桑黃之液態培養及其生理活性

梁志弘

doi:10.6845/NCHU.2009.00156

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桑黃之液態培養及其生理活性

Submerged Fermentation of Phellinus linteus and Their Physiological Activities

梁志弘 , Ph.D　　Advisor：毛正倫　　

繁體中文 DOI： 10.6845/NCHU.2009.00156

桑黃；液態培養；品質評估；抗氧化性質；抗發炎；麥角硫因； Phellinus linteus ； submerged culture ； quality evaluation ； antioxidant properties ； anti-inflammatory activity ； ergothioneine

Abstract │ Reference (278) │ Times Cited (12) │ Altmetrics

Abstract 〈TOP〉

桑黃（Phellinus linteus (Berkeley & Curtis) Teng）是一種子實體顏色鮮黃的藥用菇，常生長在桑屬植物上。桑黃之所以會引起關注主要是其抗腫瘤作用及其他藥用價值，包括免疫調節、抗血管增生、抗致突變、防癌、保肝、抗肝纖維化及降血糖等作用。本研究首先利用分子生物技術來鑑定桑黃菌種；其次探討桑黃之液態深層培養的條件，冀能提高桑黃菌絲體及胞外多醣產量，並分析培養所得菌絲體及發酵液之品質特性、抗氧化性質及對抗發炎反應之評估；最後建立菇類中麥角硫因之分析方法並針對桑黃菌絲體及發酵液麥角硫因含量進行分析。
首先利用25S rDNA及ITS rDNA序列分析，並與NCBI基因庫進行比對，以作為桑黃菌種之鑑定。實驗結果發現，桑黃之25S rDNA序列長度為876bp，將此序列與基因序列資料庫PubMed提供之軟體BLAST進行比對，其序列分數最高之前4筆為P. linteus，而最大相似度100%有1筆，相似度99%有2筆，而這3筆菌株皆是P. linteus；另外，桑黃之ITS rDNA序列長度為730bp，將此序列與基因庫進行比對，其序列分數最高之前12筆，除一筆是木層孔菌屬不知名之菌種外，其餘11筆皆是P. linteus，而最大相似度100%有11筆，P. linteus 菌株有7筆，P. baumii菌株有3筆，另一筆為木層孔菌屬不知名之菌種。綜合ITS及25S核醣體基因序列分析所得之結果及菌絲體外觀形態之觀察，確定所使用之桑黃菌種應為P. linteus。
一般人工栽培由接種到生成子實體需耗費很長的時間，應用發酵技術生產菌絲體及多醣，具有發酵時間短及品質穩定。本研究旨在探討桑黃之液態培養，以獲得生產菌絲體及胞外多醣之最適培養基組成。由結果可獲知，在10%接種量及12天的培養時間，發現使用葡萄糖為碳源和酵母抽出物為氮源，不調酸鹼值並於 30°C培養，可獲得最高菌絲體（11.53 g/L）和胞外多醣（1.19 g/L）產量。
本研究進一步針對桑黃之菌絲體及發酵液的非揮發性呈味成分及生理活性物質予以探討。結果發現，在一般組成方面，桑黃菌絲體含有較高量之粗脂肪、纖維及蛋白質含量，而發酵液則含有較高量之碳水化合物及灰份含量。在生理活性物質方面，桑黃菌絲體之麥角固醇及腺苷含量，分別為3.04和0.05 mg/g，發酵液中則未偵測到；而桑黃菌絲體及發酵液之多醣含量則分別為20.42和15.94 mg/g。在非揮發性呈味成分方面，桑黃發酵液之總可溶性糖和糖醇含量（159.84 mg/g）高於菌絲體（58.70 mg/g），其中在菌絲體及發酵液中分別以甘露糖醇（34.06 mg/g）和核醣（80.78 mg/g）含量最高。而桑黃菌絲體及發酵液之總游離胺基酸的含量分別為182.69和129.59 mg/g。另外，桑黃菌絲體（42.43 mg/g）類味精（MSG-like）成分之含量略高於發酵液（35.13 mg/g）；而無論是總5'-核苷酸或呈味5'-核苷酸含量菌絲體皆高於發酵液。以等價鮮味值來看，桑黃菌絲體及發酵液分別相當於10.16 和3.05 g的MSG。整體而言，桑黃菌絲體具有非常強的鮮味，而發酵液亦有不錯之鮮味。
桑黃之菌絲體和發酵液，針對其乙醇和熱水萃取物之抗氧化性質及所含抗氧化成分加以探討。由結果可獲知，桑黃菌絲體除熱水萃之還原力外，其餘抗氧化性質皆優於發酵液。而就萃取方式來比較，桑黃菌絲體除螯合亞鐵離子能力外，乙醇萃之效果優於熱水萃；而桑黃發酵液除清除DPPH能力外，熱水萃之效果優於乙醇萃。桑黃抗氧化性質若以EC50 值來表示，結果發現在桑黃菌絲體部分，除乙醇萃之螯合亞鐵離子能力外，桑黃菌絲體乙醇萃及熱水萃的抗氧化性質之EC50 值都小於8 mg/mL；而在桑黃發酵液部分，除乙醇萃及熱水萃之螯合亞鐵離子能力外，發酵液乙醇萃及熱水萃的抗氧化性質之EC50 值均小於4 mg/mL。因此，整體而言桑黃菌絲體和發酵液皆具有不錯之抗氧化性質。
在抗發炎試驗中，主要是以小鼠巨噬細胞RAW 264.7為實驗模式來探討桑黃菌絲體和發酵液之甲醇及乙醇萃取物對LPS誘導巨噬細胞發炎反應之影響。由結果可知，菌絲體之甲醇或乙醇萃取物在樣品濃度為250及500 μg/ml時，具有抑制巨噬細胞分泌NO之效果，而發酵液反而會促進巨噬細胞生成NO。而對LPS誘導巨噬細胞產生TNF-α抑制方面，桑黃菌絲體及發酵液之甲醇或乙醇萃取物在樣品濃度為500 μg/ml時，具有抑制巨噬細胞分泌TNF-α之效果。此外，對LPS誘導巨噬細胞產生IL-1β抑制方面，僅桑黃菌絲體之乙醇萃取物具有抑制巨噬細胞分泌IL-1β之效果，其抑制濃度為100~500 μg/ml；而其他之處理組則反而有促進發炎激素IL-1β生成之現象。而在抗發炎指標IL-10方面，除桑黃菌絲體之甲醇及乙醇萃取物在濃度為500 μg/ml外，其餘處理組皆可促進巨噬細胞分泌抗發炎激素IL-10之效果。由實驗推測，桑黃菌絲體及發酵液之甲醇或乙醇萃取物具有抗發炎之活性。
利用HPLC方法來分析麥角硫因部分，由結果可知，其標準品在10 ~50 μg/mL 的濃度範圍內，具有良好之線性關係；偵測極限為4.67 ng/mL，定量極限為15.56 ng/mL，回收率為94.87~98.04 %，而在精密度方面，不論是同日間或異日間，不同濃度點之變異係數值均小於4%，顯示具有良好之再現性。若以每克乾重含有之麥角硫因量來計算，菌絲體及發酵液之含量分別為148.5和287.7 μg/g dw。
綜合上述結果得知，桑黃之菌絲體及發酵液皆含有豐富的營養成分、必需胺基酸、鮮味物質和生理活性物質，且具有抗氧化及抗發炎活性。因此，桑黃可以用在食品中當作潛在之抗氧化劑及抗發炎物質，進而可以開發為新的膳食補充劑或保健食品。

Parallel Abstract 〈TOP〉

Phellinus linteus (Berkeley & Curtis) Teng (Hymenochaetaceae), a tan-yellow-colored medicinal mushroom, usually grows on mulberry trees.  P. linteus draws great attention because of its anti-tumor effect and other medicinal value, including immunomodulatory activity, anti-angiogenesis, antimutagenicity, prevention of cancer, hepatoprotective effect, anti-hepatic fibrosis effect as well as hypoglycemic effect.  The research reported herein was to first develop the molecular identification method for P. linteus strain.  Furthermore, the research was designed to study the submerged culture of P. linteus to produce high contents dry biomass and polysaccharide of mycelia.  In addition, the research was focused on the quality properties, antioxidant properties, and anti-inflammatory activity of mycelia and fermentation filtrate.  Finally, the research was to set up the analytical method of ergothioneine and quantify the content of ergothioneine from mycelia and fermentation filtrate.
In order to know the biological orgins of used strain, the research was to 
study molecular identification method for P. linteus based on the nucleotide 
sequences of 25S rDNA and ITS rDNA.  The results showed that total length 
of the 25S rDNA sequences from used strain was 876bp.  In BLAST search, 
the first 4 sequences that have most identity of the published sequence in gene 
bank compared with the 25S rDNA of used strain were P. linteus and they 
showed 98~100% maxium identity.  Furthermore, the total length of ITS rDNA 
sequences was 730bp.  In BLAST search, the first 12 sequences that have most 
identity of the published sequence in gene bank compared with the ITS rDNA of 
used strain were P. linteus except for one unknown strain and they showed 
98~100% maxium identity.  Based on the results and morphology of used strain, 
we could confirm the used strain was P. linteus 
Growing mushroom mycelium in liquid culture on a defined nutrient broth has been a simple and fast alternative method to produce fungal biomass.  The research reported herein was to study the optimal conditions for submerged culture of P. linteus, to evaluate the dry biomass and polysaccharides of their mycelia and fermentation filtrate.  With regard to P. linteus submerged culture, the optimal conditions were 30°C, 12 days and 10% incubation rate with carbon and nitrogen sources being 2% glucose and 0.5% yeast extract.  Contents of dry biomass and polysaccharides from P. linteus were 11.53 and 1.19 g/L.
Furthermore, the non-volatile components and physiological activity in the two forms of P. linteus mycelia and filtrate were studied.  P. linteus mycelia were high in contents of crude fat, fiber and protein and P. linteus filtrate were high in contents of carbohydrate and ash.  With regard to physiologically active components, contents of ergosterol and adenosine of P. linteus mycelia were 3.04 and 0.05 mg/g, respectively.  However, ergosterol and adenosine were not found in P. linteus filtrate.  With regard to the content of polysaccharide of P. linteus mycelia was 20.42 mg/g whereas that of P. linteus filtrate was 15.94 mg/g.  The total sugars and polyols content of P. linteus filtrate (159.84 mg/g) was higher than that of P. linteus mycelia (58.70 mg/g).  P. linteus mycelia contained high amounts of mannitol (34.06 mg/g) and P. linteus filtrate contained high amounts of ribose (80.78 mg/g).  Contents of total free amino acids were 182.69 and 129.59 mg/g for P. linteus mycelia and P. linteus filtrate, respectively. Furthermore, the content of monosodium glutamate (MSG)-like components from P. linteus mycelia (42.43 mg/g) was higher than that from P. linteus filtrate (35.13 mg/g).  Contents of total and flavor 5'-nucleotides from P. linteus mycelia were higher than those from P. linteus filtrate.  EUC values were 10.16 and 3.05 g MSG/g for P. linteus mycelia and filtrate, respectively. Overall, P. linteus mycelia and filtrate possessed highly intense umami taste.
In addition, the antioxidant properties of ethanolic and hot water extracts from P. linteus mycelia and filtrate were investigated.  The contents of potential antioxidant components in these extracts were also determined.  Ethanolic extracts were more effective than hot water extracts from P. linteus mycelia in antioxidant activity, reducing power and scavenging ability on DPPH as evidenced by lower EC50 values.  Hot water extracts were more effective than ethanolic extracts from P. linteu filtrate in antioxidant activity, reducing power and chelating ability on ferrous ions as evidenced by lower EC50 values.  For mycelia of P. linteus, most EC50 values were less than 8 mg/ml except for the ethanolic extract in chelating ability on ferrous ions.  For filtrate of P. linteus, most EC50 values were less than 4 mg/ml except for both extracts in chelating ability on ferrous ions.  Overall, both extracts from P. linteus mycelia and filtrate were good in antioxidant properties.
In the anti-inflammation activity test, we investigated the effect of methanolic and ethanolic extracts from P. linteus mycelia and filtrate on LPS-induced NO and cytokines (TNF-α, IL-1β and IL-10) production in RAW 264.7 cells.  At 250 and 500 μg/mL, the methanolic and ethanolic extracts from P. linteus mycelia had a significant inhibition on NO production in RAW 264.7 cells, but those from .P. linteus filtrate had a sightly promotion on NO production in RAW 264.7 cells.  With regard to the effect of samples on LPS-induced TNF-α production in RAW 264.7 cells.  At 500 μg/mL, the methanolic and ethanolic extracts from P. linteus mycelia and filtrate had a significant inhibition on TNF-α production in RAW 264.7 cells.  In addition, only the ethanolic extracts from P. linteus mycelia possessed a significant inhibition on IL-1β production in RAW 264.7 cells and their effective concentrations were 100~500 μg/mL.  On the contrary, other treatments would promote IL-1β production in RAW 264.7 cells.  With regard to the effect of samples on LPS-induced IL-10 production in RAW 264.7 cells, most samples would promote IL-10 production in RAW 264.7 cells except for the methanolic and ethanolic extracts at concentration of 500 μg/mL from P. linteus mycelia.  The results suggest the methanolic and ethanolic extracts from P. linteus mycelia and filtrate have an anti-inflammatory activity.
Besides, using the HPLC method was to develop the assay of ergothioneine from P. linteus mycelia and filtrate.  On the basis of the results obtained, ergothioneine was linearized in the range of 10~50 μg/mL.  Recoveries for the accuracy validation of ergothioneine ranged from 94.87~98.04%.  The limits of detection and quantitation of ergothioneine were 4.67 and 15.56 ng/mL, respectively.  A good intra-day and inter-day precisions were obtained, of which with coefficient variations to be less than 4%.  Contents of ergothioneine from P. linteus mycelia and filtrate were 148.5 and 287.7 μg/g dw.
Overall, mycelia and filtrate of P. linteus contained abundant nutritional components, essential amino acids, umami components, physiologically active components, antioxidant properties and anti-inflammatory activity. Accordingly, P. linteus might be potential antioxidants and anti- inflammatory agents for use in food products and could be developed as a new dietary supplement and functional foods.

Reference ( 278 ) 〈TOP〉

吳彩平。2006。以固態發酵製備樟芝米及其品質與抗氧化性質。國立中興大學食品暨應用生物科技學系碩士論文。台中，台灣。
連結：
林欣儀。2008。以固態發酵製備舞菇小麥及其品質與抗氧化性質。國立中興大學食品暨應用生物科技學系碩士論文。台中，台灣。
連結：
黃婉莉。2007。香杉芝之培養及其生理活性與抗氧化性質。國立中興大學食品暨應用生物科技學系碩士論文。台中，台灣。
連結：
簡韶妤。2006。雞肉絲菇之培養及其呈味性質與生理活性評估。國立中興大學食品暨應用生物科技學系碩士論文。台中，台灣。
連結：
蔡淑瑤。2006。雞腿菇和花松茸之液態培養及其生理活性。國立中興大學食品暨應用生物科技學系碩士論文。台中，台灣。
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