香杉芝之菌絲體與固態發酵產品及其生理活性物質

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香杉芝之菌絲體與固態發酵產品及其生理活性物質

Antrodia salmonea Mycelia and Solid State Fermented Products, and Their Physiological Activite Compounds

恩和 , Ph.D　　Advisor：毛正倫　　

繁體中文

香杉芝；固態發酵；品質評估；抗氧化性質；抗發炎； 28 天安全性評估；香杉芝菌絲體土司；香杉芝發酵產品土司； Antrodia salmonea ； solid state fermentation ； quality evaluation ； phytochemicals ； antioxidant properties ； anti-inflammation activity ； A. salmonea mycelium bread ； A. salmonea fermented products bread

Abstract │ Reference (260) │ Altmetrics

Abstract 〈TOP〉

香杉芝(Antrodia salmonea）為台灣特有真菌，寄生於香杉木，其新鮮子實體為鮭魚粉色，平伏貼生在香杉芝樹幹之中空內面。台灣民間早已將香杉芝與樟芝子實體用於治療腹瀉、腹部疼痛、高血壓和皮膚瘙癢、也被用當作肝癌之解毒劑等。

近年來研究發現，香杉芝子實體與菌絲體萃取物具有抗氧化、抗腫瘤和抗發炎之功效，以及能夠對人類白血球細胞產生抗氧化作用。本研究首先利用分子生物技術來鑑定香杉芝菌種；再探討香杉芝穀類(蕎麥、燕麥、胚芽米和小麥)之固態培養條件，並分析培養所得菌絲體及固態發酵產品之品質特性、抗氧化性質和對抗發炎反應之評估；另外，以香杉芝燕麥餵食小鼠，進行 28 天食用安全性試驗以確定其食用安全性。

首先利用ITS1-5.8S-ITS2序列及LSU rDNA D1-D3部分序列分析，並與NCBI基因庫進行比對，鑑定香杉芝之菌種。實驗結果發現，香杉芝之ITS1-5.8S-ITS2序列長度為808 bp，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筆之3筆為A. salmonea，而相似度99% 另外 另外進行LSU rDNA D1-D3部分序列分析以長度為913；，bp 之LR5/LROR當引子對，將此序列與基因庫進行比對，其序列分數最高之前4筆是A. salmonea，而最大相似度99%。另將本菌種之ITS及LSU序列與BCRC 36937（A. salmonea）自行定序之序列進行比對，發現其序列相似度皆為100% 根據菌落型態 分生孢子及扣子體等型態特徵及其特殊氣味。、，並依據 rDNA ITS1-5.8S-ITS2和LSU rDNA D1-D3 序列分析結果，鑑定本研究使用之菌株確為香杉芝(A. salmonea)。

一般人工栽培香杉芝，由接種到生成子實體需耗費很長的時間；若應用發酵技術生產菌絲體，其發酵時間短且品質穩定。本研究旨在探討香杉芝穀物之固態培養，並評估菌絲體之最適培養基組成條件。結果顯示，在10%接種量及28天固態培養時間之條件下，以果糖為碳源；而黃豆粉為蕎麥、燕麥和小麥之氮源，酵母抽出物為胚芽米基質的氮源、不調酸鹼值並於 25°C培養，可獲得最高菌絲體產量（香杉芝蕎麥 325.27 mg/g、香杉芝燕麥 324.97 mg/g、香杉芝胚芽米 311.31 mg/g 及香杉芝小麥 351.27mg/g）。

本研究進一步探討香杉芝菌絲體及香杉芝穀物固態發酵產品之非揮發性呈味成分和生理活性物質。結果發現，一般成分中，香杉芝菌絲體含有較高量之脂肪及蛋白質，而固態發酵產品則含有較高量之碳水化合物。在生理活性物質方面，香杉芝菌絲體之麥角固醇、麥角硫因及腺苷含量，分別為 3450、320.9 和 529.8 µg/g，而固態發酵產品方面，香杉芝蕎麥與香杉芝燕麥之麥角硫因含量(119.8 及 110.4 µg/g)高於香杉芝胚芽米(97.1 µg/g)及香杉芝小麥(81.0 µg/g)。在非揮發性呈味成分方面，香杉芝菌絲體之總可溶性糖含量（51.99 mg/g）高於香杉芝蕎麥（34.24 mg/g）、香杉芝小麥(29.4 mg/g)、香杉芝胚芽米(28.88 mg/g)及香杉芝燕麥(25.68 mg/g)。而香杉 芝 菌 絲 體 及 固 態 發 酵 產 品 之 總 游 離 胺 基 酸 的 含 量 分 別 為 13.13 和6.97~12.99 mg/g。另外，菌絲體之總 5'-核苷酸和呈味 5'-核苷酸含量，皆高於固態發酵產品。以等價鮮味值來看，香杉芝菌絲體及香杉芝燕麥分別相當於 167 和 80.83 g/100 g 的 MSG;整體而言 香杉芝菌絲體具有強的鮮味，，而香杉芝穀類發酵發酵產品亦有不錯之鮮味。

香杉芝之菌絲體和固態發酵產品，探討其熱水和 70%乙醇萃取物之抗氧化性質及所含之抗氧化成分。結果顯示，香杉芝菌絲體之抗氧化性質優於固態發酵產品。而就萃取方式來比較，香杉芝菌絲體及固態發酵產品之70%乙醇萃之抗氧化效果優於熱水萃；若以 EC50 值評估抗氧化性質，結果發現在香杉芝菌絲體部分，除了熱水萃之抗氧化能力外，香杉芝菌絲體70%乙醇萃及熱水萃的 EC50 值都小於 6 mg/mL 而固態發酵產品部分 70%；，乙醇萃的 EC50 值均小於 8 mg/mL。因此，香杉芝菌絲體和香杉芝固態發酵產品都有良好之抗氧化能力。

在抗發炎試驗中，以小鼠巨噬細胞RAW 264.7為實驗模式，探討香杉芝固態發酵產品。香杉芝蕎麥、香杉芝燕麥、香杉芝胚芽米和香杉芝小麥之70%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，70%乙醇萃 取物在 濃度500 μg/ml時 ， 抑制巨噬 細胞分泌NO之效果 是41.02~46.44%；而抑制LPS誘導之巨噬細胞分泌TNF-α，70%乙醇萃取物在濃度500 μg/ml時，抑制巨噬細胞分泌TNF-α之效果為12.55~15.41%，另外，抑制LPS誘導之巨噬細胞產生IL-1β，抑制巨噬細胞分泌IL-1β之效果為34.08~36.92%，而在IL-6方面，香杉芝固態發酵產品之70%乙醇萃取物在濃度為500 μg/ml時，抑制IL-6之效果為32.85~40.3%。由結果評估，香杉芝發酵產品之70%乙醇萃取物具有抗發炎之活性。

綜合上述結果得知，香杉芝菌絲體及香杉芝穀物固態發酵產品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，且具有良好的抗氧化及抗發炎活性。因此，香杉芝穀物之固態發酵產品具有抗氧化及抗發炎作用，且有足夠之營養成分，可開發成為新的膳食補充劑或保健食品。

香杉芝燕麥 1%、香杉芝燕麥 2.5%、香杉芝燕麥 5%及 1.66%香杉芝菌絲體取代飼料餵食 28 天後，BALB/c 小鼠之各處理組之臟器相對器官重量及肝臟、心臟、腎臟及脾臟等器官 H&E 切片結果顯示因餵食香杉芝燕麥1%、香杉芝燕麥 2.5%、香杉芝燕麥 5%及 1.66%香杉芝菌絲體取代飼料對BALB/c 小鼠並無引發組織病變。不同濃度香杉芝燕麥與香杉芝菌絲體取代飼料餵食 BALB/c 小鼠 28 天後，檢測小鼠之血清生化參數、肝與腎功能的血清化學參數之檢驗顯示香杉芝燕麥 1%、2.5%、5%及 1.66%香杉芝菌絲體取代飼料餵食 BALB/c 小鼠 28 天無產生毒性。

另外以 7% 的香杉芝菌絲體、7% 四種香杉芝發酵產品取代部分麵粉添加於麵糰中，製成香杉芝菌絲體土司、香杉芝蕎麥土司、香杉芝燕麥土司、香杉芝胚芽米土司與香杉芝小麥土司，分別進行呈味品質評估、物理性質分析及官能品評分析。結果顯示香杉芝菌絲體及香杉芝發酵產品在一般成分中因含有較高量的蛋白質及纖維，因此製成土司之後口感柔軟，並具有較高的纖維含量。在物理性質分析方面，香杉芝絲體土司及四種香杉芝發酵產品土司的比體積皆符合土司的評分標準。X 射線繞射分析發現六種土司之繞射峰的強度會隨著儲藏時間增加而逐漸增強，且繞射圖譜會由V 型光譜轉變成 B 型光譜。在分析游離胺基酸與核苷酸中，香杉芝菌絲體土司、香杉芝蕎麥土司和香杉芝燕麥土司之呈味成分皆明顯比白土司來得高。在感官品評之消費者喜好性分析上，雖然香杉芝菌絲體土司中特殊之風味及色澤降低消費者的喜好度，但香杉芝發酵產品土司在口感上與白土司無顯著差異。期望經由添加香杉芝菌絲體及香杉芝菌絲體發酵產品能提供具有健康取向的烘焙食品，以提供消費者有更多樣化的選擇。

Parallel Abstract 〈TOP〉

The medical fungus Antrodia salmonea (or Taiwanofungus salmoneus), a newly identified species of the genus Taiwanofungus, which grows on the empty rotten trunk of Cunninghamia konishii in Taiwan. The fruiting body of A. salmonea has been used in the food remedy of diarrhea, abdominal pain, hypertension, itchy skin, and liver cancer and is also used as a detoxicant in Taiwanese folk medicine. To date, there were several newly compounds were isolated from the basidiomata of A. salmonea, whose studies displayed anti-oxidative effect and anti-inflammatory activities in activated inflammatory cells. With the rapidly increased standard of living, economic growth, and The improved medical sciences/techniques, people live longer than before. concurrent chronic and age-related diseases including cardiovascular disease, osteoporosis, arthritis, type II diabetes, certain cancers, and functional decline are increasing with the senior people and thereby, geriatrics is becoming a major issue to the public. beneficial to public health. Therefore, the aim of this study is to prepare the The research reported herein was to develop the Furthermore, the new products from medicinal mushrooms using grains as substrate, which are molecular identification method for A. salmonea strain. research was designed to evaluate the optimal conditions for A. salmonea to grow in grains were found including temperature, water supplement, initial pH value, inoculation rate, nitrogen and carbon sources; and fermented products including A. salmonea fermented oat (ASFO), wheat (ASFW), embryo rice (ASFE) and buckwheat (ASFB) were prepared. The proximate composition, taste components including total soluble sugars, free amino acid and 5′-nucleotides were determined. In addition, the research was focused on the antioxidant properties, and anti-inflammatory activity of mycelia and mycelium colonized grain products. Finally, the research was to study histopathology and biochemistry analysis in a 28-day toxicological assessment of A. salmonea fermented oat in BALB/c mice.

In order to know the biological origins of used strain, the research was to study molecular identification method for A. salmonea based on the nucleotide sequences of ITS1-5.8S-ITS2 rDNA. The results showed that total length of the ITS1-5.8S-ITS2 rDNA sequences from used strain was 808bp. In BLAST search, the first 4 sequences that have most identity of the published sequence in gene bank compared with the ITS1-5.8S-ITS2 rDNA of used strain were A. salmonea and they showed 99% maximum identity, except for one of A. camphorata strain. Furthermore, the total length of LSU rDNA D1-D3 In BLAST search, the first 7 sequences that have most sequences was 913bp. identity of the published sequence in gene bank compared with the LSU rDNA D1-D3 of used strain were A. salmonea and they showed 99% maximum identity. Based on the results and morphology of used strain, we could confirm the used strain was A. salmonea.

A solid state fermentation method is provided which effective for both small and large scale fungal cultivation. evaluate the mycelial biomass. The research reported herein was to study the optimal conditions for of A. salmonea grow in the selected grains, by With regard to the optimal conditions were 25°C, 28 days and 10% incubation rate with carbon and nitrogen sources being 1% fructose and 2% soybean meal for buckwheat, oat and wheat, except 1% yeast extract for embryo rice. mg/g.

Furthermore, the non-volatile components and physiological activity in the A. salmonea mycelia and A. salmonea colonized products were studied. A. salmonea mycelia were high in contents of crude fat, ash and protein and A. salmonea coloniezed in grains products were high in contents of carbohydrate.

The total sugar content of A. salmonea mycelium (51.99 mg/g) was higher than ASFB (34.24 mg/g), ASFW (29.4 mg/g), ASFR (28.88 mg/g) and ASFO (25.68 mg/g). respectively. Contents of total free amino acids were 13.13 and 6.97~12.99 Contents of total and flavor 5'-nucleotides from A. salmonea The equivalent umami concentrations [mg mg/g for A. salmonea mycelia and A. salmonea fermented products, mycelia (9.41 and 7.74mg/g) were higher than A.salmonea fermented products (5.82~7.56 and 4.0 ~5.46 mg/g). monosodium glutamate/100 g] were found in the descending order of: mycelia (167.05) > ASCO (80.83) > ASCE (63.99) > ASCW (13.63) > ASCB (5.62). With regard to physiologically active components, contents of ergosterol , ergothioneine and adenosine of A. salmonea mycelia were 3450, 320.9 and 529.8 µg/g, respectively. The fermented products contents of three phytochemicals GABA, lovastatin and ergothioneine were 90.93-170.23, 1.06-6.60 and 81.01-119.77 mg/kg, respectively. Overall, A. salmonea mycelia and fermented products possessed highly intense umami taste.

In addition, the antioxidant properties of hot water and 70% ethanolic extracts from A. salmonea mycelia and the four fermented products were investigated. The contents of potential antioxidant components in these The 70% ethanolic extracts were more extracts were also determined. effective than hot water extracts from A. salmonea mycelia in antioxidant activity and for mycelia of A. salmonea, most EC50 values were less than 6 mg/ml except for the hot water extract in antioxidant ability. For mycelia fermented products of A.salmonea, most EC50 values were less than 8 mg/ml. Overall, both extracts from A. salmonea mycelia and mycelia fermented products were good in antioxidant properties.

In the anti-inflammation activity test, we investigated the effect of 70% ethanolic extracts from A. salmonea fermented products ASFB, ASFO, ASFR and ASFW on LPS-induced NO and cytokines (TNF-α, IL-1β and IL-6) production in RAW 264.7 cells. At 500 μg/mL, the 70% ethanolic extracts With regard to the effect of At 500 from A. salmonea fermented products (41.02~46.44%) had more effective inhibition on NO production in RAW 264.7 cells. samples on LPS-induced TNF-α production in RAW 264.7 cells. μg/mL, the 70% ethanolic extracts from A. salmonea fermented products had (12.55~15.41%) a slight inhibition on TNF-α production in RAW 264.7 cells. In addition, the mycelia fermented products (32.85~40.3%) possessed a significant inhibition on IL-1β production in RAW 264.7 cells and their effective concentrations were 500 μg/mL. With regard to the effect of samples on LPS-induced IL-6 production in RAW 264.7 cells, 70% ethanolic extracts from the products had 32.85~40.3% of inhibition effect at concentration of 500 μg/mL. activity.

Overall, mycelia and filtrate of A. salmonea mycelia and fermented products contained abundant nutritional components, essential amino acids, umami components, physiologically active components, antioxidant properties and anti-inflammatory activity. Accordingly, A. salmonea might be potential antioxidants and anti-inflammatory agents for using in food products and could be developed as a new dietary supplement and functional food. A. salmonea mycelium fermented oats 1%, 2.5%, 5% and 1.66% A. salmonea mycelium mixed into the feed fed for 28 days, the each treatment groups relative organ weight and liver, heart, kidney, spleens H&E sections showed that due to feeding

A. salmonea mycelium fermented oats 1%, 2.5%, 5% and 1.66% A. salmonea mycelium were did not lead to tissue lesions. Also the concentrations of serum chemistry parameters in BALB/c mice fed with different substituted feed of A. salmonea mycelium fermented oat and 1.66% A. salmonea mycelium for 28 days were resulted have not toxicity.

In addition to the 7% A. salmonea mycelium, 7% four kinds of fermented products to replace part of the flour added to the dough, to make A. salmonea fermented buckwheat bread (ASFB bread), A. salmonea fermented oat bread (ASFO bread), A. salmonea fermented embryo rice bread (ASFR bread) and A. salmonea fermented wheat bread (ASFW bread) thus obtained were analyzed for its proximate composition, physicochemical properties and sensory evaluation as compared to the white bread control. are significantly higher those other breads. Crude fiber of A. salmonea bread (1.69%) and ASFO bread (1.90%) in proximate composition With regard to physicochemical properties, all breads specific volume were passing grades. In the analysis of free amino acids and nucleotides, A. salmonea mycelium bread, ASFB bread and ASFO breads taste components components were significantly more than white bread. Analysis of consumer preferences on sensory evaluation, although A. salmonea bread due to have a special color and flavor to reduce preference of consumers, but the A. salmonea fermented products breads were no significant difference in the taste and also have similar acceptance with white bread. In conclusion, addition of A. salmonea mycelium and A. salmonea fermented products to dough to make bread increased nutrition values and provide a new alternative of functional baked products to consumers.

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