1242412 狄、發明說明： 【發明所屬之技術領域】 所製造之抗癌性 本發明係關於一種利用靈芝菌絲體於綠藻中培養 生機食品，以及其相關之製法。 【先前技術】 綠藻門植物體多呈綠色，故名綠藻。由於所含葉綠素a、b之 與高等植物相似，光合侧產物也驗粉，細胞壁也是纖維素所組成， 故-般認綠藻與陸生高等植物之演化有密不可分的關係。 綠藻形態變化萬千，葉綠體的形式也純當複雜。除少數種類細 胞的原生質體裸露、無細酸外，絕大錄都有細胞壁。細胞壁内層 為纖維素’外層為果膠質，|面平滑或具顆粒、孔紋、瘤刺、刺毛等 構造。細胞核-個，少數種類多個，具核仁和核膜。綠藻中，可運動 的細胞常會具二條頂生，等長的鞭毛，少數為4條，極少數為更多條 者。有的生殖細胞具-輪頂生鞭毛。在鞭毛著生的基部，—般都具有 二個伸縮泡。眼點-個，桔紅色，位於細胞的前部側面。 綠藻之色素成分與高等植物相似，有葉綠素a、b、葉黃素和胡蘿 蔔素。綠騎含有的葉綠素是植物界之冠，故其顏色呈現鮮綠色。 它在Π-20小時内可以分化4次，因此綠藻所含的核酸也是非常豐富 。綠藻中含約6G%的植物性蛋白f、2()%的複合性殿粉、11%的脂肪， 其中的82%為不飽和脂肪酸、豐富的DNA&RNA、超過幻種的維生 素及礦物質、8種必需胺基酸。綠藻除了含有上述的一般營養素外，它 也含有含有一些特殊的營養素，如細胞壁纖維、多醣體和葉綠素。其 中多醣體至少有兩類型：果膠物質或木聚糖-甘露多聽。 此外，已知種單細胞綠藻，，被使用為生物 反應之修飾幻(參知、Y· Miyazawa 等人，j〇umal 〇f Ethnopharmacology， 1242412 24(1988)，頁 135-146)。於 C57BL/6(B6)、C3H/He 及 DDD/l 小鼠中， 以腹膜内或口服方式隔曰投予已滅菌之綠藻細胞或其萃取物十曰後， 將小鼠乳癌(MM-2)或艾利希氏腹水細胞(Ehrlich ascites cells)移植於腹 腔内，另亦進行小鼠之白血病細胞(EL-4)之皮下移植。所有之對照組小 亂均於腫瘤細胞移植20日後死亡，然而有73.3-80%之經綠藻處理之小 鼠(MM-2於C3H/He以及EL-4於B6)於60日後仍存活。由於綠藻細胞 及其衍生物並為顯示出其對腫瘤或小鼠脾臟細胞具體外之胞毒活性， 是以綠藻之抗癌效應可能係藉由宿主之免疫反應而來。 靈芝是一種木質腐生菌，喜高温多濕及通風良好之環境，其分類 為：擔子菌亞門，無稽菌目，多孔菌科，靈芝屬。1881年尺咖⑶根據 具有表面漆光和菌柄的白腐型真菌jp(吻•血^ W⑸為基礎而 建立了靈芝屬(G⑽〇办rma)。靈芝在藥理研究和人工栽培時最常被使用 的學名為GWwczWwm，但常常在其中發現同名異物的情況，事實上是將 一群形態相似的G· /wc/办m complex當作同一種。在所謂的靈芝群(G /wcz麵compiex)中，其實包含有G加即e施订，G她恤讀B〇ud， G· oregonense Murr., G. resinaceum Boud.? G. pfeifferi Bres., G ame&UTorr·，GimW/ Stay”等形態相近的類緣近屬。 近年來由於人工栽培的研究成果顯示其代謝物中含有諸多具醫療 作用之活性成分，如多賴、類U機鍺化合物及腺苷等。如果 將國内外1芝之研究文獻詳加整理分析，發現靈芝具有抗癌、降金壓 三降血糖、抑制膽固醇、免疫及保肝的生理作用及藥理效果，所以靈 芝在日本早已大行其道’除醫藥學界對其積極研究探討外，各大藥廠 ^競相m但疋由於栽培成本過高，所提供之靈芝產品價位始終居 同不下，讓此種優異產品不夠普及化以廣泛嘉惠大眾之健康。 夕靈芝在經萃取的過程中，可得到分子量極大、不同麵的高分子 ^酶體’其沒有苦味，適合食用，同咖葡萄糖鍵結的方式和殿粉不 同所以不易被人體消化酵素分解成小分子的葡萄糖，而可刺激免疫 、、、田胞產生抗癌物質，故能在人體及動物體喊生多項重要的生理功能 1242412 ，幫助人體的健康。 靈芝多醣消滅癌細胞的機制為當靈芝多醣與巨噬細胞一起培養時 ，所產生之酵素會分解高分子多醣類而產生小分子的各種葡萄糖誘導 體，其會誘導巨噬細胞分泌免疫物質：干擾素(INF)-7、介白素、介 白素-2等等，使τ細胞變成殺手T細胞，殺手T細胞是具有攻擊能力 的T細胞，產生干擾素可刺激免疫細胞變為NK(Natural Killer ;天然殺 手)細胞，其對内可以抵抗癌細胞，對外可以找尋不屬於體内的生物以 攻擊之。 1998年11月台北榮民總醫院醫學研究部教授王聲遠即發表一篇「 靈芝免疫調節作用與抗腫瘤活性之關係」研究報告。他指出，使用高 純度化、高倍數的靈芝抽取液在人體内，可增強人體的免疫細胞作用 ，進而可直接或間接影響抑制到腫瘤。王聲遠表示，他將免疫細胞培 養在培養液内，發現靈芝對免疫細胞有增強的作用，而免疫細胞所產 生的激素可以抑制腫瘤的成長。研究顯示，巨嗟細胞培養液中的介白 素l(IL-l)、腫瘤壞死因子(丁仰），及介白素一6經過靈芝萃取液處理 後，將比未經處理的對照組分別多出四點一倍、九點八倍及二十九倍 ’這將可證明靈芝可以刺激τ淋巴細胞、巨噬細胞產生抗體，抑制腫 瘤細胞的生長。 鑑於靈芝之前述伽以及綠叙豐富營養成份，本發明意外地發 現利用綠藻培養真菌，特贱靈芝之_體，所培養出來的菌絲體會 ^泌各種分解多顏轉素，這轉素分解、_成份之_體後，產 他小的^办或"^6〉酶類(例如：雙聽類、半纖維素以及其 靈^ 口^體)的混合物，而發揮抗癌作用；如此竟可培養出與已知 碰絲綱之食品，甚至可提高靈芝时益人體健康之 【發明内容】 1242412 样^關於-種製造生機食品之方法，係彻賴 ，. 2㈣觀讀徵在於_駐科_其水抽岐歧 ' ^^(Gan〇dermaluc^ , 養利㈣絲體所分泌之酵素群將綠藻及/或其水抽 =收且有抗癌性之具有特殊生物學價值之食品，亦即含生= 峨财齡⑽品，此種應 本發明製法之培養條件可採用—般f用之操作方式進行；例如： 於適當容器中將賴與適量的水—起加熱抽出(可重覆抽出），並將抽出 液殺菌；冷卻後，添加菌絲體或其他真菌在2〇〜3(rc下培養$至w天 ，宜好氣性培養7至1G天’培養可於獅或振盈下進行；然後將培養 基全體冷絲燥’粉末化或進-步鍵劑化，即可食用，此產品沒有靈 芝特有之苦味缝顏色，也沒有毒性，餅低溫及/或傾度下保存 。本發明製法之殺菌可採習知方式進行，例如：加献、紫外線等。凡 熟習此微術者射依其經驗轉最適當之培養溫度''酿及時間。 又’本發明係關於利用綠藻及/或其水抽出物作為培養基原料以真 菌，特別是靈芝之菌絲體發酵培養之方法所製得之生機食品。 根據本發明製法，經肌C及離子交換層析法分析，由本發明製 法所得之產品含有下列成份之混合物： 1·靈芝蛋白質、醣質、脂質等三大營養素； 2·半纖維素之各種分餾產物；AHCC(活性六碳醣相關之化合物）； 3·肌醇六填酸(phytic acid); 4·微量成份：維生素氏、B2、E ; 5·各種有用之微置礦物質’如· Ca、Mg、Fe、Ge等· 6.抗癌物質、癌細胞生長抑制物質、免疫加強物質； 7·酵素··多類分解酵素、蛋白分解酵素。 1242412 組織淋巴瘤(diffuse histiocytic lymphoma)所獲得。於本案之實施例中， U937細胞係作為待測之腫瘤細胞’用以測試根據本發明所製得生機食 品之抗癌功效。 實驗步驟： 將U937細胞置於10公分培養盤中生長至八至九分滿後，將之以 胰蛋白酶作用，並分盤培養在含有10%FBS之DMEM之24孔培養盤 中，以不同濃度之靈芝菌絲體/綠藻培養物之水萃取液處理，在指定時 間點時吸除樣本液，並用一倍的PBS清洗一次，再加入〇·35毫升之 ΜΤΤ溶液，同時置入37χ、5%二氧化碳之培養箱中使反應三小時， 吸除未轉變之ΜΤΤ溶液後，加入〇·4毫升DMSO溶解細胞膜，避光作 用且均勻搖晃10至15分鐘，最後以ELISA讀取儀讀取波長57〇奈米 之吸光值。 ▲將U937細胞以上述步驟處理，其中分別以〇2、〇5、2與4微升/ 毫升之靈芝菌絲體/綠藻培養物之水萃取液處理，其MTT試驗之結果如 圖1所示，其中以2及4微升/毫升之靈芝菌絲體/綠藻培養物之水萃取 液處理之U937細胞分別於培養48小時與24小時後即死亡。 【圖式簡單說明】 圖1為本發明產品對U937細胞生長抑制之MTT試驗結果。 【1要部分代表符號說明】1242412 D. Description of the invention: [Technical field to which the invention belongs] The anti-cancer properties produced The present invention relates to a method for cultivating bio-food in green algae by using ganoderma mycelium. [Previous technology] Chlorophyta plants are mostly green, hence the name Chlorella. Because the chlorophyll a and b are similar to higher plants, the photosynthetic side products are also tested for powder, and the cell wall is also composed of cellulose. Therefore, it is generally recognized that green algae is closely related to the evolution of terrestrial higher plants. The morphology of green algae varies a lot, and the form of chloroplasts is pure and complicated. Except for a few types of cells, the protoplasts are bare and free of fine acids, and most of them have cell walls. The inner layer of the cell wall is made of cellulose. The outer layer is made of pectin, and the surface is smooth or has grains, holes, thorns, bristles and other structures. Nuclei-one, a few species, multiple, with nucleoli and nuclear membrane. In green algae, the mobile cells often have two terminal, equal-length flagella, a few are four, and a few are more. Some germ cells have verticillary flagella. At the base of flagellar growth, there are usually two expansion bubbles. Eyepoint-one, orange-red, located on the front side of the cell. Chlorella has similar pigment composition to higher plants, including chlorophyll a, b, lutein, and carotene. The chlorophyll contained in the green ride is the crown of the plant kingdom, so its color is bright green. It can differentiate 4 times in Π-20 hours, so the nucleic acids contained in green algae are also very rich. Chlorella contains approximately 6G% of plant protein f, 2 ()% of compound powder, and 11% of fat, of which 82% are unsaturated fatty acids, rich in DNA & RNA, vitamins and minerals that are more than phantom species. Substance, 8 essential amino acids. In addition to the general nutrients mentioned above, green algae also contains special nutrients such as cell wall fibers, polysaccharides, and chlorophyll. There are at least two types of polysaccharides: pectin substances or xylan-mannose. In addition, a single-celled green algae is known and is used as a modification of biological response (cf. Yoshi Miyazawa et al., Joumal Of Ethnopharmacology, 1242412 24 (1988), pages 135-146). In C57BL / 6 (B6), C3H / He, and DDD / l mice, sterilized green algae cells or their extracts were administered intraperitoneally or orally at intervals of ten days, and mice were subjected to breast cancer (MM- 2) Or Ehrlich ascites cells were transplanted into the abdominal cavity, and mouse leukemia cells (EL-4) were also transplanted subcutaneously. All the control group mice died 20 days after tumor cell transplantation, however, 73.3-80% of the green algae-treated mice (MM-2 at C3H / He and EL-4 at B6) survived 60 days. Because green algae cells and their derivatives do not show their specific cytotoxic activity against tumor or mouse spleen cells, the anti-cancer effect of green algae may be caused by the host's immune response. Ganoderma lucidum is a wood saprophytic fungus, which enjoys high temperature and humidity and good ventilation. Its classification is: Basidiomycotina, Agrobacterium, Poaceae, Ganoderma. In 1881, Ganoderma lucidum was established based on the white-rot fungus jp (kiss · blood ^ W⑸) with a surface varnish and a stalk. G. lucidum (G⑽〇 Office rma) was established. Ganoderma lucidum is most commonly used in pharmacological research and artificial cultivation. The scientific name used is GWwczWwm, but the case of foreign bodies with the same name is often found. In fact, a group of similar G · / wc / ban m complex is regarded as the same. In the so-called Ganoderma group (G / wcz surface compiex) In fact, it includes G plus e-booking, G she reads Bood, G. oregonense Murr., G. resinaceum Boud.? G. pfeifferi Bres., G ame & Utorr, GimW / Stay, etc. A similar genus. In recent years, due to the research results of artificial cultivation, its metabolites contain many active ingredients with medical effects, such as Dorai, U-like germanium compounds, and adenosine. The research literature has been carefully analyzed, and it has been found that Ganoderma has physiological effects and pharmacological effects of anti-cancer, lowering blood pressure, reducing blood sugar, inhibiting cholesterol, immunity, and liver protection. Therefore, Ganoderma has long been popular in Japan. , Major pharmaceutical companies However, because the cultivation cost is too high, the price of the ganoderma product provided is always the same, which makes this excellent product not popular enough to widely benefit the health of the public. Xi Lingzhi can obtain a very large molecular weight during the extraction process. The polymer ^ enzyme body with different sides has no bitterness and is suitable for consumption. It is different from the way of glucose binding with coffee and it is not easy to be broken down into small molecules of glucose by the digestive enzymes of the human body, and it can stimulate the immune cells. It produces anti-cancer substances, so it can produce a number of important physiological functions in the human body and animals 1242412, helping the health of the human body. The mechanism of ganoderma polysaccharides to destroy cancer cells is that when ganoderma polysaccharides are cultured with macrophages, the enzymes produced will Various glucose inducers that break down polymer polysaccharides to produce small molecules will induce macrophages to secrete immune substances: interferon (INF) -7, interleukin, interleukin-2, etc., making τ cells become Killer T cells, killer T cells are attacking T cells that produce interferons to stimulate immune cells to become NK (Natural Killer; natural killer) cells. Cancer cells can be resisted inside and organisms outside the body can be found to attack them. In November 1998, Wang Shengyuan, professor of the Medical Research Department of Taipei Rongmin General Hospital, published a study on the "Relationship between Ganoderma Immune Regulation and Antitumor Activity" The report. He pointed out that the use of high-purity, high-magnification ganoderma extract in the human body can enhance the body's immune cell function, which can directly or indirectly affect the suppression of tumors. Wang Shengyuan said that he cultured immune cells in the culture solution It was found that Ganoderma has an enhanced effect on immune cells, and hormones produced by immune cells can inhibit tumor growth. Studies have shown that interleukin-1 (IL-1), tumor necrosis factor (Ding Yang), and interleukin-6 in the culture medium of giant salamander cells were treated with Ganoderma lucidum extract, respectively, compared with the untreated control group. "4.1 times more, 9.8 times, and 29 times more" This will prove that Ganoderma can stimulate τ lymphocytes and macrophages to produce antibodies and inhibit tumor cell growth. In view of the rich nutritional content of the aforementioned ganoderma lucidum and ganoderma lucidum, the present invention has unexpectedly found that the fungus, the base of ganoderma lucidum, is cultivated using green algae, and the cultured mycelium will secrete various degraded polyphasin, which is broken down After the _ component of the _ body, other small enzymes (such as: dual hearing, hemicellulose and its ^ ^ ^ ^ body) mixture to play an anti-cancer effect; so It is possible to cultivate foods that are in contact with known silkworms, and even improve the health of Ganoderma lucidum. [Content of the invention] 1242412 Sample ^ About-a method for manufacturing bio-food, it is completely based. Family_its water pumping ambiguity '^^ (Gan〇dermaluc ^, the enzyme group secreted by the nutrient fibrosus extracts green algae and / or its water = collects and has anti-cancer properties of food with special biological value That is, containing health = Ecai Ling counterfeit, this kind of culture conditions according to the method of the present invention can be carried out using the general operation method; for example: in a suitable container, rely on a suitable amount of water-heat and extract (may Repeat the extraction), and sterilize the extract; after cooling, add mycelium or other The bacteria are cultured at 20 to 3 days (rc to $ w days, preferably aerobic culture 7 to 1G days). The culture can be performed under lion or vibrating; then the whole culture medium is cold-dried and powdered or further-bonded. It is ready for consumption. This product does not have the unique bitter seam color of ganoderma, nor is it toxic. The cake is stored at low temperature and / or inclination. The sterilization of the method of the present invention can be carried out in known ways, such as: addition, UV, etc. Those who are familiar with this microsurgery shoot the most suitable culture temperature according to their experience, and the time is also called. The invention also relates to the use of green algae and / or its water extract as the raw material of the culture medium to fungi, especially the mycelium of Ganoderma The organic food produced by the fermentation culture method. According to the method of the present invention, analyzed by muscle C and ion exchange chromatography, the product obtained by the method of the present invention contains a mixture of the following components: 1. Ganoderma protein, sugar, lipid and other three Macronutrients; 2. Various fractionated products of hemicellulose; AHCC (active six-carbon sugar-related compounds); 3. Phytic acid; 4. Trace ingredients: Vitamins, B2, E; 5 · Various useful micro settings Substances such as Ca, Mg, Fe, Ge, etc. 6. Anticancer substances, cancer cell growth inhibiting substances, immune-enhancing substances; 7. Enzymes ... Multi-class degrading enzymes, proteolytic enzymes. 1242412 Tissue lymphoma lymphoma). In the example of this case, the U937 cell line was used as the tumor cell to be tested to test the anti-cancer effect of the bio-food prepared according to the present invention. Experimental steps: Place U937 cells in a 10 cm culture plate After growing to eight to nine minutes, trypsinize them and culture them in a 24-well culture plate containing 10% FBS DMEM in different concentrations of ganoderma mycelium / green algae culture water. Extraction treatment, aspirate the sample solution at the specified time point, wash it once with PBS twice, add 0.35 ml of MTT solution, and place it in a 37 ×, 5% carbon dioxide incubator to react for three hours. After removing the untransformed MTT solution, 0.4 ml of DMSO was added to dissolve the cell membrane, protected from light and shaken uniformly for 10 to 15 minutes. Finally, the absorbance at a wavelength of 57 nm was read by an ELISA reader. ▲ U937 cells were treated in the above steps, among which were treated with water extracts of ganoderma mycelium / green algae culture of 〇2, 05, 2 and 4 microliters / ml. The results of the MTT test are shown in Figure 1. It was shown that U937 cells treated with 2 and 4 microliters / ml of ganoderma mycelia / green algae culture water extracts died after 48 and 24 hours in culture, respectively. [Brief description of the figure] Figure 1 shows the results of the MTT test on the growth inhibition of U937 cells by the product of the present invention. 【1Description of some representative symbols】