201238490 六、發明說明： 【發明所屬之技術領域】 [0001] 本發明係有關於—種具抗紫外線活性之液化澱粉芽 孢桿菌生產方法’尤其是指一種將特定之液化澱粉芽孢 才干菌於進行配方培養步驟後，用以產生具抗紫外線效果 者。 [0002] 【先前技術】 按’美國EPA(U.S.Envir〇nmen al Protection Agency)已核 准不少種類的芽抱桿菌屬（B a c i i } u S s p p .)微生物農藥產品上市’作為防治植物病蟲害之藥劑 ’除了一般熟知的枯草桿菌（B a e i 1 1 u s s u b t i 1 i s )之外，尚包括液化澱粉芽孢桿菌（B & C 1 1 1US subt , 1 i s var - amy 1 0 1 1 Q U e f a c i e n s )、仙人掌桿菌（B a c i 1 1 u s c e r e u s )、地衣芽孢桿菌（B a c i 1 1 u s 1 i c h p r. ί ΐ C n e 11 1 f 0 r m i s )、短小 芽孢桿菌（Bacillus pum"us)、蘇 ^(Bacillus thuringiensi S)、日本甲蟲芽孢桿菌（Bac" lus p〇p i i 1 i a e)與球形芽孢桿菌（Ba c 土 ！ ！ u s ……1…）等。芽孢桿菌已開發應用的商 品化製劑，在病害防治方面有枯草桿菌、液化搬 桿菌、仙人掌桿菌等；在蟲害防治方面為蘇力菌 甲蟲芽抱桿菌與球形芽抱桿菌等。 100111359 許多重要的植物病㈣是由土壤或 表單編號A0101 第4頁/共26頁 圈真邊所引起 1002018973-0 201238490 菌=人員利用土壤微生物間的拮抗作用，研發抑制真 革蘭氏=物防治藥劑，應用於田間防治。枯草桿菌為 革蘭氏％性’好氣性桿妝 $ ’ _錄毛及内生孢子 =為上主要特徵，此類細菌普遍存在於土壤及植物 Ο m __加物、酵素、種子減劑等生技 :、务展應用已有多年，為—般認定屬於安全性之有益 微生物種類。近來職用於土壤根圈與病㈣競爭根系 、呂養刀成為優勢菌種，進而降低病原菌的危害；也 可直接噴灑植物葉片來保護葉部真8病害，例如菜豆錢 ;亦可知用於土壤為_種子拌種處理以預防土壤病害 對多種作物之生長，尤其是根部之發育，有極為明 属之促進仙’還可做為蔬果採收後防止敗的抗真菌 劑，例如桃褐腐病與柑橘青黴病等。 Ο 液化殿粉芽抱桿菌，1 9 4 3年由曰本學者F u k 〇m〇 t 〇發現，此菌種可產生大量的a_amy i a. se及pr〇tea s e。在一開始時，由於此菌種外 觀及表現特徵和枯草桿ϋ極為相似，因此將液化殿粉芽 孢桿菌列為枯草桿菌的亞種之一。然而在1967年W e 1 ke r與c〇mpb e 1 1利用DMA雜交方法發 現括草桿菌和液化澱粉芽孢桿菌之g e n e相似度只有201238490 VI. Description of the Invention: [Technical Field of the Invention] [0001] The present invention relates to a method for producing a liquefied Bacillus amyloliquefaciens having ultraviolet-resistance activity, in particular, a method for formulating a specific liquefied starch spore-forming bacterium After the cultivation step, it is used to produce an anti-ultraviolet effect. [Prior Art] According to the 'US EPA (USEnvir〇nmen al Protection Agency) has approved a number of species of Bacillus subtilis (B acii } u S spp.) microbial pesticide products listed as a drug for controlling plant diseases and insect pests 'In addition to the well-known Bacillus subtilis (B aei 1 1 ussubti 1 is ), it also includes Bacillus licheniformis (B & C 1 1 1 US subt , 1 is var - amy 1 0 1 1 QU efaciens ), Cactus bacillus (B aci 1 1 uscereus ), Bacillus licheniformis (B aci 1 1 us 1 ichp r. ί ΐ C ne 11 1 f 0 rmis ), Bacillus pum "us, Bacillus thuringiensi S, Bacillus bacillus (Bac" lus p〇pii 1 iae) and Bacillus sphaericus (Ba c soil! ! us ...... 1...). Bacillus has been developed and applied as a commercial preparation, including Bacillus subtilis, liquefied bacillus, and cactus bacillus in disease control; in the prevention and control of insect pests, it is Bacillus licheniformis and Bacillus globosa. 100111359 Many important plant diseases (four) are caused by the soil or form number A0101 page 4 / 26 pages of the true side of the 1002018973-0 201238490 bacteria = the use of soil microbial antagonism, research and development inhibition of true Gram = physical control Medicament, applied to field control. Bacillus subtilis is a Gram's % 'aerobic makeup $' _ recording hair and endospores = the main feature, such bacteria are ubiquitous in soil and plant Ο m __ additions, enzymes, seed reducers, etc. Biotechnology: The application of the exhibition has been used for many years to identify the beneficial microbial species that are safe. Recently used for soil root ring and disease (4) competition root system, Lu Yang knife became the dominant species, thereby reducing the harm of pathogenic bacteria; can also directly spray plant leaves to protect the leaves of the true 8 diseases, such as bean money; also known for soil Seed dressing for _ seed to prevent the growth of soil diseases on a variety of crops, especially the development of roots, there is a very important promotion of immortal 'can also be used as an antifungal agent to prevent the loss of fruits and vegetables, such as peach brown rot With citrus penicillium and so on. Ο Liquefaction Temple Bacillus licheniformis, discovered in 1943 by the scholar F u k 〇m〇 t ,, this strain can produce a large number of a_amy i a. se and pr〇tea s e. In the beginning, Bacillus licheniformis was listed as one of the subspecies of Bacillus subtilis because the appearance and performance characteristics of this strain were very similar to those of the dry grass. However, in 1967, W e 1 ke r and c〇mpb e 1 1 used DMA hybridization method to find that the similarity of g e n e of Bacillus licheniformis and Bacillus liquefaciens was only
14. 7〜15. 4%之間，枯草桿菌的DNA gua nine — pius — Cytos ine 成份（G + C %)是41. 5〜43. 5%，而液化澱粉芽孢桿菌的G + C%是43. 5〜44. 9%，顯示枯草桿菌和液化澱 粉芽孢桿菌是為不同的品種。 然’可知的是液化澱粉芽孢桿菌也可使用、運用於 100111359 1002018973-0 表單編號A0101 第5頁/共26頁 201238490 農作物抑制真菌生長的生物防治藥劑範圍而在針對使用 於培養防禦範圍時，其菌株需對應農作物在日光下進行 作用，然而，陽光中的紫外線具有殺傷力，因此，菌株 在曰光下的存活率不高，相對在農作物培養與防禦上並 無法有效產生生物防治作用。 緣是，發明人有鑑於此，秉持多年該相關行業之豐 富設計開發及實際製作經驗，針對現有之結構及缺失予 以研究改良，提供一種具抗紫外線活性之液化澱粉芽孢 桿菌生產方法，以期達到更佳實用價值性之目的者。 【發明内容】 [0003] 本發明一種具抗紫外線活性之液化澱粉芽孢桿菌生 產方法的目的與功效係由以下之技術所實現： [0004] 其係將液化澱粉芽孢桿菌菌株〔包含命名為B F — 1【寄存編號：BCRC910498】、命名為BA 【寄存編號：BCRC9103 9 5】及命名為Y CM A 1【寄存編號：BCRC9 10 509】的新穎菌株 〕作為初始接種源，以將該初始接種源以基礎培養之N B培養基搖瓶加入具有碳源與氮源的培養基進行培養， 而獲取批次培養之接種源，接續，轉移至攪拌式液體發 酵槽内添加具有碳源與氮源和抗紫外線添加物的培養基 進行二次培養，而可獲得具有抗紫外線活性功能的發酵 產物。14. 7~15. 4%, Bacillus subtilis DNA gua nine — pius — Cytos ine ingredient (G + C %) is 41. 5~43. 5%, while G + C% of liquefied Bacillus amyloliquefaciens is 43. 5 to 44. 9%, showing that Bacillus subtilis and Bacillus amyloliquefaciens are different varieties. However, it is known that Bacillus amyloliquefaciens can also be used and used in 100111359 1002018973-0 Form No. A0101 Page 5 of 26 201238490 The range of biological control agents for inhibiting fungal growth of crops is The strain needs to respond to the crop in the sunlight. However, the ultraviolet light in the sunlight has the lethality. Therefore, the survival rate of the strain in the twilight is not high, and the biological control effect cannot be effectively produced in the cultivation and defense of the crop. In view of this, the inventors have been in the process of researching and improving the existing structures and defects by providing rich experience in design and development and actual production of the relevant industries for many years, and to provide a method for producing Bacillus licheniformis with anti-ultraviolet activity, in order to achieve more The purpose of good practical value. SUMMARY OF THE INVENTION [0003] The purpose and efficacy of a method for producing a Bacillus licheniformis having anti-ultraviolet activity is achieved by the following technology: [0004] It is a strain of Bacillus amyloliquefaciens (including the name BF - 1 [registration number: BCRC910498], named BA [registration number: BCRC9103 9 5] and a novel strain named Y CM A 1 [registration number: BCRC9 10 509] as the initial inoculation source to The NB medium shake flask of the basic culture is added to the medium with the carbon source and the nitrogen source for cultivation, and the inoculation source of the batch culture is obtained, and then transferred to the stirred liquid fermentation tank to add the carbon source and the nitrogen source and the ultraviolet resistant additive. The culture medium is subjected to secondary culture to obtain a fermentation product having a function of anti-ultraviolet activity.
[0005] 本發明所製得之發酵產物其菌體濃度比較C K ( L B)培養基可提升1 5倍以上，且菌體經照射U V — B ( 6 3 0 #W/cm2) 30分鐘後存活率可明顯提升。 100111359 表單編號A0101 第6頁/共26頁 1002018973-0 201238490 [0006] [0007] Ο ο [0008] 因此，本發明之發酵產物可運用於生物農藥組合物中來 抑制作物真菌與細菌病害之活性生長，亦可與殺真菌劑 、殺蟲劑、除草劑及蘇力菌搭配使用之生物防治藥劑者 本發明之批次培養之接種源濃度為i 〇 9 c f u/m 1以上，而以1〇l〇c f υ/ml為最佳者。而最終所 獲得之發酵產物含有至少i 〇 1 1 c f u/m丨孢子者。 本發明之碳源培養基進一步為〇.1%〜1%麥芽糊 精、0. 1%〜1%葡萄轉、糖蜜、〇工 /〜1 %蔗糖、〇 . 1 %〜1%可溶性殿粉其中一種或— 種以上的混合物；而其氮源培養基進一步為培養基為〇. 1 %〜1. 5%大豆分離蛋白、〇.丄％〜1 %酵母粉、 〇· 1〜1%酵母萃出物、〇.；[〜1%麥芽萃出物' 〇 1/6〜1/6玉米浸出物其中一種或一種以上的混合物； 抗紫外線添加物進一步為0. 〇 i %〜〇. 去乙醯化 80%幾丁聚醣、〇_ 〇 1%〜〇 5%糖蜜、〇 〇 〜0. 5 %水楊酸、〇· 〇 1 %〜〇. 5%腐植酸其中—種 或一種以上之混合物。 本發明另一目的係提出液化澱粉芽孢桿菌菌株〔包 含命名為B F — 1【寄存編號‘· BCRC910498 】、命名為B A【寄存編號：BCRC910395】 及命名為Y CMA1【寄存編號：BCRC91〇5〇 9】的新穎菌株〕所製得的發酵產物，用以抑制屬於下 100111359 列組成群組中至少一種真菌菌屬之生長，該病原真菌 括·番石權立枯病菌Myxo s po r i um 包 P S 1 表單編號A0101 第7頁/共26頁 1002018973-0 201238490 dii S a w a d a e t Kurosawa、赛 百合立枯絲核菌Rhizoctonia solan i、胡麻葉枯病菌Bipolaris o r y z a e (Breda d e Haan) Shoemaker 、蓮霧炭疽病菌Colletotrichum g1 oeosporioides (Penz ·) Sacc .、棗炭疽病菌Colletotrichum g1 oeospor i o i de s、檬果炭疽病菌Co 1 1 etotrichum gloeosporioid es (Penz.&Sace·)、檬果蒂腐病菌Bo tryodiplodia theobromae Pat. (Mango)、木瓜炭疽病菌Col let ot r i chum gloeosporioides Penz i g、木瓜蒂腐病菌Bo tyod i p 1 o dia theobromae Pat· (Papa ya)、木瓜疫病菌Phytophthora pa lmivora (But ler)But 1 er ·、黃 萎病菌Fusa r i um sp .、洋蔥紫根病菌Fu sarium oxysporum f · s p · c e pae、蓮霧果腐病菌Pes t a 1 ot i ops i s euginae、番石權瘡痴病菌Pes ta 1 ot iopsis psidii (Pat-)Mordu e Pestalotia psidii、甘藍黑斑 病菌 Alternaria brassicae (B ERK)Sacc·。 100111359 表單編號A0101 第8頁/共26頁 1002018973-0 201238490 L0009] 再者，本發明液化澱粉芽孢桿菌菌株〔包含命名為 B F — 1【寄存編號：BCRC910498】、命名 為BA【寄存編號：BCRC910395】及命名為 Y CMA 1【寄存編號：BCRC910509】的新 穎菌株〕所製得的發酵產物，用以對作物細菌病害有抑 制活性之植物病源至少一種細菌包含：為番茄細菌性斑 點（Q)、柑桔潰瘍病（r)、瓜類細菌性果斑（S) 〇[0005] The fermentation product prepared by the invention has a cell concentration higher than that of the CK (LB) medium by more than 15 times, and the survival rate of the cells after irradiation with UV-B (6 3 0 #W/cm2) for 30 minutes. Can be significantly improved. 100111359 Form No. A0101 Page 6 of 26 1002018973-0 201238490 [0007] [0007] Therefore, the fermentation product of the present invention can be applied to a biological pesticide composition to inhibit the activity of crop fungi and bacterial diseases. The biological control agent for growth, which can also be used together with a fungicide, an insecticide, a herbicide and a sulphide, has a concentration of the inoculum of the batch culture of the present invention of i 〇9 cfu/m 1 or more, and 1 〇 L〇cf υ/ml is the best. The resulting fermentation product contains at least i 〇 1 1 c f u/m 丨 spores. The carbon source medium of the present invention further comprises 0.1%~1% maltodextrin, 0.1%~1% grape turn, molasses, masonry/~1% sucrose, 〇. 1%~1% soluble temple powder One or more of the mixture; and the nitrogen source medium is further a medium of 〇. 1%~1. 5% soy protein isolate, 〇.丄%~1% yeast powder, 〇·1~1% yeast extract物 % % ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; 〜 〜 ; 〜 ; 〜 〜 〜 〜 〜 〜 〜 〜 〜 Deuterated 80% chitosan, 〇 _ 〇 1% ~ 〇 5% molasses, 〇〇 ~ 0. 5 % salicylic acid, 〇 · 〇 1% ~ 〇. 5% humic acid among them - one or more mixture. Another object of the present invention is to propose a strain of Bacillus amyloliquefaciens (including the name BF-1 [registered number 'BCRC910498], named BA [registered number: BCRC910395] and named Y CMA1 [registered number: BCRC91〇5〇9 Fermentation product prepared by the novel strain] for inhibiting the growth of at least one fungal genus belonging to the group consisting of the lower 100111359 column, the pathogenic fungus including the Myxo s po ri um package PS 1 form number A0101 Page 7 of 26 1002018973-0 201238490 dii S awadaet Kurosawa, Rhizoctonia solan i, Bipolaris oryzae (Breda de Haan) Shoemaker, Colletotrichum g1 oeosporioides (Penz) ·) Sacc., Jujube anthracnose Colletotrichum g1 oeospor ioi de s, Anthracnose Co 1 1 etotrichum gloeosporioid es (Penz. & Sace), Bophut rot, Bo tryodiplodia theobromae Pat. (Mango), Papaya anthrax Pathogen Col let ot ri chum gloeosporioides Penz ig, Papaya rot, Bo tyod ip 1 o dia theobromae Pat· (Papa ya), Phytophthora pa lmivora (Butler) But 1 er · Verticillium dahliae Fusa ri um sp., Fusarium oxysporum f · sp · ce pae, Pom rot fungus Pes Ta 1 ot i ops is euginae, Pes ta 1 ot iopsis psidii (Pat-) Mordu e Pestalotia psidii, Alternaria brassicae (B ERK) Sacc·. 100111359 Form No. A0101 Page 8 of 26 1002018973-0 201238490 L0009] Further, the strain of Bacillus amyloliquefaciens of the present invention (including the name of BF-1 [registered number: BCRC910498], named BA [registration number: BCRC910395] And a fermentation product prepared by the novel strain named Y CMA 1 [registration number: BCRC910509], a plant disease source having inhibitory activity against crop bacterial diseases, at least one kind of bacteria comprising: tomato bacterial spots (Q), mandarin Orange ulcer disease (r), melon bacterial fruit spot (S) 〇
0 [0010] 續，本發明液化澱粉芽孢稈菌菌株〔包含命名為B F — 1 具有獨特的]_ 6S rRNA、YyaR、It u A、I t u B、I t u C、I t u D之基因序列的新 穎菌株〕所製得的發酵產物，用以搭配殺真菌劑種類包 含：1.脂烷氮類；2.醯胺類；3.胺醯酸類；4. 苯甲醯胺類；5.苯甲醯笨胺類；6.磺胺類；7.抗 生素類；8.嗜球果傘素類；9·芳香族；1〇.笨並 咪唑類；1 1 .苯併咪唑前驅物類；i 2 .胺基甲酸鹽 〇 類；13.嗓唾類;14.三峻類；15.銅類；16 .二氣苯基二甲醯亞胺類；i 7 .二硫代胺基曱酸鹽類 ;1 8 .聚合二硫代胺基甲酸鹽類；丄9 .咪唑類；2 0 .有機磷類；2 1 .吡啶類；2 2 .嘧啶類；2 3 · 喹啉類，24 .醌類；25 .噻唑類；26 ·尿素類； 2 7 ·三。坐苯併堡唾類。 [0011] 本發明液化澱粉芽孢捍菌菌株〔包含命名為B F —[0010] Continued, the strain of the liquefied Saccharomyces cerevisiae strain of the present invention (containing a gene sequence named BF-1 having unique]_6S rRNA, YyaR, It u A, I tu B, I tu C, I tu D The fermentation product prepared by the novel strain is used to match the fungicide species: 1. alkane nitrogen; 2. guanamine; 3. amine phthalic acid; 4. benzamide; 5. benzoic acid醯 胺 amines; 6. sulfonamides; 7. antibiotics; 8. strobilurins; 9 · aromatic; 1 笨. stupid imidazoles; 1 1. benzimidazole precursors; i 2 . Amino formates; 13. sputum; 14. stern; 15. copper; 16. diphenyl phenyl dimethyl imidate; i 7. dithiol amide ;1 8. Polymeric dithiocarbamates; 丄9. Imidazoles; 20. Organophosphorus; 2 1. Pyridines; 2 2 . Pyrimidines; 2 3 · Quinolines, 24. ; 25. Thiazoles; 26 · Ureas; 2 7 · III. Take the benzo-boss saliva. [0011] The liquefied Bacillus amyloliquefaciens strain of the invention (including the name B F -
1具有獨特的16S rRNA、YyaR、ItuA 、ItuB、ItuC、ItuD之基因序列的新穎菌 100111359 表單編號A0101 第9頁/共26頁 1002018973-0 201238490 株〕所製得的發酵產物，用以搭配至少一種殺蟲劑種類 ，包含.1.三氮井系；2.有機氮與雜環類；3.氨 機曱酸鹽；4 . °比咬有機硫代碟酸鹽類；5 .聯苯類； 6 .有機氣類；7 .抗生素類；8 .新尼古丁類；9 . 、1 〇 .有機磷類·’ 1 1 ·直鏈胺有機硫代磷酸鹽類； 12.吡唑類；13.唑類。 [0012] 本發明液化澱粉芽孢桿菌菌株〔包含命名為B F_ 1具有獨特的 16S rRNA'YyaR' I tuA 、I t u B、i t u c、I i u d之基因序列的新穎菌 株〕所製得的發酵產物，用以搭配至少一種除草劑種類 ，包含·1.苯氧酸系；2.次蛾酸養；3.醯胺類； 4.聯苯醚系；5.二硝基苯胺系；6.四級胺類。 【實施方式】 [0013] 為令本發明所運用之技術内容、發明目的及其達成 之功效有更完整且清楚的揭露，兹於下詳細說明之，並 請一併參閱所揭之圖式及圖號： 丨 首先，請參閱第一圖所示，為本發明一種具抗紫外 線活性之液化澱粉芽孢桿菌生產方法，其步驟如下： (1) 取得細菌接種源一取得液化澱粉芽孢桿菌菌株〔包含 命名為BF—1【寄存編號：BCRC9l〇498】 、命名為BA【寄存編號：BCRC91〇395】及 命名為Y C MA 1【寄存編號：BCRC91〇5〇9 】的新穎菌株〕作為初始接種源； (2) 製備批次培養之接種源—以1 %比例接種用於接種源 放大培養，將初始接種源以N B培養基搖瓶加入包含碳 100111359 表單編號A0101 第10頁/共26頁 1002018973-0 201238490 源與氮源的培養基進行培養，培養1〜3天，而其中培 養條件為溫度3 0 — 3 5 °C，攪拌速率1 0 0 — 2 5 0 r pm，通氣量為 0. 2 5 — 1. ，pH 值為 5. 0 _ 7. 0，以獲取批次培養之接種源，批次培養之接種 源濃度為1 0 9 c f u/m 1以上； (3) 量產液體培養一將所獲取濃度為1 〇 9 c f u / m 1 以上培養用接種源於量產用攪拌式液體發酵槽内添加包 含碳源與氮源和抗紫外線添加物的培養基，進行液體培 養3〜6天，其中培養條件為溫度3 0 _ 3 5 °C，攪拌 速率 100 — 250rpm，通氣量為 0.25 — 1.5 vvm，pH 值為 5.0 — 7.0 ; (4) 獲取發酵產物一經上述步驟培養3〜5天後，由攪拌 式液體發酵槽收取發酵產物，為具有抗紫'外線活性功能 的發酵產物。 接續，菌株經由上述的生產培養過程，其所添加之 碳源、氮源培養基或抗紫外線添加物係可選擇採用下列 物質： 該碳源培養基為0. 1%〜1%麥芽糊精、0. 1% 〜1%葡萄糖、0. 1%〜1%糖蜜、0. 1%〜1%蔗 糖、0. 1 %〜1 %可溶性澱粉其中一種或一種以上的混 合物； 而氮源培養基為培養基為0. 1%〜1. 5%大豆分 離蛋白、0. 1 %〜1 %酵母粉、0. 1〜1 %酵母萃出 物、0. 1〜1%麥芽萃出物、0. 1%〜1%玉米浸出 物其中一種或一種以上的混合物； 其中該抗紫外線添加物為0. 0 1%〜0. 5%去乙 100111359 表單編號A0101 第11頁/共26頁 1002018973-0 201238490 醯化80%幾丁聚醣、〇.〇1%〜〇 5%糖蜜、〇 〇 l/ΰ〜〇· 5%水楊酸、〇. 〇 1%〜〇 5%腐植酸其中 一種或一種以上之混合物。 而經配方培養所取得之發酵產物經由試驗於化學農 藥中之存活率： 於LΑ培養基平板挑選一發酵產物菌落，將其接種 於3ml LB中，置於2 8t恆溫震盪培養2 4小時 後，利用光譜儀（spectroph()t〇me r ultrospec20 0 〇，p h a r m a c i a b 1 0 11 e c h ’ U S A )調整其吸收值（a )，使菌液濃度調整為1 〇 8 c ο 1.0 Π y - f ο r m ing units(cfu/ml)，取100“^ 菌液、1 0 0以1 1 0 X s t o c k的化學性藥劑 與8 0 0 # 1 d dH2O加入混合後搖晃均勻即放置於 桌上靜置1 〇分鐘，而對照組則為丄〇 〇" i菌液與9 0 0 ^ 1 ddH2〇混合，1 〇务鐘餐取工〇 〇以工 進行序列稀釋，再於稀釋液i 〇-3莖α 〇-5各取工〇 〇 β 1於L A平板上進行塗盤，之棱置於2 8它培養箱 進行2 4小時培養，2 4小時後計算菌落數，依下列公 式回推其存活率。本發明中所使用的化學性農藥有殺真 菌劑、殺細菌劑、殺蟲劑、除草劑與不同稀釋倍數之展 著劑，使用之藥劑分別列於附錄一、附錄二、附錄三， 附錄四’且藥劑稀釋倍數為建議使用濃度。 接續’針對抗紫外線配方實施在命名為BF—1〔 寄存編號：BCRC91〇498〕的液化㈣芽孢桿 菌培養方法進行測試，其UVB試驗時間為3 〇分鐘； 100111359 表單編號A0101 第12頁/共26頁 1002018973-0 201238490 在U V B照射3 0分鐘時間處理下的數據表〔請參第二 圖所示〕，於圖表中表示CK(LB)為液化澱粉芽孢 桿菌菌株未接受培育前所進行之U V B照射的反應狀態 ，相對於菌株進行步驟（2 )〔配方一〕與進行步驟（ 3 )〔配方二〕之後所作UV B照射的反應，以分別做 出比對圖表，由圖表可知，經由本發明步驟培育後的發 酵產物在UVB照射3 0分鐘之後，其存活率高於前二 者，因此，本發明製程所培育之發酵產物具有良好抗紫 外線的效果。 而於液化澱粉芽孢桿菌族群中，命名為B A【寄存 編號：BCRC910395】〔請參第三圖所示〕及 YCMA1【寄存編號：BCRC910509】〔請 參第四圖所示〕菌株可適用於本發明之製程步驟中來進 行培育，而所培育完成之發酵產物同樣具有良好抗紫外 線的效果。 另外，該液化殿粉芽孢桿菌係可與蘇力菌搭配使用 以成生物農藥組成物，請參閱第五圖所示，該圖式表示 於液化澱粉芽孢桿菌（於此採用命名為B F — 1液化澱 粉芽孢桿菌來施作）進行添加步驟（2 )〔配方一〕與 步驟（3 )〔配方二〕之後搭配蘇力菌（B t )的存活 率表；於圖表中表示CK(LB)為單純的液化澱粉芽 孢桿菌菌株及液化澱粉芽孢桿菌+蘇力菌之後的存活狀 態，相對於上述二種菌株狀態進行步驟（2 )〔配方一 〕與進行步驟（3)〔配方二〕之後的存活反應，以分 別做出比對圖表，由圖表可知，經由本發明步驟培育後 的發酵產物在搭配蘇力菌之後，其存活率高於前二者， 100111359 表單編號A0101 第13頁/共26頁 1002018973-0 201238490 因此，本發明製程所培育之發酵產物搭配蘇力菌之後， 不會被蘇力菌所壓抑。 前述之實施例或圖式並非限定本發明之結構樣態或尺寸 ，任何所屬技術領域中具有通常知識者之適當變化或修 飾，皆應視為不脫離本發明之專利範疇。 綜上所述，本發明實施例確能達到所預期之使用功效， 又其所揭露之具體構造，不僅未曾見於同類產品中，亦 未曾公開於申請前，誠已完全符合專利法之規定與要求 ，爰依法提出發明專利之申請，懇請惠予審查，並賜准 專利，則實感德便。 【圖式簡單說明】 [0014] 第一圖：本發明步驟流程圖 [0015] 第二圖：本發明液化澱粉芽孢桿菌（B F — 1菌株）抗 紫外線的比較圖 [0016] 第三圖：本發明液化澱粉芽孢桿菌（B A菌株）抗紫外 線的比較圖 [0017] 第四圖：本發明液化澱粉芽孢桿菌（YCMA1菌株） 抗紫外線的比較圖 [0018] 第五圖：本發明液化澱粉芽孢桿菌（B F — 1菌株）搭 配蘇力菌的存活數據圖 【主要元件符號說明】 100111359 表單編號A0101 第14頁/共26頁 1002018973-01 Novel bacteria 100111359 with unique gene sequences of 16S rRNA, YyaR, ItuA, ItuB, ItuC, ItuD Form No. A0101 Page 9 / 26 pages 1002018973-0 201238490 strains are used to match at least An insecticide species comprising: 1. a trinitrogen well system; 2. an organic nitrogen and a heterocyclic ring; 3. an ammonia machine citrate; a 4. ° ratio of an organic thioate salt; 5. a biphenyl 6. Organic gases; 7. Antibiotics; 8. Neo-nicotines; 9. , 1 有机. Organophosphorus · ' 1 1 · Linear amine organothiophosphates; 12. Pyrazoles; Azole. [0012] The fermentation product prepared by the strain of Bacillus amyloliquefaciens of the present invention comprising a novel strain designated as B F-1 having a unique 16S rRNA 'YyaR' I tuA , I tu B, ituc, I iud gene sequence, Used to match at least one herbicide species, including 1. phenoxy acid system; 2. humic acid; 3. guanamine; 4. diphenyl ether; 5. dinitroaniline; Amines. [Embodiment] [0013] For a more complete and clear disclosure of the technical content, the purpose of the invention and the effects thereof achieved by the present invention, the following detailed description is provided, and the drawings and Figure: 丨 First, please refer to the first figure, which is a method for producing Bacillus liquefaciens with UV resistance. The steps are as follows: (1) Obtaining a bacterial inoculation source and obtaining a strain of Bacillus amyloliquefaciens Named as BF-1 [registration number: BCRC9l〇498], named BA [registration number: BCRC91〇395] and the novel strain named YC MA 1 [registration number: BCRC91〇5〇9] as the initial inoculation source; (2) Preparation of batch culture inoculation source - inoculation at a ratio of 1% for inoculation source amplification culture, initial inoculation source added to NB medium shake flask containing carbon 100111359 Form No. A0101 Page 10 / Total 26 Page 1002018973-0 201238490 The culture medium is cultured for 1 to 3 days, wherein the culture condition is a temperature of 30 to 35 ° C, a stirring rate of 1 0 0 to 2 5 0 r pm, and aeration is 0. 2 5 - 1 . , The pH value is 5.0 _ 7. 0, in order to obtain the inoculation source of the batch culture, and the concentration of the inoculation source of the batch culture is 1 0 9 cfu/m 1 or more; (3) The concentration of the liquid culture is 1 1 〇9 cfu / m 1 The above culture is inoculated with a medium containing a carbon source and a nitrogen source and an anti-ultraviolet additive in a stirred liquid fermentation tank for mass production, and liquid culture is carried out for 3 to 6 days, wherein the culture condition is temperature. 3 0 _ 3 5 °C, stirring rate 100 - 250 rpm, aeration of 0.25 - 1.5 vvm, pH value of 5.0 - 7.0; (4) Obtaining the fermentation product after 3 to 5 days of cultivation by the above steps, by agitated liquid fermentation The tank receives the fermentation product as a fermentation product having an anti-purple 'external activity function. 1%〜1%麦麦糊糊,0。 The carbon source medium, the carbon source medium is 0. 1%~1% maltodextrin, 0, the carbon source medium, the nitrogen source medium or the anti-UV additive system may be selected from the following: 1%〜1% glucose, 0.1%~1% molasses, 0.1%~1% sucrose, 0.1%~1% soluble starch, one or more mixtures; and the nitrogen source medium is medium 0% 1%~1%% of the yeast extract, 0. 1~1% of the yeast extract, 0. 1~1% of the malt extract, 0.1% ~1% corn extracts of one or more of the mixture; wherein the UV-resistant additive is 0. 0 1%~0. 5% to B100111359 Form No. A0101 Page 11 of 26 1002018973-0 201238490 80% chitosan, 〇.〇1%~〇5% molasses, 〇〇l/ΰ~〇· 5% salicylic acid, 〇. 〇1%~〇5% humic acid one or more mixtures . The fermentation product obtained by the formula culture is tested for survival rate in the chemical pesticide: a fermentation product colony is selected from the LΑ medium plate, inoculated into 3 ml LB, and placed in a 28 t constant temperature shaking culture for 24 hours, and then utilized. The spectrometer (spectroph()t〇me r ultrospec20 0 〇, pharmaciab 1 0 11 ech ' USA ) adjusts its absorption value (a ) to adjust the bacterial concentration to 1 〇 8 c ο 1.0 Π y - f ο rm ing units( Cfu/ml), take 100"^ bacteria solution, 1 0 0 to 1 1 0 X stock chemical agent and mix 80 0 # 1 d dH2O, shake it evenly and place it on the table for 1 minute, while In the control group, 丄〇〇" i bacterial liquid was mixed with 900 ° 1 ddH2 ,, 1 〇 钟 餐 餐 取 〇〇 〇〇 〇〇 〇〇 , , , , , , 稀释 稀释 稀释 稀释 稀释 稀释 稀释 稀释 稀释 稀释 稀释 稀释 稀释Each of the 〇〇β1 was coated on the LA plate, and the ribs were placed in a 28-degree incubator for 24 hours, and the number of colonies was counted after 24 hours, and the survival rate was reversed according to the following formula. The chemical pesticides used are fungicides, bactericides, insecticides, herbicides and different For the release factor, the agents used are listed in Appendix I, Appendix II, Appendix III, Appendix IV' and the dilution ratio of the drug is the recommended concentration. The continuation of the 'anti-UV formula is named BF-1 [registration number : BCRC91〇498] liquefied (tetra) Bacillus culture method was tested, its UVB test time was 3 〇 minutes; 100111359 Form No. A0101 Page 12 / Total 26 pages 1002018973-0 201238490 Data processed under UVB irradiation for 30 minutes Table [please refer to the second figure], the chart shows that CK (LB) is the reaction state of UVB irradiation performed before the cultivation of Bacillus amyloliquefaciens strain is not carried out, and step (2) [formulation 1] is carried out with respect to the strain. The reaction with the UV B irradiation after the step (3) [formulation 2] was carried out to make a comparison chart, respectively, and the survival rate of the fermentation product after the irradiation of the present invention was 30 minutes after UVB irradiation. Higher than the former two, therefore, the fermentation product cultivated by the process of the invention has a good anti-ultraviolet effect. In the group of liquefied Bacillus amyloliquefaciens, The strain named BA [registration number: BCRC910395] (please refer to the third figure) and YCMA1 [registration number: BCRC910509] [see the fourth figure] strain can be applied to the process steps of the present invention for cultivation, and The fermented product that has been cultivated also has a good anti-ultraviolet effect. In addition, the liquefied Bacillus licheniformis can be used in combination with S. cerevisiae to form a bio-pesticide composition, as shown in the fifth figure, which is shown in the liquefied Bacillus licheniformis (here named BF-1 liquefaction) Bacillus amyloliquefaciens is used to carry out the addition step (2) [Formulation 1] and the step (3) [Formulation 2] followed by the survival rate table of Su bacterium (B t ); in the graph, CK (LB) is simple The survival state of the Bacillus aeruginosa strain and the Bacillus amyloliquefaciens + S. cerevisiae, the survival reaction after the step (2) [formulation 1] and the step (3) [formulation 2] are carried out relative to the state of the above two strains In order to make a comparison chart separately, it can be seen from the chart that the survival rate of the fermentation product cultivated by the method of the present invention after collocation with S. cerevisiae is higher than the former two, 100111359 Form No. A0101 Page 13 / Total 26 Page 1002018973 -0 201238490 Therefore, the fermentation product cultivated by the process of the present invention is not inhibited by Suri bacteria after being mixed with S. The above-described embodiments or drawings are not intended to limit the structure or the scope of the invention, and any suitable variations or modifications may be made without departing from the scope of the invention. In summary, the embodiments of the present invention can achieve the expected use efficiency, and the specific structure disclosed therein has not been seen in the same product, nor has it been disclosed before the application, and has completely complied with the requirements and requirements of the patent law. If you apply for an invention patent in accordance with the law, you are welcome to review it and grant a patent. BRIEF DESCRIPTION OF THE DRAWINGS [0014] First: Flow chart of the steps of the present invention [0015] Second figure: Comparison of ultraviolet rays resistance of Bacillus amyloliquefaciens (BF-1 strain) of the present invention [0016] Third figure: Comparative diagram of anti-ultraviolet light of the liquefied Bacillus amyloliquefaciens (BA strain) [0017] Fourth figure: Comparative diagram of anti-ultraviolet light of the liquefied Bacillus amyloliquefaciens (YCMA1 strain) of the present invention [0018] Fifth figure: The liquefied Amyloliquefaciens of the present invention ( Survival data of BF-1 strain with S. cerevisiae [Main component symbol description] 100111359 Form No. A0101 Page 14 of 26 1002018973-0