TWI819429B - Fungal fusant strain, method of manufacturing the same and composition including the same - Google Patents
Fungal fusant strain, method of manufacturing the same and composition including the same Download PDFInfo
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Abstract
Description
本發明是有關於一種真菌融合株，特別是一種樟芝及北蟲草之原生質體融合而成，且活性物質含量較高的真菌融合株、其製造方法及含其之組成物。The present invention relates to a fungal fusion strain, particularly a fungal fusion strain formed by the fusion of protoplasts of Antrodia camphorata and Cordyceps militaris and having a high content of active substances, its manufacturing method and compositions containing it.
次級代謝物(secondary metabolites)或天然產物(natural products)係初級代謝物透過特定的生合成路徑獲得之物質。這些次級代謝物不涉及產生這些次級代謝物之生物的生長、發育及/或繁殖，但可藉由改變此生物的種間關係，從而提高此生物在適應環境、防禦天敵、協助溝通及/或競爭的優勢。此外，近來的研究還發現有些次級代謝物是對人體的健康有正面影響之活性物質，而可做為保健食品、藥物或其他醫藥組成物中的有效成分。Secondary metabolites or natural products are substances obtained from primary metabolites through specific biosynthetic pathways. These secondary metabolites are not involved in the growth, development and/or reproduction of the organism that produces these secondary metabolites, but can improve the organism's ability to adapt to the environment, defend against natural enemies, assist in communication and /or competitive advantage. In addition, recent research has also found that some secondary metabolites are active substances that have a positive impact on human health and can be used as active ingredients in health foods, drugs or other pharmaceutical compositions.
許多大型真菌所含之特殊次級代謝物是有益於人體健康的活性物質，而可做為藥物或保健食品的有效成分。舉例而言，樟芝( Antrodia cinnamomea)具有多種多醣體及三萜類(triterpenoids)，且北蟲草( Cordyceps militaris)是蟲草酸(cordycepic acid)及蟲草素(cordycepin)的重要來源。目前研究發現，樟芝和北蟲草在保護肝臟、調節免疫力、抗發炎、抗腫瘤及抗老化等方面皆有顯著的正向功效。除此之外，樟芝還可抗前列腺腫大，而北蟲草可抗肥胖、降血糖、降血脂及改善慢性呼吸道疾病等。 The special secondary metabolites contained in many large fungi are active substances beneficial to human health and can be used as active ingredients in medicines or health foods. For example, Antrodia cinnamomea has a variety of polysaccharides and triterpenoids, and Cordyceps militaris is an important source of cordycepic acid and cordycepin. Current research has found that Antrodia camphorata and Cordyceps militaris have significant positive effects in protecting the liver, regulating immunity, anti-inflammation, anti-tumor and anti-aging. In addition, Antrodia camphorata can also fight prostate enlargement, while Cordyceps militaris can fight obesity, lower blood sugar, lower blood lipids, and improve chronic respiratory diseases.
然而，樟芝及北蟲草皆為寄生型菌類，其中樟芝需寄生於牛樟樹，且北蟲草寄生於鱗翅目昆蟲的蟲蛹。考量到環境保育及產業化所需的成本，如何以人工方式培養樟芝及北蟲草並提高其生物量及/或活性物質之產量成為新的研究目標。其中，細胞融合技術可以突破遺傳限制而結合兩種菌種的優勢，係新的研究方向。然而，目前沒有樟芝及北蟲草的細胞融合方法，也沒有結合樟芝及北蟲草兩種菌種優勢的真菌融合株。However, both Antrodia camphorata and Cordyceps militaris are parasitic fungi. Among them, Antrodia camphorata needs to be parasitic on the Cinnamomum camphora tree, and Cordyceps militaris is parasitic on the pupae of lepidopteran insects. Considering the costs required for environmental conservation and industrialization, how to artificially cultivate Antrodia camphorata and Cordyceps militaris and increase their biomass and/or the production of active substances has become a new research goal. Among them, cell fusion technology can break through genetic limitations and combine the advantages of two bacterial species, which is a new research direction. However, there is currently no cell fusion method for Antrodia camphorata and Cordyceps militaris, and there is no fungal fusion strain that combines the advantages of the two strains.
有鑑於此，需提供一種樟芝及北蟲草的真菌融合株，以解決上述問題。In view of this, it is necessary to provide a fungal fusion strain of Antrodia camphorata and Cordyceps militaris to solve the above problems.
因此，本發明之一態樣是提供一種真菌融合株，可人工培養，且相較於親代株，此真菌融合株的生物量及活性物質含量較高。Therefore, one aspect of the present invention is to provide a fungal fusion strain that can be artificially cultivated, and compared with the parent strain, the fungal fusion strain has higher biomass and active substance content.
本發明之另一態樣是提供一種真菌融合株之組成物，其係以真菌融合株做為有效成分。Another aspect of the present invention is to provide a composition of a fungal fusion strain, which uses the fungal fusion strain as an active ingredient.
本發明之又一態樣是提供一種真菌融合株，其中此真菌融合株可例如為樟芝( Antrodia cinnamomea)Ac-Cm-2。 Another aspect of the present invention is to provide a fungal fusion strain, wherein the fungal fusion strain can be, for example, Antrodia cinnamomea Ac-Cm-2.
本發明之再一態樣是提供一種真菌融合株，其中此真菌融合株可例如為( Cordyceps militaris)北蟲草Cm-Ac-1。 Another aspect of the present invention is to provide a fungal fusion strain, wherein the fungal fusion strain can be, for example, ( Cordyceps militaris ) Cordyceps militaris Cm-Ac-1.
本發明之又另一態樣是提供一種真菌融合株之製造方法，包含對樟芝之原生質體及北蟲草之原生質體進行細胞融合步驟，以獲得真菌融合株。Yet another aspect of the present invention provides a method for producing a fungal fusion strain, which includes performing a cell fusion step on protoplasts of Antrodia camphorata and protoplasts of Cordyceps militaris to obtain a fungal fusion strain.
根據本發明之上述態樣，提供一種真菌融合株，其可包含但不限於樟芝Ac-Cm-2及/或北蟲草Cm-Ac-1。樟芝Ac-Cm-2可例如於2020年3月3日寄存在臺灣財團法人食品工業發展研究所(Industry Research and Development Institute，FIRDI)生物資源中心(Bioresource Collection and Research Center，BCRC)，且其寄存編號為BCRC 930218。北蟲草Cm-Ac-1可例如於2019年11月6日寄存在BCRC，且其寄存編號為BCRC 930213。According to the above aspect of the present invention, a fungal fusion strain is provided, which may include but is not limited to Antrodia camphorata Ac-Cm-2 and/or Cordyceps militaris Cm-Ac-1. Antrodia camphorata Ac-Cm-2 can, for example, be deposited at the Bioresource Collection and Research Center (BCRC) of Taiwan's Industry Research and Development Institute (FIRDI) on March 3, 2020, and its The registration number is BCRC 930218. Cordyceps militaris Cm-Ac-1 can be deposited at the BCRC on November 6, 2019, for example, and its deposit number is BCRC 930213.
根據本發明之另一態樣，提出一種含真菌融合株之組成物，可包含但不限於以真菌融合株做為有效成分。According to another aspect of the present invention, a composition containing fungal fusion strains is proposed, which may include but is not limited to fungal fusion strains as active ingredients.
根據本發明之又一態樣，提出一種真菌融合株，其中真菌融合株可例如為樟芝Ac-Cm-2，且樟芝Ac-Cm-2的寄存編號可例如為BCRC 930218。根據本發明之一實施例，此真菌融合株是將親代樟芝之原生質體及親代北蟲草之原生質體融合後獲得，親代樟芝的寄存編號可例如為BCRC 35396，且親代北蟲草的寄存編號可例如為BCRC 32219。根據本發明之一實施例，相較於親代樟芝，真菌融合株之生物量含量提高75%至85%，腺苷含量提高45%至55%，多醣體含量提高45%至55%，三萜類含量提高100%至110%，且蟲草酸含量提高25%至35%。According to another aspect of the present invention, a fungal fusion strain is proposed, wherein the fungal fusion strain can be, for example, Antrodia camphorata Ac-Cm-2, and the registration number of Antrodia camphorata Ac-Cm-2 can be, for example, BCRC 930218. According to one embodiment of the present invention, the fungal fusion strain is obtained by fusing the protoplasts of the parent Antrodia camphorata and the protoplasts of the parent Cordyceps militaris. The registration number of the parent Antrodia camphorata can be, for example, BCRC 35396, and the parent Cordyceps militaris has a registration number of BCRC 35396. The registration number of Cordyceps sinensis can be, for example, BCRC 32219. According to one embodiment of the present invention, compared with the parent Antrodia camphorata, the biomass content of the fungal fusion strain is increased by 75% to 85%, the adenosine content is increased by 45% to 55%, and the polysaccharide content is increased by 45% to 55%. The content of triterpenes is increased by 100% to 110%, and the content of cordycepic acid is increased by 25% to 35%.
根據本發明之再一態樣，提出一種真菌融合株，其中真菌融合株可例如為北蟲草Cm-Ac-1，且北蟲草Cm-Ac-1的寄存編號可例如為BCRC 930213。根據本發明之一實施例，此真菌融合株是將親代樟芝之原生質體及親代北蟲草之原生質體融合後獲得，親代樟芝的寄存編號可例如為BCRC 35396，且親代北蟲草的寄存編號可例如為BCRC 32219。根據本發明之一實施例，相較於親代北蟲草，真菌融合株之生物量含量提高95%至105%，腺苷含量提高20%至30%，多醣體含量提高200%至210%，三萜類含量提高210%至220%，且蟲草酸含量提高55%至65%。根據本發明之一實施例，此真菌融合株含有蟲草素。According to yet another aspect of the present invention, a fungal fusion strain is proposed, wherein the fungal fusion strain can be, for example, Cordyceps militaris Cm-Ac-1, and the registration number of Cordyceps militaris Cm-Ac-1 can be, for example, BCRC 930213. According to one embodiment of the present invention, the fungal fusion strain is obtained by fusing the protoplasts of the parent Antrodia camphorata and the protoplasts of the parent Cordyceps militaris. The registration number of the parent Antrodia camphorata can be, for example, BCRC 35396, and the parent Cordyceps militaris has a registration number of BCRC 35396. The registration number of Cordyceps sinensis can be, for example, BCRC 32219. According to one embodiment of the present invention, compared with the parent Cordyceps militaris, the biomass content of the fungal fusion strain is increased by 95% to 105%, the adenosine content is increased by 20% to 30%, and the polysaccharide content is increased by 200% to 210%. The triterpene content increased by 210% to 220%, and the cordycepic acid content increased by 55% to 65%. According to an embodiment of the present invention, the fungal fusion strain contains cordycepin.
根據本發明之又另一態樣，提供一種真菌融合株之製造方法，包含將樟芝之原生質體及北蟲草之原生質體進行細胞融合步驟，以獲得真菌融合株。樟芝的寄存編號可例如為BCRC 35396，北蟲草的寄存編號可例如為BCRC 32219。在一實施例中，樟芝之原生質體與北蟲草之原生質體之一者係經熱處理，另一者係經光處理。熱處理可例如為以55°C至65°C加熱10分鐘至30分鐘，且光處理可例如為以25瓦至35瓦、200 nm至300 nm的紫外線照射4分鐘至19分鐘。According to yet another aspect of the present invention, a method for producing a fungal fusion strain is provided, which includes performing a cell fusion step of protoplasts of Antrodia camphorata and protoplasts of Cordyceps militaris to obtain a fungal fusion strain. The registration number of Antrodia camphorata can be, for example, BCRC 35396, and the registration number of Cordyceps militaris can be, for example, BCRC 32219. In one embodiment, one of the protoplasts of Antrodia camphorata and the protoplasts of Cordyceps militaris has been heat-treated, and the other has been light-treated. The heat treatment may be, for example, heating at 55°C to 65°C for 10 to 30 minutes, and the light treatment may be, for example, irradiation with ultraviolet rays of 25 to 35 watts and 200 to 300 nm for 4 to 19 minutes.
應用本發明之真菌融合株，經人工培育後可提升此真菌融合株之腺苷、三萜類、多醣體、蟲草酸及蟲草素等活性物質的含量，可做為各種組成物的有效成分。Using the fungal fusion strain of the present invention, after artificial cultivation, the content of active substances such as adenosine, triterpenoids, polysaccharides, cordycepic acid and cordycepin in the fungal fusion strain can be increased, and can be used as active ingredients in various compositions.
承上所述，本發明提供一種真菌融合株，可包含但不限於樟芝Ac-Cm-2及北蟲草Cm-Ac-1，其中樟芝Ac-Cm-2係於2020年3月3日寄存於臺灣財團法人食品工業發展研究所(Industry Research and Development Institute，FIRDI)生物資源中心(Bioresource Collection and Research Center，BCRC)(地址：臺灣新竹市東區食品路331號，郵遞區號：300193)，寄存編號為BCRC 930218。北蟲草Cm-Ac-1係於2019年11月6日寄存於BCRC，且寄存編號為BCRC 930213。Based on the above, the present invention provides a fungal fusion strain, which may include but is not limited to Antrodia camphorata Ac-Cm-2 and Cordyceps militaris Cm-Ac-1, wherein Ac-Cm-2 of Antrodia camphorata was obtained on March 3, 2020. Deposited at the Bioresource Collection and Research Center (BCRC) of Taiwan's Industry Research and Development Institute (FIRDI) (Address: No. 331, Shishi Road, East District, Hsinchu City, Taiwan, Postal Area Code: 300193), deposited The number is BCRC 930218. Cordyceps militaris Cm-Ac-1 was deposited with BCRC on November 6, 2019, and the deposit number is BCRC 930213.
上述真菌融合株可例如對第一親代菌株與第二親代菌株之原生質體進行細胞融合步驟後獲得。第一親代菌株及第二親代菌株可為同種不同菌株、同屬不同種、同科不同屬，或是同界不同門之真菌菌株。本發明之真菌融合株可例如以同界不同門之菌種做為第一親代菌株及第二親代菌株。在一實施例中，以擔子菌門之樟芝( Antrodia cinnamomea)及子囊菌門之北蟲草( Cordyceps militaris)之一者為第一親代菌株，另一者為第二親代菌株，例如：以樟芝為第一親代菌株，並以北蟲草為第二親代菌株。 The above-mentioned fungal fusion strain can be obtained, for example, by subjecting protoplasts of the first parent strain and the second parent strain to a cell fusion step. The first parent strain and the second parent strain may be different strains of the same species, different species of the same genus, different genera of the same family, or fungal strains of the same kingdom and different phyla. The fungal fusion strain of the present invention can, for example, use strains from different phyla in the same world as the first parent strain and the second parent strain. In one embodiment, one of Antrodia cinnamomea from Basidiomycota and Cordyceps militaris from Ascomycota is the first parent strain, and the other one is the second parent strain, for example: Antrodia camphorata was used as the first parent strain, and Cordyceps militaris was used as the second parent strain.
上述原生質體可例如對樟芝及北蟲草之菌絲體進行細胞壁移除步驟後獲得。細胞壁移除步驟的方法不限，可包含但不限於離心、震盪、超音波、電穿孔及高壓滲透等物理處理及/或酵素法。上述酵素法可例如為利用細胞溶解酶分解細胞壁，其中細胞溶解酶可例如為消解酶、溶壁酶(lywallzyme)、蝸牛酶(snailase)及/或纖維素酶(cellulose)。上述細胞溶解酶之作用條件(如：使用量、作用時間及溫度)不限，可依據酵素種類、第一親代菌株及第二親代菌株之菌種進行調整。在一實施例中，細胞壁移除步驟可例如為在30°C至34°C、0.5 M至0.7 M的KCl下，以細胞溶解酶處理樟芝或北蟲草之菌絲體達1.5小時至2.5小時，從而獲得樟芝或北蟲草之原生質體。上述細胞溶解酶可例如由1.5重量%至2.0重量%之溶壁酶、1.0重量%之蝸牛酶、1.0重量%至1.5重量%之纖維素酶及上述任意組合所組成之一族群。在一具體例中，細胞壁移除步驟係在32°C、0.6 M之KCl下，以2.0重量%之溶壁酶及1.0重量%蝸牛酶處理樟芝或北蟲草之菌絲體達2.0小時。The above-mentioned protoplasts can be obtained, for example, by subjecting the mycelium of Antrodia camphorata and Cordyceps militaris to a cell wall removal step. The method of the cell wall removal step is not limited, and may include, but is not limited to, physical treatments such as centrifugation, shaking, ultrasound, electroporation, and high-pressure osmosis, and/or enzymatic methods. The above-mentioned enzymatic method may, for example, utilize a cell lytic enzyme to decompose the cell wall, wherein the cell lytic enzyme may be, for example, a lytic enzyme, a lywallzyme, a snailase, and/or a cellulase. The action conditions of the above-mentioned cell lytic enzymes (such as usage amount, action time and temperature) are not limited and can be adjusted according to the enzyme type, the first parent strain and the second parent strain. In one embodiment, the cell wall removal step may be, for example, treating the mycelium of Antrodia camphorata or Cordyceps militaris with a cytolytic enzyme at 30°C to 34°C and 0.5 M to 0.7 M KCl for 1.5 hours to 2.5 hours. hours to obtain protoplasts of Antrodia camphorata or Cordyceps militaris. The above-mentioned cell lytic enzyme may, for example, be a group consisting of 1.5 to 2.0% by weight of wall-lytic enzyme, 1.0% by weight of helicase, 1.0 to 1.5% by weight of cellulase, and any combination of the above. In a specific example, the cell wall removal step involves treating the mycelium of Antrodia camphorata or Cordyceps militaris with 2.0% by weight of wall-lytic enzyme and 1.0% by weight of helicase at 32°C and 0.6 M KCl for 2.0 hours.
在細胞壁移除步驟後，可包含對第一親代菌株及第二親代菌株之原生質體進行抑制處理步驟，以抑制原生質體之生長。值得說明的是，分別對第一親代菌株及第二親代菌株之原生質體進行不同的抑制處理步驟，可有利於真菌融合株之篩選。詳細而言，抑制處理步驟可分為熱處理及光處理，其中熱處理可使原生質體進入休眠狀態，且光處理可使原生質體之DNA片段化。因此，當對第一親代菌株的原生質體進行熱處理，且對第二親代菌株的原生質體進行光處理時，第一親代菌株的原生質體進入休眠狀態，第二親代菌株的原生質體之DNA片段化，兩者皆無法形成菌落。然而，經熱處理的第一親代菌株的原生質體的DNA仍保持完整，且經光處理的第二親代菌株的原生質體未進入休眠狀態，兩者在生理上可互補，因此如果融合兩者，獲得之真菌融合株可形成菌落。換言之，同時培養經熱處理的第一親代菌株的原生質體、經光處理的第二親代菌株的原生質體及真菌融合株，可形成菌落者為真菌融合株。因此，先對第一親代菌株之原生質體進行熱處理，且對第二親代菌株之原生質體進行光處理，可有利真菌融合株之篩選。After the cell wall removal step, an inhibition treatment step may be performed on the protoplasts of the first parent strain and the second parent strain to inhibit the growth of the protoplasts. It is worth noting that performing different inhibition treatment steps on the protoplasts of the first parental strain and the second parental strain can be beneficial to the screening of fungal fusion strains. In detail, the inhibition treatment steps can be divided into heat treatment and light treatment. Heat treatment can make the protoplasts enter a dormant state, and light treatment can fragment the DNA of the protoplasts. Therefore, when the protoplasts of the first parent strain are heat-treated and the protoplasts of the second parent strain are light-treated, the protoplasts of the first parent strain enter a dormant state and the protoplasts of the second parent strain enter a dormant state. The DNA is fragmented and neither can form colonies. However, the DNA of the protoplasts of the heat-treated first parent strain remains intact, and the protoplasts of the light-treated second parent strain do not enter a dormant state. The two are physiologically complementary, so if they are fused , the obtained fungal fusion strain can form colonies. In other words, the heat-treated protoplasts of the first parent strain, the light-treated protoplasts of the second parent strain, and the fungal fusion strain are cultured at the same time, and those that can form colonies are considered fungal fusion strains. Therefore, heat treatment of the protoplasts of the first parental strain and light treatment of the protoplasts of the second parental strain can facilitate the screening of fungal fusion strains.
熱處理的方法不限，可例如對第一親代菌株之原生質體以55°C至65°C加熱10分鐘至30分鐘。光處理的條件不拘，但需使第二親代菌株之DNA片段化而不損壞其胞器之效果。在一實施例中，光處理可例如以25瓦至35瓦、200 nm至300 nm的紫外線照射第二親代菌株之原生質體達4分鐘至19分鐘，其中第二親代菌株之原生質體與光源間距5公分至15公分，如10公分。The method of heat treatment is not limited. For example, the protoplasts of the first parent strain can be heated at 55°C to 65°C for 10 to 30 minutes. The conditions of light treatment are not restricted, but the effect must be to fragment the DNA of the second parent strain without damaging its organelles. In one embodiment, the light treatment may be, for example, irradiating the protoplasts of the second parent strain with ultraviolet light of 25 watts to 35 watts and 200 nm to 300 nm for 4 minutes to 19 minutes, wherein the protoplasts of the second parent strain are The distance between light sources is 5 cm to 15 cm, such as 10 cm.
在一實施例中，第一親代菌株為樟芝，且第二親代菌株為北蟲草，其中熱處理是對樟芝之原生質體以55°C加熱10分鐘，且光處理是對北蟲草之原生質體以30瓦、254 nm的紫外線照射4分鐘。In one embodiment, the first parent strain is Antrodia camphorata, and the second parent strain is Cordyceps militaris, wherein the heat treatment is to heat the protoplasts of Antrodia camphorata at 55°C for 10 minutes, and the light treatment is to treat Cordyceps militaris. Protoplasts were irradiated with 30 W, 254 nm UV for 4 minutes.
在另一實施例中，第一親代菌株是北蟲草，且第二親代菌株是樟芝，其中熱處理是對北蟲草之原生質體以55°C加熱10分鐘，且光處理是對樟芝之原生質體以30瓦、254 nm的紫外線照射7分鐘。In another embodiment, the first parent strain is Cordyceps militaris, and the second parent strain is Antrodia camphorata, wherein the heat treatment is to heat the protoplasts of Cordyceps militias at 55°C for 10 minutes, and the light treatment is to treat Antrodia camphorata The protoplasts were irradiated with 30 W, 254 nm ultraviolet light for 7 minutes.
上述細胞融合步驟係指突破細胞膜之限制，融合第一親代菌株及第二親代菌株的原生質體，從而獲得真菌融合株之技術。細胞融合步驟之方法不限，可例如：聚乙二醇(Polyethylene Glycol，PEG)等化學方法、電融合或激光誘導等物理方法，或者病毒誘導等生物方法。在一實施例中，聚乙二醇化學法使用之聚乙二醇的分子量不限，可例如為200至10000。在一實施例中，細胞融合步驟是以含35體積%至44體積%之PEG 2000及0.03 mol/L至0.12 mol/L之鈣離子溶液(pH值為6.2至8.0)於30°C至40°C下進行15至30分鐘。在一實施例中，細胞融合步驟的第一親代菌株之及第二親代菌株之原生質體的濃度分別為10 7CFU/mL。在一具體例中，細胞融合步驟是以含35體積%之PEG 2000及0.09 mol/L之鈣離子溶液(pH值為7.3)於34.60°C下進行10分鐘。 The above-mentioned cell fusion step refers to a technology that breaks through the limitations of the cell membrane and fuses the protoplasts of the first parent strain and the second parent strain to obtain a fungal fusion strain. The method of the cell fusion step is not limited, and may include chemical methods such as polyethylene glycol (PEG), physical methods such as electrofusion or laser induction, or biological methods such as viral induction. In one embodiment, the molecular weight of the polyethylene glycol used in the polyethylene glycol chemical method is not limited, and can be, for example, 200 to 10,000. In one embodiment, the cell fusion step is performed with a calcium ion solution (pH value of 6.2 to 8.0) containing 35% to 44% by volume of PEG 2000 and 0.03 to 0.12 mol/L at 30°C to 40 °C for 15 to 30 minutes. In one embodiment, the protoplast concentrations of the first parent strain and the second parent strain in the cell fusion step are 10 7 CFU/mL respectively. In a specific example, the cell fusion step is performed with a calcium ion solution (pH value 7.3) containing 35% by volume of PEG 2000 and 0.09 mol/L at 34.60°C for 10 minutes.
利用上述方法獲得之真菌融合株的基因型(如：18S rRNA的序列)及表現型(如：拮抗作用、菌落型態)與第一親代菌株較為相似，與第二親代菌株較不相似，但真菌融合株可產生之生物量及活性物質皆高於第一親代菌株。此外，上述真菌融合株的基因體穩定性高，其中第10代與第1代的真菌融合株的18S rRNA之相似度為至少98.2%，且第10代與第1代的真菌融合株的活性物質之含量在統計上沒有顯著差異。The genotype (e.g., 18S rRNA sequence) and phenotype (e.g., antagonism, colony morphology) of the fungal fusion strain obtained by the above method are relatively similar to the first parent strain, but less similar to the second parent strain. , but the biomass and active substances that the fungal fusion strain can produce are higher than those of the first parent strain. In addition, the genome stability of the above-mentioned fungal fusion strain is high, the similarity of the 18S rRNA between the 10th generation fungal fusion strain and the 1st generation fungal fusion strain is at least 98.2%, and the activity of the 10th generation fungal fusion strain and the first generation fungal fusion strain is There was no statistically significant difference in substance content.
補充說明的是，特定的培養基培養真菌融合株，獲得之活性物質含量較高。樟芝Ac-Cm-2之特定的培養基可包含但不限於0.2重量%至0.3重量%之K 2HPO 4、0.050重量%至0.060重量%之KH 2PO 4、0.05重量%至0.20重量%之MgSO 4、0.9重量%至2.1重量%之麥芽萃取物、0.8重量%至1.4重量%之蛋白腖及平衡量的水，且其初始pH值可例如為5.0至5.5。值得注意的是，在培養7至13天後，可此特定的培養基中加入0.1重量%至0.7重量%之檸烯。在一具體例中，此特定的培養基包含0.275重量%之K 2HPO 4、0.055重量%之KH 2PO 4、0.1重量%之MgSO 4、0.9重量%之麥芽萃取物、1.4重量%之蛋白腖及平衡量的水，且此特定的培養基的初始pH值為5.2。在一具體例中，在樟芝Ac-Cm-2培養13天後，可此特定的培養基中加入0.6重量%之檸烯。 It should be added that when the fungal fusion strain is cultured in a specific medium, the content of active substances obtained is higher. The specific culture medium of Antrodia camphorata Ac-Cm-2 may include, but is not limited to, 0.2 to 0.3 wt% K 2 HPO 4 , 0.050 to 0.060 wt% KH 2 PO 4 , 0.05 to 0.20 wt% MgSO 4 , 0.9 to 2.1% by weight of malt extract, 0.8 to 1.4% by weight of proteinaceous and a balance of water, and the initial pH value may be, for example, 5.0 to 5.5. It is worth noting that after 7 to 13 days of culture, 0.1 to 0.7% by weight of limonene can be added to this specific medium. In a specific example, this specific culture medium contains 0.275% by weight of K 2 HPO 4 , 0.055% by weight of KH 2 PO 4 , 0.1% by weight of MgSO 4 , 0.9% by weight of malt extract, and 1.4% by weight of proteinaceous protein. and a balancing amount of water, and the initial pH of this particular culture medium was 5.2. In a specific example, after culturing Antrodia camphorata Ac-Cm-2 for 13 days, 0.6% by weight of limonene can be added to this specific medium.
北蟲草Cm-Ac-1之特定的培養基可包含但不限於0.2重量%至0.3重量%之K 2HPO 4、0.050重量%至0.060重量%之KH 2PO 4、0.05重量%至0.20重量%之MgSO 4、0.5重量%至1.3重量%之甘胺酸、1.0重量%至2.0重量%之葡萄糖、0.01重量%至0.06重量%之維生素B1、1.0重量%至1.8重量%的酵母萃取物及平衡量的水，且第二改良培養液的初始pH值可例如為5.0至5.5。在一具體例中，此特定的培養基包含0.275重量%之K 2HPO 4、0.055重量%之KH 2PO 4、0.1重量%之MgSO 4、0.9重量%之甘胺酸、2.0重量%之葡萄糖、0.06重量%之維生素B1、1.8重量%的酵母萃取物及平衡量的水，且此特定的培養基的初始pH值為5.2。 The specific culture medium of Cordyceps militaris Cm-Ac-1 may include, but is not limited to, 0.2 to 0.3 wt% K 2 HPO 4 , 0.050 to 0.060 wt% KH 2 PO 4 , 0.05 to 0.20 wt% MgSO 4 , 0.5% to 1.3% by weight glycine, 1.0% to 2.0% by weight glucose, 0.01% to 0.06% by weight vitamin B1, 1.0% to 1.8% by weight yeast extract and a balance of water, and the initial pH value of the second modified culture solution may be, for example, 5.0 to 5.5. In a specific example, this specific culture medium contains 0.275% by weight K 2 HPO 4 , 0.055% by weight KH 2 PO 4 , 0.1% by weight MgSO 4 , 0.9% by weight glycine, 2.0% by weight glucose, 0.06% by weight of vitamin B1, 1.8% by weight of yeast extract and a balance of water, and the initial pH of this particular culture medium was 5.2.
經實驗證實，在相同條件下進行培養，相較於親代樟芝，樟芝Ac-Cm-2之生物量含量可提高75%至85%，腺苷含量可提高45%至55%，多醣體含量可提高45%至55%，三萜類含量可提高100%至110%，且蟲草酸含量可提高25%至35%。其次，在相同條件下進行培養，相較於親代北蟲草，北蟲草Cm-Ac-1之生物量含量提可高95%至105%，腺苷含量可提高20%至30%，多醣體含量可提高200%至210%，三萜類含量可提高210%至220%，且蟲草酸含量可提高55%至65%。再者，北蟲草Cm-Ac-1還可以產生約0.01 g/L至0.10 g/L之蟲草素。Experiments have confirmed that when cultured under the same conditions, compared with the parent Antrodia camphorata, the biomass content of Antrodia camphorata Ac-Cm-2 can be increased by 75% to 85%, the adenosine content can be increased by 45% to 55%, and the polysaccharide content can be increased by 45% to 55%. The body content can be increased by 45% to 55%, the triterpene content can be increased by 100% to 110%, and the cordycepic acid content can be increased by 25% to 35%. Secondly, when cultured under the same conditions, compared with the parent Cordyceps militaris, the biomass content of Cordyceps militaris Cm-Ac-1 can be increased by 95% to 105%, and the adenosine content can be increased by 20% to 30%. The content can be increased by 200% to 210%, the triterpene content can be increased by 210% to 220%, and the cordycepic acid content can be increased by 55% to 65%. Furthermore, Cordyceps militaris Cm-Ac-1 can also produce approximately 0.01 g/L to 0.10 g/L cordycepin.
本發明另提供一種含真菌融合株之組成物，包含以上述真菌融合株做為有效成分。在一實施例中，上述組成物可例如為藥品、保健食品、機能性食品、營養補充食品或特殊營養食品。上述組成物可選擇性包含食品或藥學上可接受的載體、乳化劑、懸浮劑、崩解劑、黏合劑、安定劑、螫合劑、稀釋劑、膠凝劑、防腐劑、潤滑劑及/或吸收延緩劑等。The present invention also provides a composition containing a fungal fusion strain, including the above-mentioned fungal fusion strain as an active ingredient. In one embodiment, the above-mentioned composition may be, for example, medicine, health food, functional food, nutritional supplement food or special nutritional food. The above composition may optionally include food or pharmaceutically acceptable carriers, emulsifiers, suspending agents, disintegrants, binders, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants and/or Absorption delaying agents, etc.
以下利用數個實施例以說明本發明之應用，然其並非用以限定本發明，本發明技術領域中具有通常知識者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾。 實施例一、製備真菌融合株 Several examples are used below to illustrate the application of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. polish. Example 1. Preparation of fungal fusion strains
在本發明中，以寄存編號為BCRC 35396的樟芝及寄存編號為BCRC 32219的北蟲草之其中一者為第一親代菌株，並以另一者為第二親代菌株。上述樟芝(寄存編號：BCRC 35396，又稱為菌株Ac-Cm-2)及北蟲草(寄存編號：BCRC 32219，又稱為菌株Cm-Ac-1)皆可自財團法人食品工業發展研究所(FIRDI)生物資源中心(BCRC)購得。 1. 製備原生質體 In the present invention, one of the Antrodia camphorata with the registration number BCRC 35396 and the Cordyceps militaris with the registration number BCRC 32219 is the first parent strain, and the other one is the second parent strain. The above-mentioned Antrodia camphorata (registration number: BCRC 35396, also known as strain Ac-Cm-2) and Cordyceps militaris (registration number: BCRC 32219, also known as strain Cm-Ac-1) can be obtained from the Food Industry Development Institute Purchased from (FIRDI) Biological Resource Center (BCRC). 1. Preparation of protoplasts
將上述第一親代菌株及第二親代菌株以馬鈴薯葡萄糖瓊脂(Potato Dextrose Agar，PDA，Difco，美國)於25°C下進行液態培養5天(北蟲草)或14天(樟芝)，以獲得菌絲體。取500 mg的菌絲體，並利用蒸餾水清洗3次後，以無菌濾紙吸乾水份。接著，利用細胞溶解酶進行細胞壁移除步驟，從而獲得原生質體。The above-mentioned first parent strain and second parent strain were cultured in liquid state on potato dextrose agar (PDA, Difco, USA) at 25°C for 5 days (Cordyceps militaris) or 14 days (Antrodia camphorata), to obtain mycelium. Take 500 mg of mycelium, wash it three times with distilled water, and absorb the water with sterile filter paper. Next, a cell wall removal step is performed using cytolytic enzymes to obtain protoplasts.
為評估細胞壁移除步驟的理想作用條件，以不同條件之因子進行測試，其中條件之因子可分為：細胞溶解酶的組成、KCl濃度及溫度及時間，並將上述因子再分為三個水準，如表1所示。In order to evaluate the ideal conditions for the cell wall removal step, tests were carried out with factors under different conditions. The factors of conditions can be divided into: composition of cell lytic enzymes, KCl concentration, temperature and time, and the above factors are further divided into three levels. ,As shown in Table 1.
表1
以表1所述之不同因子之不同水準做為條件進行細胞壁移除步驟，如表2所示。然後，於25°C下，以含0.6 mol/L之甘露醇(做為穩滲劑)之PDA培養原生質體，觀察原生質體是否可再生，並計算原生質體生成數(以下稱為y i值，n=3)。接著，計算各種因子不同水準之y i值的平均，舉例而言，KCl濃度水準1的y i值的平均係表2中條件1、條件4及條件7的y i值之平均。將結果記錄於表2中。 The cell wall removal step was performed with different levels of different factors described in Table 1 as conditions, as shown in Table 2. Then, culture the protoplasts with PDA containing 0.6 mol/L mannitol (as a permeability stabilizer) at 25°C, observe whether the protoplasts can be regenerated, and calculate the number of protoplasts generated (hereinafter referred to as the yi value , n=3). Next, calculate the average of the yi values at different levels of various factors. For example, the average of the yi values of the KCl concentration level 1 is the average of the yi values of condition 1, condition 4 and condition 7 in Table 2. Record the results in Table 2.
表2
如表2所示，溶壁酵素為2.0重量%之溶壁酶及1.0重量%蝸牛酶(水準1)時的yi值之平均較高，KCl濃度為0.6 M(水準2)時的yi值之平均較高，溫度為32°C(水準2)時的yi值之平均較高，且時間為2.0小時(水準2)時的yi值之平均較高。因此，細胞壁移除步驟之理想的作用條件是於32°C、0.6 M之KCl，以含有2.0重量%之溶壁酶及1.0重量%蝸牛酶之溶壁酵素分別處理第一親代菌株及第二親代菌株的菌絲體達2.0小時，從而獲得第一原生質體及第二原生質體。 2. 對原生質體進行熱處理及光處理 As shown in Table 2, the average yi value is higher when the wall-lytic enzyme is 2.0% by weight of wall-lytic enzyme and 1.0% by weight of helicase (level 1). The yi value is higher when the KCl concentration is 0.6 M (level 2). The average yi value is higher when the temperature is 32°C (Level 2), and the average yi value is higher when the time is 2.0 hours (Level 2). Therefore, the ideal conditions for the cell wall removal step are to treat the first parental strain and the third parent strain respectively with a wall-lytic enzyme containing 2.0% by weight of wall-lytic enzyme and 1.0% by weight of helicase at 32°C and 0.6 M KCl. The mycelium of the second parental strain was maintained for 2.0 hours, thereby obtaining the first protoplast and the second protoplast. 2. Heat and light treatment of protoplasts
接著，對第一原生質體進行熱處理，並對第二原生質體進行光處理。以不同溫度(50°C、55°C、60°C及65°C)及不同時間(10分鐘、20分鐘及30分鐘)為熱處理的測試條件，再將經熱處理的第一原生質體培養於PDA上達14至21天(樟芝)或5至10天(北蟲草)。計算PDA上的菌落數，以菌落數為0之測試條件為熱處理的有效條件。結果顯示，以樟芝為第一原生質體，熱處理的有效條件為50°C、30分鐘或至少55°C、至少10分鐘，而以北蟲草為第一原生質體，熱處理的有效條件為至少55°C、至少10分鐘。為避免過度抑制原生質體之活性，後續實驗以55°C、10分鐘進行熱處理。Next, the first protoplast is subjected to heat treatment, and the second protoplast is subjected to light treatment. Using different temperatures (50°C, 55°C, 60°C and 65°C) and different times (10 minutes, 20 minutes and 30 minutes) as heat treatment test conditions, the heat-treated first protoplasts were cultured in On PDA it lasts 14 to 21 days (Antrodia camphorata) or 5 to 10 days (Cordyceps militaris). Calculate the number of colonies on the PDA, and the test condition where the number of colonies is 0 is the effective condition for heat treatment. The results show that with Antrodia camphorata as the first protoplast, the effective conditions for heat treatment are 50°C and 30 minutes or at least 55°C and at least 10 minutes, while with Cordyceps militaris as the first protoplast, the effective conditions for heat treatment are at least 55 °C, at least 10 minutes. In order to avoid over-inhibiting the activity of protoplasts, subsequent experiments were heat treated at 55°C for 10 minutes.
光處理是以30瓦、波長254 nm的紫外線做為光源，且光源距離第二原生質體10公分。以不同時間(4分鐘、7分鐘、10分鐘、13分鐘、16分鐘及19分鐘)為測試條件進行光處理，再將經光處理的第二原生質體培養於PDA上達14至21天(樟芝)或5至10天(北蟲草)。計算PDA上的菌落數，並以菌落數為0之測試條件為光處理的有效條件。結果顯示，以樟芝為第二原生質體，光處理的有效條件為至少7分鐘，而以北蟲草為第二原生質體，光處理的有效條件至少4分鐘。為避免傷害原生質體的其他胞器，後續實驗之光處理係以30瓦、波長254 nm的紫外線照射樟芝之原生質體7分鐘，或以30瓦、波長254 nm的紫外線照射北蟲草4分鐘。 3. 對原生質體進行細胞融合步驟 The light treatment uses 30 watts of ultraviolet light with a wavelength of 254 nm as the light source, and the light source is 10 cm away from the second protoplast. Light treatment was performed at different times (4 minutes, 7 minutes, 10 minutes, 13 minutes, 16 minutes and 19 minutes) as test conditions, and then the light-treated second protoplasts were cultured on PDA for 14 to 21 days (Antrodia camphorata ) or 5 to 10 days (Cordyceps militaris). Count the number of colonies on the PDA, and use the test condition where the number of colonies is 0 as the effective condition for light treatment. The results showed that using Antrodia camphorata as the second protoplast, the effective conditions for light treatment were at least 7 minutes, while using Cordyceps militaris as the second protoplast, the effective conditions for light treatment were at least 4 minutes. In order to avoid damaging other organelles of the protoplast, the light treatment in subsequent experiments was to irradiate the protoplasts of Antrodia camphorata with 30 watts of ultraviolet light at a wavelength of 254 nm for 7 minutes, or to irradiate Cordyceps militaris with 30 watts of ultraviolet light at a wavelength of 254 nm for 4 minutes. 3. Cell fusion step for protoplasts
將經熱處理的第一原生質體及經光處理的第二原生質體以1：1之比例混合後，加入含有Ca 2+及PEG 6000的溶液中，以進行細胞融合步驟，從而獲得真菌融合株。以不同第一原生質體及第二原生質體濃度(10 6CFU/mL、10 7CFU/mL或10 8CFU/mL)、溫度(30°C至40°C)、Ca 2+濃度(0.03 mol/L至0.12 mol/L)、pH值(6.2至8.0)、PEG 6000濃度(35體積%至44體積%)及時間(10分鐘至30分鐘)做為測試條件進行細胞融合步驟，並將獲得之真菌融合株以0.6 mol/L之KCl溶液清洗三次，再以0.6 mol/L之KCl溶液稀釋(稀釋倍率為100、10、1)，然後於25°C下以PDA進行培養，並於培養的第7天起，每2日觀察並計數菌落數，從而獲得融合率，其中融合率是PDA上的菌落數對第一原生質體數及第二原生質體數之總和的比值。結果顯示，以上述測試條件進行細胞融合步驟，融合率可達至少1.53×10 -6。當溫度係40°C、第一原生質體(樟芝)及第二原生質體(北蟲草)之濃度分別係10 7CFU/mL、Ca 2+之濃度係0.1 mol/L、pH值係8.0、PEG 6000濃度係44體積%、時間係27.2分鐘時，融合率可達較佳的7.04×10 -5。當溫度係40°C、第一原生質體(北蟲草)及第二原生質體(樟芝)之濃度分別係10 7CFU/mL、Ca 2+之濃度係0.09 mol/L、pH值係7.3、PEG 6000濃度係35體積%、時間係10分鐘時，融合率可達較佳的8.24×10 -5。 After the heat-treated first protoplast and the light-treated second protoplast are mixed in a ratio of 1:1, they are added to a solution containing Ca 2+ and PEG 6000 to perform the cell fusion step to obtain a fungal fusion strain. With different first protoplast and second protoplast concentrations (10 6 CFU/mL, 10 7 CFU/mL or 10 8 CFU/mL), temperature (30°C to 40°C), Ca 2+ concentration (0.03 mol /L to 0.12 mol/L), pH value (6.2 to 8.0), PEG 6000 concentration (35 volume % to 44 volume %) and time (10 minutes to 30 minutes) as test conditions for the cell fusion step, and will obtain The fungal fusion strain was washed three times with 0.6 mol/L KCl solution, diluted with 0.6 mol/L KCl solution (dilution ratio 100, 10, 1), and then cultured with PDA at 25°C, and incubated From the 7th day onwards, observe and count the number of colonies every 2 days to obtain the fusion rate, where the fusion rate is the ratio of the number of colonies on the PDA to the sum of the number of first protoplasts and the number of second protoplasts. The results show that when the cell fusion step is performed under the above test conditions, the fusion rate can reach at least 1.53×10 -6 . When the temperature is 40°C, the concentrations of the first protoplast (Antrodia camphorata) and the second protoplast (Cordyceps militaris) are 10 7 CFU/mL respectively, the concentration of Ca 2+ is 0.1 mol/L, the pH value is 8.0, When the PEG 6000 concentration is 44% by volume and the time is 27.2 minutes, the fusion rate can reach an optimal 7.04×10 -5 . When the temperature is 40°C, the concentrations of the first protoplast (Cordyceps militaris) and the second protoplast (Antrodia camphorata) are 10 7 CFU/mL respectively, the concentration of Ca 2+ is 0.09 mol/L, the pH value is 7.3, When the concentration of PEG 6000 is 35% by volume and the time is 10 minutes, the fusion rate can reach an optimal 8.24×10 -5 .
篩選真菌融合株中菌落生長速率較快者，以進行後續評估，其編號分別為Ac-Cm-1、Ac-Cm-2、Cm-Ac-1及Cm-Ac-2，其中菌株Ac-Cm-1及菌株Ac-Cm-2係以樟芝為第一親代菌株，以北蟲草為第二親代菌株，且菌株Cm-Ac-1及菌株Cm-Ac-2係以北蟲草為第一親代菌株，並以樟芝為第二親代菌株。 實施例二、評估真菌融合株與親代菌株的基因型差異 The fungal fusion strains with the fastest colony growth rate were screened for subsequent evaluation. Their numbers are Ac-Cm-1, Ac-Cm-2, Cm-Ac-1 and Cm-Ac-2, among which strain Ac-Cm -1 and strain Ac-Cm-2 use Antrodia camphorata as the first parent strain and Cordyceps militaris as the second parent strain, and strain Cm-Ac-1 and strain Cm-Ac-2 use Cordyceps militaris as the third parent strain. One parent strain, and Antrodia camphorata as the second parent strain. Example 2. Assessment of genotypic differences between fungal fusion strains and parental strains
抽取上述菌株Ac-Cm-1、菌株Ac-Cm-2、菌株Cm-Ac-1及菌株Cm-Ac-2之RNA，反轉錄成cDNA後，再以引子1及引子2進行聚合脢鏈反應(Polymerase Chain Reaction，PCR)。引子1為18S rRNA基因的上游引子，其序列係如序列辨識碼(SEQ ID NO)：1所示；引子2為18S rRNA基因的下游引子，其序列係如SEQ ID NO)：2所示。然後，進行基因定序，以獲得18S rRNA序列，其中菌株Ac-Cm-2的18S rRNA之序列是如SEQ ID NO：3所示，且菌株Cm-Ac-1的18S rRNA之序列是如SEQ ID NO：4所示。Extract the RNA of the above-mentioned strains Ac-Cm-1, strain Ac-Cm-2, strain Cm-Ac-1 and strain Cm-Ac-2, reverse-transcribe into cDNA, and then use primer 1 and primer 2 to perform polymerization chain reaction. (Polymerase Chain Reaction, PCR). Primer 1 is the upstream primer of the 18S rRNA gene, and its sequence is shown in SEQ ID NO: 1; Primer 2 is the downstream primer of the 18S rRNA gene, and its sequence is shown in SEQ ID NO: 2. Then, gene sequencing is performed to obtain the 18S rRNA sequence, wherein the sequence of the 18S rRNA of the strain Ac-Cm-2 is as shown in SEQ ID NO: 3, and the sequence of the 18S rRNA of the strain Cm-Ac-1 is as shown in SEQ ID NO: 4.
然後，利用BLAST 2 SEQUENCES RESULTS VERSION BLASTP 2.2.26軟體比對上述真菌融合株(菌株Ac-Cm-1、菌株Ac-Cm-2、菌株Cm-Ac-1及菌株Cm-Ac-2)、樟芝及北蟲草之18S rRNA相似度，並將結果紀錄於表3。Then, use BLAST 2 SEQUENCES RESULTS VERSION BLASTP 2.2.26 software to compare the above fungal fusion strains (strain Ac-Cm-1, strain Ac-Cm-2, strain Cm-Ac-1 and strain Cm-Ac-2), camphor The similarity of 18S rRNA between Zhizhi and Cordyceps militaris, and the results are recorded in Table 3.
表3
如表3所示，相較於第二親代菌株，真菌融合株與第一親代菌株的18S rRNA相似度較高，且真菌融合株與第二親代菌株之18S rRNA相似度近似於第一親代菌株與第二親代菌株之18S rRNA相似度。由此證實，光處理確實可使第二代菌株之DNA片段化，使得真菌融合株在基因型上近似於第一代菌株。因此，鑑定以樟芝為第一親代菌株之菌株Ac-Cm-2為樟芝，且以北蟲草為第一親代菌株之菌株Cm-Ac-1為北蟲草。然而，真菌融合株與第一親代菌株基因體仍存有差異，可視為新的菌株。As shown in Table 3, compared with the second parent strain, the 18S rRNA similarity between the fungal fusion strain and the first parent strain is higher, and the 18S rRNA similarity between the fungal fusion strain and the second parent strain is close to that of the second parent strain. 18S rRNA similarity between the first parent strain and the second parent strain. This confirmed that light treatment can indeed fragment the DNA of the second-generation strain, making the fungal fusion strain genotypically similar to the first-generation strain. Therefore, the strain Ac-Cm-2 with Antrodia camphorata as the first parent strain was identified as Antrodia camphorata, and the strain Cm-Ac-1 with Cordyceps militaris as the first parent strain was identified as Cordyceps militaris. However, the fungal fusion strain still has genome differences from the first parent strain and can be regarded as a new strain.
利用引子3至6(序列係如SEQ ID NOs：5至8所示)對樟芝、北蟲草、菌株Ac-Cm-1、菌株Ac-Cm-2、菌株Cm-Ac-1及菌株Cm-Ac-2進行PCR及電泳，以進行隨機擴增多態DNA (Random Amplification of Polymorphic DNA，RAPD)分析。Use primers 3 to 6 (sequences are shown in SEQ ID NOs: 5 to 8) to detect Antrodia camphorata, Cordyceps militaris, strain Ac-Cm-1, strain Ac-Cm-2, strain Cm-Ac-1 and strain Cm- Ac-2 performs PCR and electrophoresis to perform Random Amplification of Polymorphic DNA (RAPD) analysis.
請參閱圖1A至圖1F，其係顯示根據本發明一實施例之樟芝(圖1A)、北蟲草(圖1B)、菌株Ac-Cm-1(圖1C)、菌株Ac-Cm-2(圖1D)、菌株Cm-Ac-1(圖1E)及菌株Cm-Ac-2(圖1F)之電泳圖，其中泳道(lanes)由左至右為DNA分子量標準(marker)、引子3至引子6的RAPD結果及利用引子1及引子2獲得之18s rRNA基因片段。Please refer to Figures 1A to 1F, which show antrodia camphorata (Figure 1A), Cordyceps militaris (Figure 1B), strain Ac-Cm-1 (Figure 1C), strain Ac-Cm-2 (Figure 1C) according to an embodiment of the present invention. Figure 1D), electrophoresis patterns of strain Cm-Ac-1 (Figure 1E) and strain Cm-Ac-2 (Figure 1F), in which the lanes from left to right are DNA molecular weight standards (markers), primer 3 to primer The RAPD results of 6 and the 18s rRNA gene fragment obtained using primer 1 and primer 2.
如圖1A至圖1E所示，樟芝(圖1A)、北蟲草(圖1B)與真菌融合株(圖1C至圖1F)之間的電泳圖不同，顯示真菌融合株與第一親代菌株基因體仍存有差異，而可視為不同的菌株。 實施例三、評估真菌融合株與親代菌株之表現型的差異 1. 菌落外觀 As shown in Figure 1A to Figure 1E, the electrophoresis patterns between Antrodia camphorata (Figure 1A), Cordyceps militaris (Figure 1B) and the fungal fusion strain (Figure 1C to Figure 1F) are different, showing that the fungal fusion strain is different from the first parent strain. There are still differences in the genomes and they can be considered different strains. Example 3. Evaluating the phenotypic differences between the fungal fusion strain and the parent strain 1. Colony appearance
將帶有樟芝、北蟲草或真菌融合株之菌絲體的PDA切下(以下稱為原膠)，並同時接種至新鮮的PDA上，再於25°C的避光環境下，共培養樟芝、北蟲草或真菌融合株，從而獲得共培養物。觀察並記錄共培養物，如圖2A至圖2C所示。Cut out the PDA containing the mycelium of Antrodia camphorata, Cordyceps militaris or fungal fusion strain (hereinafter referred to as original glue), and inoculate it onto fresh PDA at the same time, and then co-culture in a light-proof environment at 25°C. Antrodia camphorata, Cordyceps militaris or fungal fusion strains to obtain co-cultures. Observe and record the co-cultures as shown in Figure 2A to Figure 2C.
圖2A至2C分別係顯示根據本發明一實施例之樟芝、菌株Ac-Cm-1及菌株Ac-Cm-2的共培養物(圖2A)，北蟲草、菌株Ac-Cm-1、菌株Ac-Cm-2及菌株Cm-Ac-1的共培養物(圖2B)及北蟲草、菌株Ac-Cm-1、菌株Ac-Cm-2及菌株Cm-Ac-2的共培養物(圖2C)的照片，其中C、A、AC1、AC2、CA1及CA2分別代表北蟲草、樟芝、菌株Ac-Cm-1、菌株Ac-Cm-2、菌株Cm-Ac-1及菌株Cm-Ac-2，圖2A的培養時間係28天，圖2B及圖2C的培養時間係14天，且虛線圓圈標示原膠的位置。Figures 2A to 2C respectively show the co-culture of Antrodia camphorata, strain Ac-Cm-1 and strain Ac-Cm-2 according to an embodiment of the present invention (Figure 2A), Cordyceps militaris, strain Ac-Cm-1, strain Co-cultures of Ac-Cm-2 and strain Cm-Ac-1 (Fig. 2B) and co-cultures of Cordyceps militaris, strain Ac-Cm-1, strain Ac-Cm-2 and strain Cm-Ac-2 (Fig. 2C) photo, in which C, A, AC1, AC2, CA1 and CA2 represent Cordyceps militaris, Antrodia camphorata, strain Ac-Cm-1, strain Ac-Cm-2, strain Cm-Ac-1 and strain Cm-Ac respectively. -2, the culture time in Figure 2A is 28 days, the culture time in Figures 2B and 2C is 14 days, and the dotted circle marks the position of the original glue.
如圖2A所示，菌株Ac-Cm-1及菌株Ac-Cm-2與樟芝之菌落型態相似，具有圓滑(round)的菌落邊緣與完整的菌落表面，且三者無明顯節抗作用。如圖2B及圖2C所示，菌株Cm-Ac-1、菌株Cm-Ac-2及北蟲草之菌落型態相似，皆具有不規則(irregular)的菌落邊緣及波浪般的(undulate)菌落表面。其次，比較原膠位於菌落的位置可知，北蟲草、菌株Cm-Ac-1及菌株Cm-Ac-2可對其他菌株產生拮抗作用，其中菌株Cm-Ac-1及菌株Cm-Ac-2的拮抗作用較強。此外，相較於菌株Ac-Cm-1及菌株Ac-Cm-2，菌株Cm-Ac-1及菌株Cm-Ac-2的菌落較大，說明在此培養條件下，菌株Cm-Ac-1及菌株Cm-Ac-2的生長速度較快。由上述結果可知，相較於第二親代菌株，真菌融合株的菌落與第一親代菌株的菌落在形態上較為相近。 2.生物量及活性物質含量 As shown in Figure 2A, strain Ac-Cm-1 and strain Ac-Cm-2 are similar to the colony morphology of Antrodia camphorata, with round colony edges and complete colony surfaces, and the three have no obvious antagonistic effect. . As shown in Figure 2B and Figure 2C, the colony shapes of strain Cm-Ac-1, strain Cm-Ac-2 and Cordyceps militaris are similar, with irregular colony edges and undulate colony surfaces. . Secondly, by comparing the position of the original glue in the colony, it can be seen that Cordyceps militaris, strain Cm-Ac-1 and strain Cm-Ac-2 can produce antagonistic effects on other strains. Among them, strain Cm-Ac-1 and strain Cm-Ac-2 have antagonistic effects on other strains. The antagonistic effect is strong. In addition, compared with strain Ac-Cm-1 and strain Ac-Cm-2, strain Cm-Ac-1 and strain Cm-Ac-2 have larger colonies, indicating that under these culture conditions, strain Cm-Ac-1 And the growth rate of strain Cm-Ac-2 is faster. From the above results, it can be seen that compared with the second parent strain, the colonies of the fungal fusion strain are morphologically similar to the colonies of the first parent strain. 2. Biomass and active substance content
將樟芝、北蟲草及四株真菌融合株(Ac-Cm-1、Ac-Cm-2、Cm-Ac-1及Cm-Ac-2)之菌絲體接種於馬鈴薯葡萄糖肉汁(Potato Dextrose Broth，PDB)中，並於25°C、震盪速率120 rpm下培養5天(Cm-Ac-1、Cm-Ac-2及北蟲草)或14天(Ac-Cm-1、Ac-Cm-2及樟芝)，從而獲得醱酵物。將醱酵物以8000×g之轉速離心5分鐘，以分離菌絲體及醱酵液。然後，對菌絲體進行冷凍乾燥、研磨及烘乾，從而獲得菌絲體粉末，並將醱酵液在50°C下減壓濃縮至原先體積1/20，再進行冷凍乾燥、研磨及烘乾成醱酵液粉末，從而獲得醱酵液粉末。接著，測量菌絲體粉末的重量，以獲得生物量含量，並分別檢測菌絲體粉末及醱酵液粉末的活性物質(腺核苷、多醣體、三萜類、蟲草素、蟲草酸)含量，藉以計算醱酵物的活性物質含量。The mycelium of Antrodia camphorata, Cordyceps militaris and four fungal fusion strains (Ac-Cm-1, Ac-Cm-2, Cm-Ac-1 and Cm-Ac-2) were inoculated into Potato Dextrose Broth. , PDB) and cultured at 25°C with a shaking speed of 120 rpm for 5 days (Cm-Ac-1, Cm-Ac-2 and Cordyceps militaris) or 14 days (Ac-Cm-1, Ac-Cm-2 and Antrodia camphorata), thereby obtaining fermentation. Centrifuge the fermentation material at a speed of 8000×g for 5 minutes to separate the mycelium and fermentation liquid. Then, the mycelium is freeze-dried, ground and dried to obtain mycelium powder, and the fermentation liquid is concentrated under reduced pressure at 50°C to 1/20 of the original volume, and then freeze-dried, ground and dried. Dry fermentation liquid powder to obtain fermentation liquid powder. Then, measure the weight of the mycelium powder to obtain the biomass content, and detect the content of active substances (adenosine, polysaccharides, triterpenes, cordycepin, and cordycepic acid) in the mycelium powder and fermentation liquid powder respectively. , to calculate the active substance content of the fermentation product.
分別取0.5 g的菌絲體粉末及醱酵液粉末，並加入20 mL的去離子水，於60°C下進行超音波震盪30分鐘，靜置冷卻後，再加水定量到25 mL，並以5500×g之轉速離心3分鐘後，取上清液過濾。接著，利用臺灣衛生福利部食品藥物管理署(Taiwan Food and Drug Administration，FDA)於2017年12月08日公告之〈膠囊與錠狀食品中腺核苷及蟲草素之檢驗方法〉的方法檢測腺核苷及蟲草素的含量，在此不另贅述。Take 0.5 g of mycelium powder and fermentation liquid powder respectively, add 20 mL of deionized water, conduct ultrasonic vibration at 60°C for 30 minutes, let it cool, then add water to measure to 25 mL, and add After centrifugation at 5500×g for 3 minutes, filter the supernatant. Next, the adenosine was detected using the method "Testing Methods for Adenosine and Cordycepin in Capsules and Tablets" announced by the Taiwan Food and Drug Administration (FDA) of the Ministry of Health and Welfare on December 8, 2017. The content of nucleosides and cordycepin will not be described again here.
多醣體的含量是參閱Dubois等人於1956年發表於《化學(Chemistry)》期刊之〈Colorimetric method for determination of sugars and related substances〉一文所述的方法測量，且三萜類的含量是參閱2015年出版的《中華人民共和國藥典》所述的方法測量，在此不另贅述。The content of polysaccharides was measured according to the method described in the article "Colorimetric method for determination of sugars and related substances" published by Dubois et al. in the journal "Chemistry" in 1956, and the content of triterpenes was measured according to the method published in 2015. The measurement methods described in the published "Pharmacopoeia of the People's Republic of China" will not be described again here.
以甲醇萃取醱酵液粉末，並以8000×g之轉速離心15分鐘，從而獲得醱酵液甲醇萃取物。以熱水(45°C、5 mL)萃取菌絲體粉末，並以8000×g之轉速離心10分鐘，從而獲得菌絲體熱水萃取物。接著，以楊爽等人於2014年發表於《藥物食品分析(Journal of Food and Drug Analysis)》期刊第22卷第468頁至第476頁的〈Optimization of fermentation process of Cordyceps militarisand antitumor activities of polysaccharides in vitro〉一文所述之方法測量蟲草酸之含量，在此不另贅述。 The fermentation liquid powder was extracted with methanol and centrifuged at 8000×g for 15 minutes to obtain the methanol extract of the fermentation liquid. Extract the mycelium powder with hot water (45°C, 5 mL) and centrifuge at 8000×g for 10 minutes to obtain the hot water extract of mycelium. Next, take "Optimization of fermentation process of Cordyceps militaris and antitumor activities of polysaccharides" published by Yang Shuang et al. in 2014 on pages 468 to 476 of Volume 22 of Journal of Food and Drug Analysis. The content of cordycepic acid was measured using the method described in the article "in vitro" and will not be described again here.
將菌絲體粉末與醱酵液粉末的各活性物質(腺苷、多醣體、三萜類及蟲草酸)之含量分別加總，以獲得醱酵物之各活性物質含量，其中含量是以每公升的PDB所含之活性物質的重量表示(單位：g/L)。上述結果是顯示於圖3A至圖3E中。值得說明的是，上述菌絲體粉末與醱酵液粉末之蟲草素含量小於檢測極限，故未繪示於圖中。Add the contents of each active substance (adenosine, polysaccharides, triterpenes and cordycepic acid) of the mycelium powder and fermentation broth powder respectively to obtain the content of each active substance of the fermentation broth. The content is expressed per liter. The weight of the active substance contained in the PDB is expressed (unit: g/L). The above results are shown in Figures 3A to 3E. It is worth noting that the cordycepin content of the above-mentioned mycelium powder and fermentation liquid powder is less than the detection limit, so it is not shown in the figure.
圖3A至圖3E分別顯示根據本發明之一實施例的醱酵物之生物量含量(圖3A)、腺苷含量(圖3B)、多醣體含量(圖3C)、三萜類含量(圖3D)及蟲草酸含量(圖3E)的直條圖，其中橫軸表示組別，縱軸表示含量(單位：g/L)，實施例1、實施例2、實施例3、實施例4、對照例1及對照例2分別代表培養於PDB的菌株Ac-Cm-1、菌株Ac-Cm-2、菌株Cm-Ac-1、菌株Cm-Ac-2、樟芝及北蟲草，字母a、b、c及d分別表示以單因子變異數分析(Analysis of Variance，ANOVA)分析後用最小顯著差異法(Least Significant Difference，LDS)分析的統計組別，其中相同字母表示統計上不具有顯著差異，且不同字母表示統計上具有顯著差異(p＜0.05)。Figures 3A to 3E respectively show the biomass content (Figure 3A), adenosine content (Figure 3B), polysaccharide content (Figure 3C), and triterpenoid content (Figure 3D) of the fermentation product according to one embodiment of the present invention. and cordycepic acid content (Figure 3E), in which the horizontal axis represents the group and the vertical axis represents the content (unit: g/L), Example 1, Example 2, Example 3, Example 4, and Comparative Example 1 and Comparative Example 2 respectively represent strain Ac-Cm-1, strain Ac-Cm-2, strain Cm-Ac-1, strain Cm-Ac-2, Antrodia camphorata and Cordyceps militaris cultivated in PDB, letters a, b, c and d respectively represent the statistical groups analyzed by single-factor analysis of variation (Analysis of Variance, ANOVA) and then analyzed by the least significant difference method (Least Significant Difference, LDS), where the same letter indicates that there is no statistically significant difference, and Different letters indicate statistically significant differences (p<0.05).
如圖3A所示，對照例2的生物量含量係高於對照例1。比較真菌融合株及其第一親代菌株，實施例1及實施例2的生物量含量係顯著高於對照例1，分別比對照例1多40.4%、33.3%，且實施例3及實施例4的生物量含量係顯著高於對照例2，分別比對照例2多98.7%及101.1%，說明真菌融合株的生物量含量係高於第一親代菌株之生物量含量。As shown in Figure 3A, the biomass content of Comparative Example 2 was higher than that of Comparative Example 1. Comparing the fungal fusion strain and its first parent strain, the biomass content of Examples 1 and 2 was significantly higher than that of Control Example 1, 40.4% and 33.3% more than Control Example 1 respectively, and Examples 3 and 2 The biomass content of 4 was significantly higher than that of control example 2, 98.7% and 101.1% more than control example 2 respectively, indicating that the biomass content of the fungal fusion strain was higher than that of the first parent strain.
如圖3B所示，對照例1的腺苷含量係高於對照例2。比較真菌融合株及其第一親代菌株，其中對照例1、實施例1及實施例2的腺苷含量沒有統計上的顯著差異，但實施例2的腺苷含量比對照例1多31.6%。實施例3及實施例4的腺苷含量係顯著係低於對照例2，分別比對照例2少40.18%及41.98%。As shown in Figure 3B, the adenosine content of Comparative Example 1 is higher than that of Comparative Example 2. Comparing the fungal fusion strain and its first parent strain, there is no statistically significant difference in the adenosine content of Control Example 1, Example 1 and Example 2, but the adenosine content of Example 2 is 31.6% more than that of Control Example 1 . The adenosine content of Examples 3 and 4 is significantly lower than that of Comparative Example 2, being 40.18% and 41.98% less than Comparative Example 2 respectively.
如圖3C所示，對照例1的多醣體含量係顯著高於對照例2。比較真菌融合株及其第一親代菌株，對照例1、實施例1及實施例2的多醣體含量沒有統計上的顯著差異，但實施例3及實施例4顯著高於對照例2，分別比對照例2多89.34%及86.90%。As shown in Figure 3C, the polysaccharide content of Comparative Example 1 was significantly higher than that of Comparative Example 2. Comparing the fungal fusion strain and its first parent strain, there is no statistically significant difference in the polysaccharide content of Control Example 1, Example 1 and Example 2, but Examples 3 and 4 are significantly higher than Control Example 2, respectively. 89.34% and 86.90% more than control example 2.
如圖3D所示，對照例1及對照例2的三萜類含量沒有顯著差異。比較真菌融合株及其第一親代菌株，實施例1與對照例1之間沒有統計上的顯著差異，但實施例2係顯著高於對照例1，比對照例1多68.00%。實施例3及實施例4顯著高於對照例2，分別比對照例2多65.38%及86.54%。此結果顯示相較第一親代菌株，真菌融合株具有較高的三萜類含量。As shown in Figure 3D, there is no significant difference in the triterpenoid content of Comparative Example 1 and Comparative Example 2. Comparing the fungal fusion strain and its first parent strain, there is no statistically significant difference between Example 1 and Control Example 1, but Example 2 is significantly higher than Control Example 1, 68.00% more than Control Example 1. Examples 3 and 4 are significantly higher than Comparative Example 2, 65.38% and 86.54% more than Comparative Example 2 respectively. This result shows that the fungal fusion strain has a higher triterpene content compared to the first parent strain.
如圖3E所示，對照例2的蟲草酸含量較高，其次為實施例3及實施例4，而實施例1、實施例2及對照例1的蟲草酸含量較低。As shown in Figure 3E, the cordycepic acid content of Comparative Example 2 is higher, followed by Example 3 and Example 4, while the cordycepic acid content of Example 1, Example 2 and Comparative Example 1 is lower.
分別以25°C、震盪速率120 rpm的環境培養菌株Ac-Cm-2及樟芝達14天，並以相同條件培養菌株Cm-Ac-1及北蟲草達5天，再進行高效能液相層析法(High Performance Liquid Chromatography，HPLC)。The strains Ac-Cm-2 and Antrodia camphorata were cultured at 25°C and a shaking rate of 120 rpm for 14 days, and the strains Cm-Ac-1 and Cordyceps militaris were cultured under the same conditions for 5 days, and then high-performance liquid phase was performed. Chromatography (High Performance Liquid Chromatography, HPLC).
圖4A及圖4B分別是根據本發明之一實施例之菌株Ac-Cm-2與樟芝(圖4A)及菌株Cm-Ac-1與北蟲草(圖4B)的HPLC圖譜，其中橫軸表示時間，縱軸表示折射率強度，且折線401、403、405及407分別表示菌株Ac-Cm-2、樟芝、菌株Cm-Ac-1及北蟲草。如圖4A所示，相較於樟芝，菌株Ac-Cm-2在滯留時間為27分鐘的範圍內，物質種類及其含量較高。如圖4B所示，相較於北蟲草，菌株Cm-Ac-1在滯留時間為27分鐘的範圍內，物質種類及其含量較高。由上述結果可知，相較於第二代菌株，真菌融合株所含之物質組成和第一代菌株較相近，但物質種類及其含量較高。 3. 利用改良培養基獲得之生物量含量及活性物質含量 Figure 4A and Figure 4B are respectively the HPLC patterns of strain Ac-Cm-2 and Antrodia camphorata (Figure 4A) and strain Cm-Ac-1 and Cordyceps militaris (Figure 4B) according to one embodiment of the present invention, in which the horizontal axis represents Time, the vertical axis represents the refractive index intensity, and the broken lines 401, 403, 405 and 407 represent strain Ac-Cm-2, Antrodia camphorata, strain Cm-Ac-1 and Cordyceps militaris respectively. As shown in Figure 4A, compared with Antrodia camphorata, strain Ac-Cm-2 had higher substance types and contents within the retention time of 27 minutes. As shown in Figure 4B, compared with Cordyceps militaris, strain Cm-Ac-1 has higher substance types and contents within the retention time of 27 minutes. From the above results, it can be seen that compared with the second-generation strain, the substance composition of the fungal fusion strain is similar to that of the first-generation strain, but the substance types and contents are higher. 3. Biomass content and active substance content obtained by using modified culture medium
於25°C、震盪速率120 rpm的條件，利用第一改良培養液培養菌株Ac-Cm-2達14天，其中第一改良培養液初始pH值為5.2，且第一改良培養液含有0.275重量%之K 2HPO 4、0.055重量%之KH 2PO 4、0.1重量%之MgSO 4。其次，第一改良培養液還含有檸烯(limonene)麥芽萃取物及蛋白腖及平衡量的水。檸烯、麥芽萃取物及蛋白腖的含量及檸烯添加時間與菌株Ac-Cm-2之生物量及活性物質含量有關，其中表4是記錄獲得菌株Ac-Cm-2之較佳生物量及活性物質含量較佳配方。 The strain Ac-Cm-2 was cultured for 14 days using the first modified culture medium at 25°C and a shaking rate of 120 rpm, wherein the initial pH value of the first modified culture medium was 5.2, and the first modified culture medium contained 0.275 wt. % of K 2 HPO 4 , 0.055% by weight of KH 2 PO 4 , and 0.1% by weight of MgSO 4 . Secondly, the first improved culture solution also contains limonene malt extract and proteinaceous protein and a balanced amount of water. The contents of limonene, malt extract and proteinaceous as well as the addition time of limonene are related to the biomass and active substance content of strain Ac-Cm-2. Table 4 records the optimal biomass and active substance content of strain Ac-Cm-2. Formula with better active substance content.
表4
舉例而言，如欲獲得較佳腺苷含量，第一改良培養液的麥芽萃取物含量為0.9重量%、蛋白腖含量為1.4重量%，且需於培養後第13天，於第一改良培養液加入0.6重量%之檸烯。For example, if you want to obtain better adenosine content, the malt extract content of the first modified culture solution is 0.9% by weight and the proteinaceous content is 1.4% by weight, and it is necessary to start the first modified culture solution on the 13th day after culture. 0.6% by weight of limonene was added to the solution.
於25°C、震盪速率120 rpm的條件，利用第二改良培養液培養菌株Cm-Ac-1達5天，其中第二改良培養液的初始pH值為5.2，且第二改良培養液包含0.275重量%之K 2HPO 4、0.055重量%之KH 2PO 4、0.1重量%之MgSO 4。其次，第二改良培養液還含有甘胺酸、葡萄糖、維生素B1、酵母萃取物及平衡量的水，且菌株Cm-Ac-1的菌齡是84小時，接種量為8體積%。甘胺酸、葡萄糖、維生素B1、酵母萃取物的含量與菌株Cm-Ac-1之生物量及活性物質含量有關，其中表5是記錄獲得菌株Cm-Ac-1之較佳生物量及活性物質含量較佳配方。 The strain Cm-Ac-1 was cultured for 5 days using a second modified culture medium at 25°C and a shaking rate of 120 rpm, wherein the initial pH value of the second modified culture medium was 5.2, and the second modified culture medium contained 0.275 % by weight of K 2 HPO 4 , 0.055% by weight of KH 2 PO 4 , 0.1% by weight of MgSO 4 . Secondly, the second modified culture medium also contains glycine, glucose, vitamin B1, yeast extract and a balanced amount of water, and the bacterial age of strain Cm-Ac-1 is 84 hours, and the inoculum amount is 8% by volume. The contents of glycine, glucose, vitamin B1, and yeast extract are related to the biomass and active substance content of strain Cm-Ac-1. Table 5 records the optimal biomass and active substances of strain Cm-Ac-1. Better content formula.
表5
舉例而言，如欲獲得較佳腺苷含量，第二改良培養液的甘胺酸含量為0.9重量%、葡萄糖含量為2.0重量%、維生素B1含量為0.06重量%，酵母萃取物含量為1.8重量%。For example, to obtain a better adenosine content, the glycine content of the second modified culture medium is 0.9% by weight, the glucose content is 2.0% by weight, the vitamin B1 content is 0.06% by weight, and the yeast extract content is 1.8% by weight. %.
以培養菌株Ac-Cm-2之相同條件培養樟芝，且以培養菌株Cm-Ac-1之相同條件培養北蟲草，並利用上述方法檢測上述醱酵物的生物量含量、腺苷含量、多醣體含量、三萜類含量及蟲草酸含量。將結果記錄於圖5A至圖5F中。Cultivation of Antrodia camphorata under the same conditions as the culture strain Ac-Cm-2, and cultivation of Cordyceps militaris under the same conditions as the culture strain Cm-Ac-1, and using the above method to detect the biomass content, adenosine content, and polysaccharide of the fermentation product content, triterpenoid content and cordycepic acid content. The results are reported in Figures 5A to 5F.
圖5A至圖5F係分別係繪示本發明之一實施例以不同培養基培養不同菌株獲得之生物量含量(圖5A)、腺苷含量(圖5B)、多醣體含量(圖5C)、三萜類含量(圖5D)、蟲草酸含量(圖5E)及蟲草素含量(圖5F)的直條圖，其中橫軸表示組別，縱軸表示含量(單位：g/L)，實施例2及實施例3分別係利用PDB培養菌株Ac-Cm-2及菌株Cm-Ac-1，實施例2a及對照例1a分別係利用第一改良培養基培養菌株Ac-Cm-2及樟芝，實施例3b及對照例2b分別係利用第一改良培養基培養菌株Cm-Ac-1及北蟲草，且直條上的字母a、b、c及d表示經ANOVA及LDS分析的統計組別，其中相同字母表示統計上不具有顯著差異，且不同字母表示統計上具有顯著差異(p＜0.05)。Figures 5A to 5F respectively illustrate the biomass content (Figure 5A), adenosine content (Figure 5B), polysaccharide content (Figure 5C), triterpene content obtained by culturing different strains in different media according to one embodiment of the present invention. Bar graph of the content (Figure 5D), cordycepic acid content (Figure 5E) and cordycepin content (Figure 5F), in which the horizontal axis represents the group and the vertical axis represents the content (unit: g/L), Example 2 and Example 3 uses PDB to culture strain Ac-Cm-2 and strain Cm-Ac-1 respectively. Example 2a and Comparative Example 1a use the first modified medium to culture strain Ac-Cm-2 and Antrodia camphorata respectively. Example 3b and Control Example 2b respectively used the first modified medium to culture strains Cm-Ac-1 and Cordyceps militaris, and the letters a, b, c and d on the bars represent the statistical groups analyzed by ANOVA and LDS, where the same letters represent There is no statistically significant difference, and different letters indicate statistically significant differences (p<0.05).
如圖5A所示，實施例2a的生物量含量係顯著高於實施例2(提高0.83倍)及對照例1a(提高83.33%)，實施例3b的生物量含量係顯著高於實施例3(提高0.31倍)及對照例2b(提高100%)，證實利用改良培養基，真菌融合株可產生較多的生物量含量，且真菌融合株的生物量含量多於以相同條件下培養的第一親代菌株。As shown in Figure 5A, the biomass content of Example 2a is significantly higher than that of Example 2 (increased by 0.83 times) and Comparative Example 1a (increased by 83.33%), and the biomass content of Example 3b is significantly higher than that of Example 3 ( (increased by 0.31 times) and control example 2b (increased by 100%), it is confirmed that using the modified medium, the fungal fusion strain can produce more biomass content, and the biomass content of the fungal fusion strain is more than that of the first parent cultured under the same conditions. generation strains.
如圖5B所示，實施例2a的腺苷含量係顯著高於實施例2(提高5倍)及對照例1a(提高50.00%)，實施例3b的腺苷含量係顯著高於實施例3(提高43倍)及對照例2b(提高22.22%)，顯示利用改良培養基，真菌融合株可產生較多的腺苷含量，且真菌融合株的腺苷含量係多於以相同條件下培養的第一親代菌株。As shown in Figure 5B, the adenosine content of Example 2a is significantly higher than that of Example 2 (increased by 5 times) and Comparative Example 1a (increased by 50.00%), and the adenosine content of Example 3b is significantly higher than that of Example 3 ( (increased by 43 times) and control example 2b (increased by 22.22%), showing that using the modified medium, the fungal fusion strain can produce more adenosine content, and the adenosine content of the fungal fusion strain is higher than that of the first strain cultured under the same conditions. Parental strain.
如圖5C所示，實施例2a的多醣體含量係顯著高於實施例2(提高9.66倍)及對照例1a(提高52.38%)，實施例3b的多醣體含量係顯著高於實施例3(提高9.75倍)及對照例2b(提高207.14%)，顯示利用改良培養基，真菌融合株可產生較多的多醣體含量，且真菌融合株的多醣體含量係多於以相同條件下培養的第一親代菌株。As shown in Figure 5C, the polysaccharide content of Example 2a is significantly higher than that of Example 2 (increased by 9.66 times) and Comparative Example 1a (increased by 52.38%), and the polysaccharide content of Example 3b is significantly higher than that of Example 3 ( Increased by 9.75 times) and Control Example 2b (increased by 207.14%), show that using the modified culture medium, the fungal fusion strain can produce more polysaccharide content, and the polysaccharide content of the fungal fusion strain is higher than that of the first strain cultured under the same conditions. Parental strains.
如圖5D所示，實施例2a的三萜類含量係顯著高於實施例2(提高16.5倍)及對照例1a(提高105.88%)，實施例3b的三萜類含量係顯著高於實施例3(提高11.5倍)及對照例2b(提高212.50%)，顯示利用改良培養基，真菌融合株可產生較多的三萜類含量，且真菌融合株的三萜類含量係多於以相同條件下培養的第一親代菌株。As shown in Figure 5D, the triterpenoid content of Example 2a is significantly higher than that of Example 2 (increased by 16.5 times) and Comparative Example 1a (increased by 105.88%). The triterpenoid content of Example 3b is significantly higher than that of Example 2a. 3 (increased by 11.5 times) and Control Example 2b (increased by 212.50%), show that using the modified culture medium, the fungal fusion strain can produce more triterpenoid content, and the triterpenoid content of the fungal fusion strain is higher than that under the same conditions. The first parental strain in culture.
如圖5E所示，實施例2a的蟲草酸含量係顯著高於實施例2(提高21.5倍)及對照例1a(提高32.35%)，實施例3b的蟲草酸含量係顯著高於實施例3(提高12.33倍)及對照例2b(提高60.00%)，顯示利用改良培養基，真菌融合株可產生較多的蟲草酸含量，且真菌融合株的蟲草酸含量係多於以相同條件下培養的第一親代菌株。As shown in Figure 5E, the cordycepic acid content of Example 2a is significantly higher than that of Example 2 (increased by 21.5 times) and Comparative Example 1a (increased by 32.35%), and the cordycepic acid content of Example 3b is significantly higher than that of Example 3 ( (increased by 12.33 times) and control example 2b (increased by 60.00%), showing that using the modified culture medium, the fungal fusion strain can produce more cordycepic acid content, and the fungal fusion strain has more cordycepic acid content than the first strain cultured under the same conditions. Parental strain.
如圖5F所示，實施例2、實施例3、實施例2a、對照例1a及對照例2b皆無法測得蟲草素，但實施例3b含有約0.08 g/L的蟲草素。As shown in Figure 5F, cordycepin cannot be detected in Example 2, Example 3, Example 2a, Comparative Example 1a, and Comparative Example 2b, but Example 3b contains approximately 0.08 g/L of cordycepin.
由上述結果可知，相較於利用PDB，利用第一改良培養基培養菌株Ac-Cm-2或利用第二改良培養基培養菌株Cm-Ac-1，可獲得較高的生物量及活性物質含量。其次，利用第一改良培養基及第二改良培養基進行培養，相較於第一親代菌株(樟芝或北蟲草)，真菌融合株(菌株Ac-Cm-2或菌株Cm-Ac-1)可獲得較高的生物量及活性物質含量。 實施例四、評估真菌融合株之基因體穩定性 It can be seen from the above results that compared with using PDB, culturing strain Ac-Cm-2 using the first modified medium or cultivating strain Cm-Ac-1 using the second modified medium can obtain higher biomass and active substance content. Secondly, using the first modified medium and the second modified medium for cultivation, compared with the first parent strain (Antrodia camphorata or Cordyceps militaris), the fungal fusion strain (strain Ac-Cm-2 or strain Cm-Ac-1) can Obtain higher biomass and active substance content. Example 4. Assessment of genome stability of fungal fusion strains
於25°C、震盪速率120 rpm的條件，分別培養菌株Ac-Cm-1、Ac-Cm-2、Cm-Ac-1及Cm-Ac-2於PDB中，每10天繼代一次，持續培養10代。比較第1代、第5代及第10代的18S rRNA之差異，並比較第1代及第10代的活性物質之差異(n=2)。世代之間的相似度之結果是紀錄於表6及表7。Culture strains Ac-Cm-1, Ac-Cm-2, Cm-Ac-1 and Cm-Ac-2 in PDB at 25°C and shaking rate of 120 rpm, and subculture once every 10 days. Cultivated for 10 generations. Compare the differences in 18S rRNA between the 1st, 5th and 10th generations, and compare the differences in active substances between the 1st and 10th generations (n=2). The results of similarity between generations are recorded in Table 6 and Table 7.
表6
表7
如表6所示，真菌融合株之第1代、第5代及第10代之18S rRNA差異小，顯示真菌融合株具基因體穩定性。如表7所示，真菌融合株之第1代及第10代產生之活性物質的含量沒有顯著上的統計差異(P＜0.05)，說明真菌融合株具有基因體穩定性。As shown in Table 6, the differences in the 18S rRNA of the 1st, 5th and 10th generations of the fungal fusion strain were small, indicating that the fungal fusion strain has genome stability. As shown in Table 7, there is no significant statistical difference in the content of active substances produced by the 1st and 10th generations of the fungal fusion strain (P<0.05), indicating that the fungal fusion strain has gene body stability.
綜合上述，應用本發明的真菌融合株(樟芝Ac-Cm-2及/或北蟲草Cm-Ac-1)，可獲得較高的生物量及活性物質含量，其中真菌融合株是以牛樟菌及北蟲草之任一者做為第一親代菌株，另一者做為第二親代菌株，且活性物質是選自於由腺苷、多醣體、三萜類、蟲草酸、蟲草素及上述任意組合所組成的一族群。Based on the above, higher biomass and active substance content can be obtained by applying the fungal fusion strain of the present invention (Antrodia camphorata Ac-Cm-2 and/or Cordyceps militaris Cm-Ac-1), in which the fungal fusion strain is based on Cinnamomum camphora. Either one of Bacillus miltiorrhiza or Cordyceps militaris is used as the first parent strain, and the other is used as the second parent strain, and the active substance is selected from the group consisting of adenosine, polysaccharides, triterpenes, cordycepic acid, and cordycepin. and a group composed of any combination of the above.
需補充的是，本發明雖以特定的方法、特定的菌株、特定的培養基及/或特定的評估方法做為例示，說明本發明之真菌融合株、其製造方法及含其之組成物，惟本發明所屬技術領域中任何具有通常知識者可知，本發明並不限於此，在不脫離本發明之精神和範圍內，本發明亦可使用其他方法、其他菌株、其他培養基及/或其他評估方法說明本發明之真菌融合株、其製造方法及含其之組成物。It should be added that although the present invention uses specific methods, specific strains, specific culture media and/or specific evaluation methods as examples to illustrate the fungal fusion strains of the present invention, their manufacturing methods and compositions containing them, only Anyone with ordinary knowledge in the technical field to which the present invention belongs will know that the present invention is not limited thereto. The present invention can also use other methods, other strains, other culture media and/or other evaluation methods without departing from the spirit and scope of the present invention. The fungal fusion strain of the present invention, its manufacturing method and compositions containing it are described.
雖然本發明已以數個實施例揭露如上，然其並非用以限定本發明，在本發明所屬技術領域中任何具有通常知識者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。Although the present invention has been disclosed in several embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can make various modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended patent application scope.
401,403,405,407:折線401,403,405,407: Polyline
為讓本發明之上述和其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之詳細說明如下： [圖1A]至[圖1F]係顯示根據本發明一實施例之樟芝、北蟲草、菌株Ac-Cm-1、菌株Ac-Cm-2、菌株Cm-Ac-1及菌株Cm-Ac-2之電泳圖。 [圖2A]至[圖2C]分別顯示根據本發明一實施例之樟芝、菌株Ac-Cm-1及菌株Ac-Cm-2的共培養物，北蟲草、菌株Ac-Cm-1、菌株Ac-Cm-2及菌株Cm-Ac-1的共培養物及北蟲草、菌株Ac-Cm-1、菌株Ac-Cm-2及菌株Cm-Ac-2的共培養物的照片。 [圖3A]至[圖3E]係分別顯示根據本發明之一實施例的醱酵物之生物量含量、腺苷含量、多醣體含量、三萜類含量及蟲草酸含量的直條圖。 [圖4A]及[圖4B]係分別顯示根據本發明之一實施例之菌株Ac-Cm-2與樟芝及菌株Cm-Ac-1與北蟲草的HPLC圖譜 [圖5A]至[圖5F]係分別係繪示本發明之一實施例以不同培養基培養不同菌株獲得之生物量含量、腺苷含量、多醣體含量、三萜類含量、蟲草酸含量及蟲草素含量的直條圖。 In order to make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the detailed description of the accompanying drawings is as follows: [Figure 1A] to [Figure 1F] show Antrodia camphorata, Cordyceps militaris, strain Ac-Cm-1, strain Ac-Cm-2, strain Cm-Ac-1 and strain Cm-Ac- according to one embodiment of the present invention. 2. Electropherogram. [Figure 2A] to [Figure 2C] respectively show the co-culture of Antrodia camphorata, strain Ac-Cm-1 and strain Ac-Cm-2 according to an embodiment of the present invention, Cordyceps militaris, strain Ac-Cm-1, strain Photographs of co-cultures of Ac-Cm-2 and strain Cm-Ac-1 and co-cultures of Cordyceps militaris, strain Ac-Cm-1, strain Ac-Cm-2 and strain Cm-Ac-2. [Figure 3A] to [Figure 3E] are bar graphs respectively showing the biomass content, adenosine content, polysaccharide content, triterpenoid content and cordycepic acid content of the fermentation product according to one embodiment of the present invention. [Figure 4A] and [Figure 4B] respectively show the HPLC patterns of strain Ac-Cm-2 and Antrodia camphorata and strain Cm-Ac-1 and Cordyceps militaris according to one embodiment of the present invention. [Figure 5A] to [Figure 5F] respectively illustrate the biomass content, adenosine content, polysaccharide content, triterpenoid content, cordycepic acid content and cordyceps obtained by culturing different strains in different media according to one embodiment of the present invention. Bar graph of element content.
國內寄存資訊(請依寄存機構、日期、號碼順序註記) 樟芝Ac-Cm-2係於2020年3月3日寄存於臺灣財團法人食品工業發展研究所生物資源中心(BCRC，地址：臺灣新竹市東區食品路331號，郵遞區號：300193)，寄存編號為BCRC 930218。 北蟲草Cm-Ac-1係於2019年11月6日寄存於BCRC，寄存編號為BCRC 930213。 Domestic storage information (please note in order of storage institution, date and number) The Ac-Cm-2 series of Antrodia camphorata was deposited at the Biological Resource Center of the Taiwan Food Industry Development Research Institute (BCRC, address: No. 331, Shishi Road, East District, Hsinchu City, Taiwan, Postal Area Code: 300193) on March 3, 2020, with the deposit number for BCRC 930218. Cordyceps militaris Cm-Ac-1 was deposited at BCRC on November 6, 2019, with the deposit number BCRC 930213.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266101A (en) * | 2013-04-18 | 2013-08-28 | 天津师范大学 | Fusion method for protoplast of Cordyceps sinensis and Cordyceps militaris |
| CN108410854A (en) * | 2018-03-21 | 2018-08-17 | 福建师范大学 | A method of preparing cordyceps and hickory chick fusant bacterial strain |
| CN110387333A (en) * | 2018-04-20 | 2019-10-29 | 云南云百草实验室有限公司 | A method for cultivating Antrodia camphorata with Lanping Cordyceps fungus powder |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266101A (en) * | 2013-04-18 | 2013-08-28 | 天津师范大学 | Fusion method for protoplast of Cordyceps sinensis and Cordyceps militaris |
| CN108410854A (en) * | 2018-03-21 | 2018-08-17 | 福建师范大学 | A method of preparing cordyceps and hickory chick fusant bacterial strain |
| CN110387333A (en) * | 2018-04-20 | 2019-10-29 | 云南云百草实验室有限公司 | A method for cultivating Antrodia camphorata with Lanping Cordyceps fungus powder |
Non-Patent Citations (2)
| Title |
|---|
| 期刊 陳美杏, et al., "台灣菇類育種現況與展望", 農業試驗所特刊, 第155號, 農業試驗所, October 2011, pages 79-92; * |
| 網路文獻 曾顯雄, et al., "牛樟芝三萜類生合成基因之選殖、特性界定及其表現", 科技部補助專題研究計畫成果報告期末報告, 2014, https://www.grb.gov.tw/search/planDetail?id=3092828 * |
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