201211258 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種發酵液及其製造方法，特別是關於一種具 有高濃度超氧化歧化酵素(SOD)之排毒發酵液及其製造方法。 【先前技術】 發酵技術可應用的範圍極廣，包括從極高單價的醫藥級產品 到工業用的酵素，以及食品用的有益微生物，農業用的生物殺蟲 劑，甚至從動物飼料藥劑或直接添加在飼料中的微生物等到廢棄 物回收，都屬於發酵技術的應用範圍。近年來，除農業與食品以 外’發酵技術也延伸應用在保健食品的產業上，包括腸道益生菌、 食藥用真菌類等等。例如以乳酸菌與酵母菌作為菌元，將蔬菜汁 或果菜汁發酵可生產許多種有益健康的物質，如有機酸、菌種多 醣體以及菌體中所產生之維生素營養物質，亦或是含有可排毒、 抗氧化的超氧歧化酵素(SUperoxide dismutases，SOD)。然而，目前 一般已知的乳酸菌與酵母菌菌種所發酵的蔬果汁發酵液，其中的 超氧歧化酵素含量最高只能達1000Units/mL。 【發明内容】 如先前技術所述，目前-般已知的減菌與酵母_種所發 酵的蔬果汁發酵液，其中的超氧歧化酵素含量最高只能達1000 Units/ mL。因此，如何利用發酵技術得到高濃度超氧歧化酵素之 發酵液，仍是一亟待解決的課題。 緣此，本發明之一目的即是提供一種超氧化歧化酵素⑽切 201211258 排毒發酵液之製造方法。 本發明之另-目的即是提供i具有高濃度超氧化歧 (SOD)之排毒發酵液。 、 —本發縣解決f知技狀_所採狀技術手段係為一種超 氧化歧化酵素(SOD)排毒發酵液之製造方法，其步驟包括： (A) &供胚芽乳酸才干n「尤⑽^础沿冰咐㈣历)以及一酿酒酵母 {Saccharomyce cerevisie) ⑼取-縣單位量之該胚芽乳酸桿菌以及該釀酒酵母接種入— 具有預定糖度成分之植物性原料中； (C) 在-預定溫度範Βτ進行通氣觸靜置發酵培養_預定時間； (D) 產生一具有超氧歧化酵素(gj〇D)之發酵液。 母係=梅步一乳酸桿⑽_ _====!：__輸驗讓 水果=地，前職造方法之步驟⑼之植物性·係為蔬菜汁或 2_較|^，。前述製造方法之步驟(C)之預定溫度範圍係為攝氏 較佳地，前述製造方法之步驟(C)之預定時_為2_3天。 ⑻===(D)之發酵液中的超氧歧化酵素 201211258 經由上述製造方法及在所述較佳條件之下，即可製得具有高 濃度超氧化歧化酵素(SOD)之排毒發酵液，其中排毒發酵液所含之 超氧歧化酵素(SOD)係大於3000 Units/ mL。 經由本發明所採用之技術手段，經由有機梅子表皮所薛選出 之胚芽礼酸桿菌（k伽坤训如^)以及釀酒酵母 ㈣myce cerev加e)兩種菌種所發酵之發酵液可將超氧歧化酵 素提升至3000 Units/ mL以上。同時經由測試此高濃度超氧歧化 酵素之發酵液，發現其可有效抑制紫外光照射、氧化毒素所生成 自由基以及高溫炸油與燒烤焦炭之因傷害，並於抗突變試驗中 φ 可防止毒素所導致的突變發生。經由此些已經證實的功效性，在 未來可將本發明之發酵液應用於化妝品、食品、化工等領域，製 備各種具有排毒、抗氧化功效性的產品。 【實施方式】 本發明之超氧化歧化酵素(SOD)排毒發酵液之製造方法將可 由以下的實施例說明而得到充分瞭解，使得熟習本技藝之人士可 以據以完成之’然而本案之實施並非可由下列實施例而被限制其# 實施型態，熟習本技藝之人士仍可依據除既揭露之實施例的精神 推演出其他實施例，該等實施例皆當屬於本發明之範圍。 名詞定義 超氧歧化酵素(SOD):超氧化物歧化酶（Super〇xide此腹㈣， SOD)又稱過氧化物歧化酶，屬於金屬酶’按照結合金屬離子種 類不同’有以下三種：含銅與鋅超氧化物歧化酶（Cu_Zn_s〇D)、 6 201211258 含猛超氧化物歧化酶（Mn-SOD )和含鐵超氧化物歧化酶 (Fe-SOD>三種超氧化物歧化酶都催化超氧化物陰離子自由基歧 化為過氧化氫與氧氣。 抗基因傷害及抗致突變·在此主要係指透過超氧歧化酵素(S〇D) 對抗各種環境或外來因子所導致的基因傷害甚至是基因發生突變 的現象’特別是指利用彗星分析法(comet assay)及安姆試驗(Ames test) ’測試其抗基因傷害及抗致突變的能力。 • 製造方法 參閱第1圖，其係為本發明超氧化歧化酵素排毒發酵液之製 造方法流程圖。 首先長_供一胚芽乳酸桿菌(LactobaciHus pZantarum)以反一酿 酒酵母(步驟101)。在此所使用的胚芽乳酸 桿菌以及釀酒酵母，皆是由有機梅子表皮所篩選出。 接著，取一預定單位量(1〇9 CFU/ mL)之該胚芽乳酸桿菌以及 籲該酿酒酵母接種入一具有預定糖度成分(可視原料種類及所需作適 當調整)之植難原射(轉丨Q2)。在此制雜雛原料可以是 蔬菜汁或水料，其種類絲限定，主要為-般仙性蔬果類。 再者’在一預定溫度範圍下(較佳為攝氏20-30度之間)進行通 氣持續靜置發酵培養一預定時間(大、約2-3天)(步驟1〇3)。 最終，疋成上述發酵步驟後，即可產生一具有超氧歧化酵素 (S〇D)之發酵液。為了測試其SOD活性測定、抗基因傷害及抗致 突變等功效’進行-系酬試如後。 201211258 實施例ι·超氧歧化酵素(super0Xide dismutase，S0D)活性之測定 本實施例中係採用商業分析套組(SUperoxide dismutase assay kit’ Cayman’ Ann Arbor，MI, USA)分析 SOD 之活性。可於波長 450 nm下測定其吸光值，依據s〇D之標準活性曲線計算出發酵液中 超氧歧化酵素的含量單位以Uint/mL表示。在此分別進行A、B、 C、D共四個組別，a發酵液為使用乳酸菌American Type Culture201211258 VI. Description of the Invention: [Technical Field] The present invention relates to a fermentation broth and a method for producing the same, and more particularly to a detoxification fermentation broth having a high concentration of superoxide dismutase (SOD) and a method for producing the same. [Prior Art] Fermentation technology can be applied in a wide range of applications, from extremely high-priced pharmaceutical-grade products to industrial enzymes, to beneficial microbes for food, biological insecticides for agriculture, and even from animal feed agents or directly The addition of microorganisms in the feed to waste recycling is within the scope of application of fermentation technology. In recent years, in addition to agriculture and food, fermentation technology has also been extended to the health food industry, including intestinal probiotics, edible and medicinal fungi, and the like. For example, lactic acid bacteria and yeast are used as fungi, and vegetable juice or fruit juice can be fermented to produce a variety of health-promoting substances, such as organic acids, polysaccharides, and vitamins produced in bacteria, or Detoxification, antioxidant superoxide dismutases (SOD). However, currently known lactic acid bacteria and yeast strains are fermented with vegetable juices, and the superoxide dismutase content can only reach 1000 Units/mL. SUMMARY OF THE INVENTION As described in the prior art, currently known as bacteriostatic and yeast-fermented vegetable juice fermentation broths, the superoxide dismutase content can only be up to 1000 Units/mL. Therefore, how to obtain a fermentation broth of high concentration superoxide dismutase by using fermentation technology is still an urgent problem to be solved. Accordingly, it is an object of the present invention to provide a method for producing a super-oxidative dismutase (10) cut 201211258 detoxification fermentation broth. Another object of the present invention is to provide a detoxified fermentation broth having a high concentration of superoxide disintegration (SOD). - Benfa County solves the problem of knowing the state of technology. The method of manufacturing is a method for producing superoxide dismutase (SOD) detoxification fermentation liquid, the steps of which include: (A) & for germ lactic acid talent "" (10) ^Based on the hail (four) calendar) and a brewing yeast {Saccharomyce cerevisie) (9) Take the county unit amount of the lactobacillus of the germ and the inoculation of the brewing yeast into the plant material with the predetermined sugar content; (C) in-predetermined Temperature Βτ is subjected to aeration and immersion fermentation culture for a predetermined time; (D) A fermentation broth with superoxide dismutase (gj〇D) is produced. Maternal = Meibu lactic acid rod (10) _ _====!:_ _Inspection of fruit = ground, the planting of step (9) of the predecessor method is vegetable juice or 2_ _, ^. The predetermined temperature range of step (C) of the above manufacturing method is preferably Celsius, The predetermined step (C) of the above manufacturing method is _3 days. (8) === (D) The superoxide dismutase in the fermentation broth 201211258 can be produced by the above manufacturing method and under the preferred conditions. Detoxification fermentation broth with high concentration of superoxide dismutase (SOD), including detoxification fermentation broth The superoxide dismutase (SOD) system is greater than 3000 Units/mL. According to the technical means adopted by the present invention, the genus Escherichia coli (k kankan training) and the Saccharomyces cerevisiae (4) myce cerev plus are selected by the organic plum epidermis. e) The fermentation broth fermented by the two strains can raise the superoxide dismutase to more than 3000 Units/mL. At the same time, it is found that it can effectively inhibit ultraviolet light irradiation and oxidize toxin by testing the fermentation liquid of the high concentration superoxide dismutase. The generated free radicals and the high temperature oil and barbecue coke damage, and in the anti-mutation test φ can prevent the occurrence of mutations caused by toxins. Through the proven efficacy, the fermentation broth of the present invention can be applied in the future. Various products having detoxification and antioxidation efficacy are prepared in the fields of cosmetics, food, chemicals, etc. [Embodiment] The method for producing a superoxide dismutase (SOD) detoxification fermentation liquid of the present invention can be obtained by the following examples. Fully understood that the person skilled in the art can do so. However, the implementation of this case is not limited by the following examples. # 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Oxide dismutase (Super〇xide (4), SOD), also known as superoxide dismutase, belongs to the metalloenzyme 'as a function of the type of bound metal ion'. There are three types: copper and zinc superoxide dismutase (Cu_Zn_s〇D) ), 6 201211258 Containing superoxide dismutase (Mn-SOD) and iron-containing superoxide dismutase (Fe-SOD> Three superoxide dismutases catalyze the disproportionation of superoxide anion radicals to hydrogen peroxide and oxygen . Anti-genetic damage and anti-mutagenicity. Here mainly refers to the phenomenon of genetic damage or even gene mutation caused by super-oxygen dismutase (S〇D) against various environmental or foreign factors, especially the use of comet analysis ( Comet assay) and Ames test 'test its ability to resist genetic damage and anti-mutagenicity. • Manufacturing method Referring to Fig. 1, it is a flow chart of a method for producing a superoxide dismutase detoxification fermentation broth according to the present invention. First, a Lactobacillus (Lactobaci Hus pZantarum) is used to make a yeast (Step 101). The Lactobacillus brevis and the Saccharomyces cerevisiae used herein are all selected from the epidermis of organic plum. Next, taking a predetermined unit amount (1〇9 CFU/mL) of the lactobacillus of the germ and inviting the brewing yeast to inoculate a planting primitive having a predetermined sugar content (visible material type and suitable adjustment)丨Q2). In this case, the raw materials for the weeds may be vegetable juices or water materials, and the types thereof are limited by silk, and are mainly-like fruits and vegetables. Further, the mixture is subjected to continuous gas fermentation for a predetermined period of time (large, about 2-3 days) under a predetermined temperature range (preferably between 20 and 30 degrees Celsius) (step 1〇3). Finally, after the above fermentation step, a fermentation broth having superoxide dismutase (S〇D) can be produced. In order to test its SOD activity assay, anti-gene damage and anti-mutagenic effects, the system was tested as follows. 201211258 Example 1 Measurement of super0Xide dismutase (SOD) activity In this example, the activity of SOD was analyzed using a commercial analysis kit (SUperoxide dismutase assay kit' Cayman' Ann Arbor, MI, USA). The absorbance can be measured at a wavelength of 450 nm, and the unit of superoxide dismutase in the fermentation broth is expressed in terms of Uint/mL according to the standard activity curve of s〇D. There are four groups of A, B, C, and D, respectively. The fermentation broth is lactic acid bacteria American Type Culture.
Collection (ATCC)14917，B 發酵液為使用乳酸菌 American Type Culture Collection (ATCC)8014 與市售酵母(澳洲 Coopers 公司），C 發酵液為使用乳酸菌 American Type Culture Colleetion (ATCC)14917與市售酵母(澳洲c00pers公司），而d組為本發明之 SOD排毒發酵液。 其結果如第2圖所示，a、B、C發酵液分別含890、280、950 Uints/ mL 之 SOD，SOD 排毒發酵液含 3500 Units/ mL 之 SOD。 一般而言，已知的乳酸菌與酵母菌菌種所發酵的蔬果汁發酵液其 超氧歧化酵素含量最高只能達1〇〇〇Units/mL，然而經由本發明製 造方法所得之發酵液’其中的超氧歧化酵素(s〇D)含量明顯高於習 知發酵液的超氧歧化酵素含量。 實施例2.細胞基因傷害測試 本實施例之細胞基因傷害測試，是採用彗星分析法(c〇met assay ’ Aderson et al” 1998) ’並以肝癌細胞HepG2作為觀察標的， 此方法可將DNA所受觸害程度呈現不關形。影像分析與計算 為隨機取50個彗星影像，於螢光顯微鏡下觀察，並計算尾部長度 百分比(％ of tail length) ’尾部區域長度百分比（％ 〇ftail leng% %TL)=(尾部DNA長度/彗星影像全長)χ1〇〇。第3圖中各組別名稱 201211258 縮寫含義如下：SOD為本發明SOD排毒發酵液、UVB表照射紫 外光、S9為小鼠肝臟活化酵素、2AF為致癌劑2_Aminifluorene、Collection (ATCC) 14917, B fermentation broth using lactic acid bacteria American Type Culture Collection (ATCC) 8014 and commercially available yeast (Coopers, Australia), C fermentation broth using lactic acid bacteria American Type Culture Colleetion (ATCC) 14917 and commercially available yeast (Australia) C00pers company), and group d is the SOD detoxification fermentation broth of the invention. The results are shown in Figure 2. The a, B, and C fermentation broths contain 890, 280, and 950 Uints/mL of SOD, and the SOD detoxification broth contains 3,500 Units/mL of SOD. In general, the vegetable juice fermentation broth fermented by the known lactic acid bacteria and the yeast strain has a superoxide dismutase content of up to 1 Units/mL, but the fermentation broth obtained by the production method of the present invention The content of superoxide dismutase (s〇D) is significantly higher than the superoxide dismutase content of the known fermentation broth. Example 2. Cellular Gene Injury Test The cell gene injury test of this example was carried out by using the comet assay (c〇met assay 'Aderson et al" 1998) and using the hepatoma cell HepG2 as an observation target. The degree of damage is not visible. Image analysis and calculation are randomly taken 50 comet images, observed under a fluorescent microscope, and the percentage of tail length is calculated (% of tail length) '% of tail area length (% 〇ftail leng% %TL)=(tail DNA length/full length of comet image)χ1〇〇. In Figure 3, each group name 201211258 has the following abbreviations: SOD is the SOD detoxification fermentation broth of the invention, UVB is irradiated with ultraviolet light, and S9 is mouse liver. Activase, 2AF is a carcinogen 2_Aminifluorene,
4NQO 為致癌劑 4-nittroquinoline-N-oxide、HO 表高溫炸油、HC 表碳烤焦黑碎削。在此共分為空白組(培養液）、SOD對照組(SOD 排毒發酵液）、18 組實驗組:UVB、UVB+SOD、2FA、2FA+SOD、 2FA+S9、2FA+S9+SOD、4NQO、4NQO+SOD、4NQO+S9、 4NQO+S9+SOD、HO、HO+SOD、HO+S9、HO+S9+SOD、HC、 HC+SOD、HC+S9、HC+S9+SOD。 結果如第3圖所示，其顯示出本發明之超氧歧化酵素排毒發 酵液具有抑制紫外線、2-aminifluorene 及 4-nitroquinoline-N-oxide 致癌劑以及高溫炸油、碳烤焦黑碎削所造成的基因傷害現象。 實施例3.抗致突變測試 本實施例採用的安姆試驗(Ames test)，是短期遺傳毒物檢測系 統中最常偵測致突變物質的方法之一。本實驗採用的微生物為 Salmonella typhimurium組胺酸合成缺陷株(His-)，觀察其產生回復 突變菌落數的數目，可以判定受試物質的致突變作用。第4圖中 各組別名稱縮寫含義如下：SOD表本發明SOD排毒發酵液、UVB 表照射紫外光、S9為小鼠肝臟活化酵素、2AF為致癌劑4NQO is a carcinogen 4-nittroquinoline-N-oxide, HO table high temperature frying oil, HC table carbon scorched black crushed. It is divided into blank group (culture medium), SOD control group (SOD detoxification fermentation broth), and 18 experimental groups: UVB, UVB+SOD, 2FA, 2FA+SOD, 2FA+S9, 2FA+S9+SOD, 4NQO 4NQO+SOD, 4NQO+S9, 4NQO+S9+SOD, HO, HO+SOD, HO+S9, HO+S9+SOD, HC, HC+SOD, HC+S9, HC+S9+SOD. The results are shown in Fig. 3, which shows that the superoxide dismutase detoxification fermentation broth of the present invention has the effects of inhibiting ultraviolet rays, 2-aminifluorene and 4-nitroquinoline-N-oxide carcinogens, as well as high temperature frying oil and carbon scorch black crushing. The phenomenon of genetic damage. Example 3. Anti-mutation test The Ames test used in this example is one of the most frequently detected methods for detecting mutagenic substances in short-term genetic toxicology detection systems. The microorganism used in this experiment was Salmonella typhimurium histidine-deficient strain (His-), and the number of revertant mutants was observed to determine the mutagenic effect of the test substance. The meanings of the abbreviations of each group name in Fig. 4 are as follows: SOD table SOD detoxification fermentation broth of the invention, UVB meter irradiation ultraviolet light, S9 is mouse liver activation enzyme, 2AF is carcinogen
2-Aminifluorene、4NQO 為致癌劑 4-nittroquinoline-N-oxide、HO 表高溫炸油、HC表碳烤焦黑碎削。在此共分為空白組(培養液）、 SOD對照組(SOD排毒發酵液）、18組實驗組:uvb、uyb+sOD、 2FA、2FA+SOD、2FA+S9、2FA+S9+SOD、4NQO、4NQO+SOD、 4NQO+S9、4NQO+S9+SOD、HO、HO+SOD、HO+S9、 HO+S9+SOD ' HC ' HC+SOD ' HC+S9 ' HC+S9+SOD 〇 201211258 結果如第4圖所示，其顯示出本發明之超氧歧化酵素排毒發 酵液具有抑制紫外線、2-aminifluorene 及 4-nitroquinoliiie_：Ni_〇xide 致癌劑以及高溫炸油、碳烤焦黑碎削所造成的基因突變傷害現象。 經由上述實施例說明’經由本發明之製造方法所製得之發酵 液’除了其中的超氧歧化酵素(SOD)含量明顯高於習知發酵液的超 氧歧化酵素含量外，且進一步證實其具有顯著的抗基因傷害及抗 致突變等功效，未來可應用於化妝品、食品、化工等領域。 【圖式簡單說明】 第1圖係為本發明超氧化歧化酵素排毒發酵液之製造方法流程圖； 第2圖係顯示第一實施例之超氧歧化酵素活性測定結果； 第3圖係顯示第二實施例之細胞基因傷害測試結果； 第4圖係顯示第三實施例之抗致突變測試。 【主要元件符號說明】2-Aminifluorene, 4NQO is a carcinogen 4-nittroquinoline-N-oxide, HO table high temperature frying oil, HC table carbon scorched black crushed. It is divided into blank group (culture medium), SOD control group (SOD detoxification fermentation broth), and 18 experimental groups: uvb, uyb+sOD, 2FA, 2FA+SOD, 2FA+S9, 2FA+S9+SOD, 4NQO , 4NQO+SOD, 4NQO+S9, 4NQO+S9+SOD, HO, HO+SOD, HO+S9, HO+S9+SOD 'HC ' HC+SOD ' HC+S9 ' HC+S9+SOD 〇201211258 As shown in Fig. 4, it shows that the superoxide dismutase detoxification fermentation broth of the present invention has the effects of inhibiting ultraviolet rays, 2-aminifluorene and 4-nitroquinoliiie_:Ni_〇xide carcinogens, as well as high-temperature frying oil and carbon scorch black crushing. Gene mutation damage phenomenon. The above description shows that the fermentation broth prepared by the production method of the present invention has a superoxide dismutase (SOD) content higher than that of the conventional fermentation broth, and further confirms that it has Significant anti-genetic damage and anti-mutagenic effects, can be applied to cosmetics, food, chemical and other fields in the future. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing a method for producing a superoxide dismutase detoxification fermentation liquid of the present invention; Fig. 2 is a graph showing the results of superoxide dismutase activity measurement in the first embodiment; The results of the cellular gene injury test of the second embodiment; Fig. 4 shows the anti-mutagenic test of the third embodiment. [Main component symbol description]