti-photobacterium infection of sea cans still have many defects, which are not a good designer, but need to be improved. In many studies, 'daily 3 line sang alae baeteria, LAB' has been used as probiotics. The organic acids and bacteriocins produced by lactic acid bacteria (bacteri〇dn are sufficient to inhibit or directly kill many microorganisms (ErC〇lani et al., 1976; Mathur et al, 2〇〇5), and the ability of lactic acid bacteria to resist acid and anti-biliary The survival rate of the bacterium in the gastrointestinal tract of the animal (Iri_ et d, fiber) can be increased. In addition, previous studies have shown that 'about the intestinal tract of m to have the potential to become a probiotic (Sugita et al., 2002) » In view of the above-mentioned practices, the inventors of the present invention used the various methods derived from the method of resisting luminescent bacillus infection by sea bream and the possibility of yin as a probiotic _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 'Finally succeeded in the development and completion of this new 辄 株 ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' The aim of the present invention is to provide a novel lactic acid strain, Pediococcus lactici, which is screened from the intestinal tract of sea lice three days after starvation. The second object of the present invention system To provide a novel lactic acid bacteria strain containing lactic _ pentosaceus the composition, the composition has a growth inhibition and Photobacterium _ / ae) and other pathogens, the immune system and enhance the effect of cobia. Another object of the present invention is to provide a composition comprising a novel lactic acid strain, Pediococcus pentreatus (/^•〇咖〇^邮0娜咖), which can be used as a fish feed or a house vaccine. Additives to enhance the ability of fish to resist pathogenic bacteria such as luminescent bacilli. A novel lactic acid strain, a composition comprising the same, and a use thereof, comprising: a novel lactobacillus sphaeroides, or a subcultured progeny of the strain; the strain is screened from a jellyfish intestinal sample And the acid-resistant and bile-salt-resistant sieve 201242517, and the challenge test after feeding the sea otter, can obtain the lactic acid strain which can help the sea lice resist Pdp infection of the luminescent bacterium. The new lactic acid pentosaceus strain (P pertiosacew·?) has been deposited in the Bioresource Conservation and Research Center of the Hsinchu Food Industry Development Research Institute, and the strain numbered BCRC910480 was deposited. The storage period was July 22, 2010. The cultured suspension of Pediococcus lactis (the 40 Π strain culture suspension of the present invention) was cultured with the luminescent bacterium φ φ, and the results showed that the novel lactic acid pentosaceus 4012 strain of the present invention can inhibit the growth of the pathogenic bacterium γ. The Pseudomonas pentoxide (/! pewtoyacews) 4012 strain of the present invention was mixed with a commercially available fish feed to feed sea otters, and the results showed that the P. pentosus lactic acid (p p(9) along) 4012 strain of the present invention can significantly promote the growth rate of sea bream. With the vaccination with luminescent bacillus, the resistance of sea bream to Pdp infection of luminescent bacillus can be increased, and the relative survival rate is significantly higher than that of the vaccinated fish and the control group. The present invention further provides pentose gluconate containing the present invention. a composition of Pediococcus (a 4012 strain for inhibiting the growth of luminescent bacillus; the composition may be in the form of an animal feed supplement or a medical composition for animals, etc. wherein the composition may further comprise a A pharmaceutically acceptable carrier comprising, but not limited to, a solvent, an emulsifier, a suspending ag Ent), decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent Preservatives, lubricants, surfactants, adjuvants, and other carriers similar or suitable for use in the present invention. The present invention further provides an enhancer immunity against luminescent bacterium The method comprises the steps of: administering an effective amount of Pediococcus pentreatus CP (9) tosaceus 4012 strain, or a composition comprising the same, and a luminescent vaccine to a fish to enhance immunity against the luminescent bacterium; The Pseudomonas pentose CR 4012 strain can be further mixed with a feed or animal for feeding fish with the 201242517 medical composition, and vaccinated with the luminescent bacterium to increase its immunity against the luminescent bacterium. Further, the present invention also provides The aforementioned P. lactis (P / pewtosacew??) 4012 strain is used for preparing a method or use for protecting fish from infection with luminescent bacterium. The novel milk of the present invention The Pediococcus pentosace 4012 strain should also contain its subcultured progeny, or mutant strain, but still have the same characteristics, genomic, or functional properties as described in the present invention. "This means that, in the amount of the individual, the amount can effectively enhance and improve the immunity of the individual against the luminescent bacterium, thereby preventing and protecting the individual from infection with the luminescent bacterium and related diseases thereof. The term "preventing, protecting, meaning that, compared to a vaccine not using the present invention, the vaccine of the present invention can effectively enhance its immunity against luminescent bacilli, increase its survival rate, and thereby prevent and protect it. The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. Example 1 kelp intestinal lactic acid bacteria Screening and Isolation of the Plants 1. The lactic acid bacteria in the intestines of the sea bream was collected and collected from the ternary snails of the genus Sankyo, in the __ thorn base (a 69966), cultured at 37 ° C in an anaerobic environment (De Man heart touch The isolated lactic acid bacteria were inferior in vitro and _ salt test, and after the _ sex test, three strains of lactic acid were strained. Among them, the milk strain with gamma 4G12 was the most tolerant to gastric acid and bile salts. Therefore, it was selected as a test material for feeding sea lice. 201242517 2. Identification of lactic acid bacteria of sea bream by 16S rDNA sequencing to identify the lactic acid bacteria selected by smuggling, and PCR reaction was carried out with the following primer pairs ( Weisburg e t al., 1991). Forward primer: 5,-agagtttgatcatggctcag-3' (SEQIDNo.: 1) Reverse primer: 5,-aaggaggtgatccagcc-31 (SEQ ID No.: 2) with PCR System 2700 (Applied Biosystems) The pcR reaction was carried out, and the pcR product was sequenced by Am 3730 (AppUed Bi〇systems), followed by sequence alignment using the BLAST function on the Ncm website. According to the comparison result, the side strain belongs to Pediococcus pentosaceus {Pediococcus pentosaceus). ° Example 2 Effect of lactic acid strain of sea bream on anti-photobacterium Ρ φ 1. Growth of lactic acid strain 4012 inhibits growth of luminescent bacillus φ φ The metabolite of the isolated sputum strain 4 〇 12 was tested to inhibit luminescent bacilli Φ The effect of the illuminating rod i Ρφ strain Ρ40 is provided by the professor of Guzheng Fishing, National Color Lake University, and cultured at 37 C in MRS liquid medium to absorbance 〇〇6. 2, then 5, 〇〇〇xgr After centrifugation for 15 minutes, the supernatant was collected (pH value was added to the Bffi liquid medium (Becton, Dickinson and Company). The mixing ratio of the BHI medium and the supernatant was 4··1, and the record was issued. Growth curve of Bacillus households do in this mixed medium. ΒΗΙ negative control line to mix the liquid medium MRS liquid medium (pH value 6_2) culturing Photobacterium households φ,
而正對照組則是以BHI液體培養基混合調整過pH值的MRS液體培養基(pH 值為5)來培養發光桿菌户Φ ;兩個對照組的培養基混合比例皆為4:1。在此 試驗中’分別將〇.lml的發光桿菌户φ菌液(吸光值〇£>6〇〇為加入—各 組培養基中，於28。(：下培養，並以吸光值〇D600測量發光桿菌Ρφ的生長 曲線。 201242517 發光桿菌Pdp的生長曲線如圖一所示。有添加乳酸菌株4〇12培養上清 液之Ρφ生長曲線遠低於沒有添加乳酸菌培養上清液的兩個負對照組。 實施例二乳酸戊糖片球菌(户菌株4〇〗2餵食海鱺後對海罐免 疫力提升的效果分析 隨機將魚隻(體重為4.6 g)分為餵食乳酸菌組與對照組，每組含有17隻 魚。乳酸菌餵食劑量為l〇9CFU/g，每曰餵食量為魚隻體重的5%，共餵食^ 週。傲食期間結束後，每組選出2隻魚來採血以進行分析；接著進行攻毒 試驗。 ’ 餵食期間過後，所有的魚隻都以浸泡方式進行攻毒試驗，於2〇()c下將 魚隻浸泡在含有2xl01 2 3 CFU/ml發光桿菌的海水中2分鐘。收集死亡魚隻並 分離腎臟與脾臟中的微生物樣本以確認死因，並計算攻毒後1〇天内魚隻的 累積死亡率》 ά 1. 海鱺體重增長率的分析 比較餵食乳酸菌14天後的試驗組海總與對照組海鱺的體重，結果如圖 二所示。傲食乳酸菌搬的海鱺體重有明顯增長，該結果顯*，餵食乳酸 菌株4012可以提高魚隻12%的生長率。 8 1 海嫌呑噬細胞瓦解病原體能力的分析 根據Choudhmy等人於細年提出的方法，以硝基藍四氮哇⑽ tetrax〇Hum，NBT)分析法測定血液中吞嗔細胞瓦解病原菌的能力，又稱為呼 吸爆(respiratory burst，RB)。簡言之，取96孔盤，每孔加入5叫血液樣本， 2 並於37Τ下培養1小時，使吞_胞_於盤上。接著，去除上清液，並 以PBS清洗二次後，加入50μ1 2。/〇確基藍四兔0坐⑽τ)，於37〇c下再培養 1小時。之後加入50μ1 100%曱醇作用3分鐘，並以3〇%甲醇清洗三次。待 3 96孔盤風乾後，每孔加入_ 2Ν氫氧化钟㈣assium hydr〇xide)以及_ 201242517 二曱基亞颯(dimethyl sulphoxide，DMSO)，待所有試劑反應後，以盤式酵素 免疫分析儀(ELISA reader，MRX II，Dynex Techn〇i〇gies 公司)讀取吸光值 OD490的讀值》 海嫌吞嗔細胞瓦解病原體能力的分析結果如圖三所^結果顯示假食 乳酸菌的海鱺呑噬細胞瓦解病原體的能力明顯高於對照組(?<〇 〇5)。 3.攻毒試驗結果 進行攻毒試驗後，海纖的死亡率如圖四所示。餵食乳酸菌(試驗組)的海 鱺相對存活率(RPS)為74.3。所有死亡的魚隻確定死因皆為感染發光桿菌 βφ所引起，餵食較對照組高出60%。 實施例四接種死毒疫苗配合餵食乳酸戊糖片球菌(jR 菌 株4012對海鱺免疫力提升的效果分析 隨機將魚隻(體重為35g)分為3組(接種疫苗組、接種疫苗加傲食乳酸菌 組、無接種疫苗無餵食乳酸菌之對照組）。每組含有12隻魚。所有的試驗皆 為二重複’攻毒試驗方式與實施例三相同，但攻毒劑量改為1 5xl〇5 CFu/mi。 除了對照組魚隻外，所有魚隻於試驗開始時皆進行户φ死毒疫苗初次 免疫(primary vaccination)，並於三週後進行第二次追加免疫》初次免疫及追 加免疫的免疫劑量為0.333 mg發光桿菌户命/魚體重(g)。 攻毒試驗結果如圖五所示，只接種疫苗沒有飯食乳酸菌株4012的海 鱺’其相對存活率(RPS)為59 ;配合餵食乳酸菌株4012與接種疫苗的海鱺 死亡率最低(如圖五所示），則存活率(RPS)提高為95.5。 本發明所提供之新穎乳酸菌株⑼tosacewj)、含彼之組合物 及其用途’與其他習用技術相互比較時，更具有下列之優點： 1·本發明所提供之乳酸戊糖片球菌株4012 ⑽strain 4012)係 201242517 自海鱺腸道分離而來，該菌具有耐酸性及耐膽鹽等特性，製成口服劑型餵 食魚隻後，可存活在魚隻腸胃道内。 2. 餵食本發明所提供之乳酸戊糖片球菌株4012 〇R strain 4012)的魚隻，在同一生長時間内，其體重較未餵食該菌株之魚隻重；可見 本發明之菌株可增加魚隻的生長率。In the positive control group, the MRS liquid medium (pH 5) adjusted by pH-mixed BHI liquid medium was used to culture the luminescent bacteria Φ; the mixing ratio of the two control groups was 4:1. In this test, '1 ml of luminescent bacteria φ bacterium solution (absorbance value &£>6 〇〇 was added to each group of medium, at 28. (under: culture, and measured by absorbance 〇D600) Growth curve of luminescent bacillus φ φ 201242517 The growth curve of luminescent bacterium Pdp is shown in Figure 1. The growth curve of Ρ φ with the culture supernatant of lactic acid strain 4〇12 was much lower than that of the two negative control without lactic acid culture supernatant. Example 2. Analysis of the effect of Pediococcus lactis (family strain 4〇) 2 on the immunity of sea cans after feeding sea lice. The fish (body weight 4.6 g) was randomly divided into feeding lactic acid bacteria group and control group, each The group contains 17 fish. The feeding dose of lactic acid bacteria is l〇9CFU/g, and the feeding amount per carp is 5% of the body weight of the fish, which is fed for 2 weeks. After the end of the arrogant period, 2 fish are selected for each group to collect blood for analysis. Then, the challenge test was carried out. 'After the feeding period, all the fish were tested for soaking in the soaking method. The fish was immersed in seawater containing 2xl01 2 3 CFU/ml of Photobacterium at 2〇()c. Minutes. Collect dead fish and separate kidney and spleen Microbial samples in order to confirm the cause of death and calculate the cumulative mortality of fish within 1 day after challenge. ά 1. Analysis of the growth rate of jellyfish weight compared with the control group of 14 days after feeding lactic acid bacteria and the control group Body weight, the results are shown in Figure 2. The weight of the sea otter moved by the lactic acid bacteria has increased significantly, and the result is *, feeding the lactic acid strain 4012 can increase the growth rate of the fish by 12%. 8 1 The suspicion of the phlegm cells to disintegrate the pathogen According to the method proposed by Choudhmy et al. in the fine years, the ability of phagocytic cells to disintegrate pathogens in blood is determined by nitroblue tetrazolium (10) tetrax〇Hum, NBT) analysis, also known as respiratory burst (RB). In short, take a 96-well plate, add 5 blood samples per well, 2 and incubate for 1 hour at 37 , to make the cells _ on the plate. Then, remove the supernatant and wash it twice with PBS. Thereafter, 50 μl 2 / 〇 基 蓝 四 四 兔 兔 坐 坐 坐 坐 10 10 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 加入 加入 加入 50 50 50 After the 96-well plate is air-dried, add _ 2 Ν Ν (each) to each well. Ium hydr〇xide) and _ 201242517 dimethyl sulphoxide (DMSO), after all reagents were reacted, read by ELISA reader (MRX II, Dynex Techn〇i〇gies) The reading value of the absorbance value OD490" The results of the analysis of the ability of the swallowed cells to disintegrate the pathogens are shown in Fig. 3. The results show that the ability of the sea cucumber cells of the fake lactic acid bacteria to disintegrate the pathogen is significantly higher than that of the control group (?<〇〇5 ). 3. Results of the challenge test After the challenge test, the mortality rate of the sea fiber is shown in Figure 4. The relative survival rate (RPS) of the larvae fed to the lactic acid bacteria (test group) was 74.3. All the dead fish were determined to be caused by infection with photoreceptor βφ, which was 60% higher than the control group. Example 4 Inoculation of a deadly vaccination vaccine with feeding of Pediococcus lactis (the effect of jR strain 4012 on the immunity of sea otters) The fish (weight 35 g) were randomly divided into 3 groups (vaccination group, vaccination plus arrogance) Lactic acid bacteria group, no vaccination, no control group for feeding lactic acid bacteria. Each group contains 12 fish. All tests are two repeated 'attack test methods are the same as in the third example, but the attack dose is changed to 1 5xl〇5 CFu /mi. Except for the control fish, all fish were initially vaccinated at the beginning of the trial and a second boost after three weeks. Initial immunization and immunization immunization The dose is 0.333 mg of luminescent bacterium life/fish weight (g). The results of the challenge test are shown in Figure 5. The vaccination is not vaccinated with lactic acid strain 4012. The relative survival rate (RPS) is 59; with the feeding of lactic acid bacteria The strain 4012 and the vaccinated sea otter have the lowest mortality (as shown in Figure 5), and the survival rate (RPS) is increased to 95.5. The novel lactic acid strain (9) tosacewj), the composition containing the same and the use thereof are provided by the present invention. When the conventional techniques are compared with each other, the following advantages are obtained: 1. The lactic acid pentosaceus strain 4012 (10) strain 4012) provided by the present invention is isolated from the intestinal tract of the sea bream, and the bacterium has acid resistance and bile salt resistance. Characteristics, after being prepared as an oral dosage form, can survive in the gastrointestinal tract of fish. 2. Feeding the fish of the present invention, the strain of C. glutamate 4012 〇R strain 4012), the body weight is heavier than that of the fish not fed the same strain during the same growth time; it can be seen that the strain of the present invention can increase the fish Only the growth rate.
3. 本發明所提供之礼酸戊糖片球菌株4012 (/! pewiosaceMi strain 4012)的 培養懸浮液可以抑制發光桿菌Pdp的生長速度；而且以發光桿菌pdp攻毒 後’餵食該菌株的魚隻的相對存活率較對照組高出60。/〇 ;此外，餵食本發 明菌株的魚隻’其呑嗤細胞瓦解病原體的能力(reSpirat〇ry burst)明顯比對照 組的吞噬細胞來的高；是以，本發明之乳酸戊糖片球議株4〇12 (A strain4012)可作為益生菌，幫助魚隻抵抗發光桿菌。 4. 本發明所提供之含乳酸戊糖片球菌株4〇12的飼料，經與户办死毒疫 苗搭配使職，可㈣賴或其他魚騎抗發光㈣的免魏力，且其效 果相較於單獨使用疫苗者，餵食該飼料及搭配疫苗接種者，其相對存活率 大幅提高。 上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例 並非用以關本發明之專概圍，凡未脫離本發明技藝精神所為之等效實 施或變更，均應包含於本案之專利範圍中。 综上所述’本案所提供之乳酸菌株不但為—新賴菌株，並具有上述多 項功效，應已充分符合新酿及進步性之法定發明專利要件，爰依法提出 申晴’ 貴局核准本件發明專利㈣案，以勵發明，至感德便。 201242517 【圖式簡單說明】 圖一、為海鱺腸道乳酸菌株抑制發光桿菌户办生長的曲線圖；取乳酸戊糖 片球菌(戶⑽)菌株4012培養懸浮液加入發光桿菌ρφ生長 培養基中培養該菌，並觀察發光桿菌Ρφ的生長量；負對照組係以 ΒΗΙ液體培養基混合MRS液體培養基(pH值為6.2)培養發光桿菌 Ρφ ’而正對照組則是以Bm液體培養基混合調整過pH值的遠3 液體培養基(pH值為5)培養發光桿菌;吸光值〇D6gq代表發光桿 菌户办的生長量。 圖二、為比較傲食乳酸菌株(4012)14天後的試驗組海鱺與對照組海鱺的體 重，圖二A為海鱺體重分析圖；圖二B為海鱺生長速率分析圖。 圖三、為比較餵食乳酸菌株4012(乳酸菌餵食試驗組)海鱺與未餵食乳酸菌 (對照組)海纖的吞'&細胞瓦解病原體的能力分析圖；吸光值〇D595代 表吞嗤細胞瓦解病原體的能力。 圖四、為針對餵食乳酸菌株4012(乳酸菌餵食試驗組）海鱺與未餵食乳酸菌 (對照組)海鱺，進行發光桿菌坤攻毒試驗的結果分析圖。 圖五、為針對接種發光桿菌疫苗之海鱺’進行發光桿菌户办攻毒試驗的結 果分析圖；兩組試驗組魚隻均接種發光桿菌疫苗，其中一組試驗組 並配合餵食乳酸菌株4012(疫苗接種及乳酸菌餵食組），另一組試驗 組則只餵食市售魚飼料(僅疫苗接種組）；對照組則未接種疫苗，並且 只餵食市售魚飼料。 【主要元件符號說明】 201242517 序列表 <11〇>生合生物科技股份有限公司 國立澎湖科技大學 <120>新穎乳酸菌株、含彼之組合物及其用途 <160〉 3 <210〉1 <211>20 <212>DNA <213>人工序列 <220> <223>正向引子 <400〉1 agagtttgat catggctcag 203. The culture suspension of the P. oleracea strain 4012 (/! pewiosaceMi strain 4012) provided by the present invention can inhibit the growth rate of the luminescent Bacillus Pdp; and the fish fed the strain after the photobacterium bacillus pdp challenge The relative survival rate was 60 higher than the control group. In addition, the ability of the fish fed the strain of the present invention to resolve the pathogen (reSpirat〇ry burst) is significantly higher than that of the control group, and is based on the pentose sugar tablet of the present invention. Strain 4〇12 (A strain4012) can be used as a probiotic to help fish resist luminescent bacteria. 4. The feed containing the lactic acid pentosaceus strain 4〇12 provided by the invention is used in combination with the household-running dead vaccination vaccine, and can be used for (4) Lai or other fish riding anti-lighting (4), and the effect is Compared with those who use the vaccine alone, the relative survival rate of the feed and the vaccinated person is greatly increased. The detailed description of the present invention is intended to be illustrative of the preferred embodiments of the invention, and is not intended to In the scope of the patent in this case. In summary, the lactic acid strain provided in this case is not only the Xinlai strain, but also has the above-mentioned multiple functions. It should have fully complied with the statutory invention patent requirements of the new brewing and progressive, and proposed Shen Qing's approval of this invention. The patent (4) case, in order to invent invention, to the sense of virtue. 201242517 [Simplified description of the diagram] Figure 1. The graph of inhibition of luminescent bacteria growth by the lactic acid strain of the jellyfish intestinal tract; taking the culture suspension of Pediococcus lactis (10) strain 4012 into the growth medium of γ γ ρφ growth medium The bacteria were observed for the growth of γ-ray Ρφ; the negative control group was cultured with RS liquid medium mixed with MRS liquid medium (pH 6.2) to culture luminescent bacillus Ρφ' while the positive control group was adjusted with Bm liquid medium to adjust the pH value. The photobacterium was cultured in a far 3 liquid medium (pH 5); the absorbance value 〇D6gq represents the growth amount of the luminescent bacterium. Figure 2 shows the body weight of the sea otter in the experimental group and the control group after 14 days of comparison with the lactic acid strain (4012). Figure 2A shows the weight analysis of sea otter; Figure 2B shows the growth rate of sea otter. Figure 3 is a comparison of the ability of the lactic acid bacteria 4012 (lactic acid bacteria feeding test group) sea otter and unfed lactic acid bacteria (control group) sea fiber to swallow '& cells to disintegrate pathogens; absorbance value 595 D595 represents swallowing cell disintegration pathogen Ability. Fig. 4 is a graph showing the results of a luminescent bacillus challenge test for sea otters fed with lactic acid bacteria 4012 (lactic acid bacteria feeding test group) and sea otters without feeding lactic acid bacteria (control group). Figure 5 is an analysis of the results of the luminescent bacillus household vaccination test for the vaccination of the luminescent bacillus vaccine; the two groups of the test group were vaccinated with the luminescent bacillus vaccine, and one of the experimental groups was combined with the lactic acid strain 4012 ( In the vaccination and lactic acid bacteria feeding group, the other group was fed only commercial fish feed (vaccination group only); the control group was not vaccinated and only the commercial fish feed was fed. [Main component symbol description] 201242517 Sequence Listing <11〇> Shenghe Biotechnology Co., Ltd. National Wuhu University of Science and Technology<120> Novel lactic acid strain, composition containing the same and its use <160> 3 <210 〉1 <211>20 <212>DNA <213>Artificial sequence<220><223> Forward introduction <400>1 agagtttgat catggctcag 20
<210〉2 <211> 17 <212〉DNA <213>人工序列 <220〉 <223>反向引子 <400> 2 aaggaggtga tccagcc 17 1 201242517 <210>3 <211> (16S rDNA序列長 1507bp)<210>2 <211> 17 <212>DNA <213>Artificial sequence<220><223> Reverse introduction<400> 2 aaggaggtga tccagcc 17 1 201242517 <210>3 <211> (16S rDNA sequence length 1507bp)
<212> DNA <213> 乳酸戊糖片球菌株pewtoracews) <400〉3 gcggcgcgtg ctatacatgc agtcgaacga acttccgtta attgattatg acgtacttgt 60 actgattgag attttaacac gaagtgagtg gcgaacgggt gagtaacacg tgggtaacct 120 gcccagaagt aggggataac acctggaaac agatgctaat accgtataac agagaaaacc 180 gcatggtttt cttttaaaag atggctctgc tatcacttct ggatggaccc gcggcgtatt 240 agctagttgg tgaggtaaag gctcaccaag gcagtgatac gtagccgacc tgagagggta 300 atcggccaca ttgggactga gacacggccc agactcctac gggaggcagc agtagggaat 360 cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa gggtttcggc 420 tcgtaaagct ctgttgttaa agaagaacgt gggtaagagt aactgtttac ccagtgacgg 480 tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540 aagcgttatc cggatttatt gggcgtaaag cgagcgcagg cggtctttta agtctaatgt 600 gaaagccttt cggctcaacc gaagaagtgc attggaaact gggagacttg agtgcagaag 660 aggacagtgg aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg 720 gcgaaggcgg ctgtctggtc tgcaactgac gctgaggctc gaaagcatgg gtagcgaaca 780 ggattagata ccctggtagt ccatgccgta aacgatgatt actaagtgtt ggagggtttc 840 cgcccttcag tgctgcagct aacgcattaa gtaatccgcc tggggagtac gaccgcaagg 900 ttgaaactca aaagaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 960 aagctacgcg aagaacctta ccaggtcttg acatcttctg acagtctaag agattagagg 1020 ttcccttcgg ggacagaatg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat 1080 gttgggttaa gtcccgcaac gagcgcaacc cttattacta gttgccagca ttaagttggg 1140 cactctagtg agactgccgg tgacaaaccg gaggaaggtg gggacgacgt caaatcatca 1200 tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga gtcgcgaaac 1260 cgcgaggtta agctaatctc ttaaaaccat tctcagttcg gactgtaggc tgcaactcgc 1320 ctacacgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1380 gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa gccggtgggg 1440 taacctttta ggagctagcc gtctaaggtg ggacagatga ttagggtgaa gtcgtaacaa 1500 gagcccc 1507≪ 212 > DNA < 213 > acid pentosaceus sphere strain pewtoracews) < 400> 3 gcggcgcgtg ctatacatgc agtcgaacga acttccgtta attgattatg acgtacttgt 60 actgattgag attttaacac gaagtgagtg gcgaacgggt gagtaacacg tgggtaacct 120 gcccagaagt aggggataac acctggaaac agatgctaat accgtataac agagaaaacc 180 gcatggtttt cttttaaaag atggctctgc tatcacttct ggatggaccc gcggcgtatt 240 agctagttgg tgaggtaaag gctcaccaag gcagtgatac gtagccgacc tgagagggta 300 atcggccaca ttgggactga gacacggccc agactcctac gggaggcagc agtagggaat 360 cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa gggtttcggc 420 tcgtaaagct ctgttgttaa agaagaacgt gggtaagagt aactgtttac ccagtgacgg 480 tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540 aagcgttatc cggatttatt gggcgtaaag cgagcgcagg cggtctttta agtctaatgt 600 gaaagccttt cggctcaacc gaagaagtgc attggaaact gggagacttg agtgcagaag 660 aggacagtgg Aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg 720 gcgaaggcgg ctgtctggtc tgcaactgac gctgaggctc gaaagcatgg gtagcgaaca 780 ggattagata ccc tggtagt ccatgccgta aacgatgatt actaagtgtt ggagggtttc 840 cgcccttcag tgctgcagct aacgcattaa gtaatccgcc tggggagtac gaccgcaagg 900 ttgaaactca aaagaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 960 aagctacgcg aagaacctta ccaggtcttg acatcttctg acagtctaag agattagagg 1020 ttcccttcgg ggacagaatg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat 1080 gttgggttaa gtcccgcaac gagcgcaacc cttattacta gttgccagca ttaagttggg 1140 cactctagtg agactgccgg tgacaaaccg gaggaaggtg gggacgacgt caaatcatca 1200 tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga gtcgcgaaac 1260 cgcgaggtta agctaatctc ttaaaaccat tctcagttcg gactgtaggc tgcaactcgc 1320 ctacacgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1380 gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa gccggtgggg 1440 taacctttta ggagctagcc gtctaaggtg ggacagatga ttagggtgaa gtcgtaacaa 1500 gagcccc 1507