樟芝菌絲體量產技術開發及相關功能性之探討

Studies on the Mass Production of Antrodia cinnamomea Mycelia and Its Related Biological Activities

張謙裕(Chien-Yu Chang)

指導教授 ： 潘子明

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.01493

摘要

利用核醣體RNA基因 [包括ITS1、ITS2、5.8S與18S核醣體RNA基因] 的序列為指標，配合特殊引子聚合酶鏈反應 (Polymerase chain reaction)之分子生物學鑑定技術，來鑑別從台灣南部六龜山區所採集之樟芝子實體並經分離與純化後之樟芝菌株，(編號ACTS1與AC0623)是否學名為Antrodia cinnamomea之物種，並與其他的相關菌株包括不同屬之菌株(Antrodiella semisupina, Antrodiella romellii 與 Trametes versicolor)進行序列比對與鑑別，結果證實從子實體分離之樟芝菌株ACTS1與AC0623與食品工業發展研究所生物資源保存及研究中心之菌株BCRC 35396與BCRC 35398應屬同種之相似菌株。本研究以樟芝AC0623菌株為主，利用反應曲面法探討液體浸沉培養之菌絲體生成之最適培養條件，以提高菌體與三萜類的產量，結果指出，搖瓶之液體浸沉培養於28℃，起始pH 5.5及攪拌速率105 rpm下，乾菌體量與三萜類成分分別可增至3.2% (w/w)及31.8 mg/g。本實驗繼續擴大至100-L與700-L槽之培養，根據RSM，經由控制培養基組成及培養條件，於700-L槽之培養，可得到高的三萜類成分（63 mg/g），總多醣含量亦達5.59% (w/w)，另一方面，乾菌體量2.0% (w/w)實際上也已經達到工業量產的規模。本研究繼續利用高效液相層析儀，針對樟芝A. cinnamomea AC0623菌株，探討於5公升與100公升醱酵槽培養時，菌體之腺苷、蟲草素等各成分之變化趨勢，同時亦以A. cinnamomea AC0623菌株進行700-L槽培養，經噴霧乾燥處理之乾菌體量，其腺苷、蟲草素及麥角固醇的含量分別為15.70 mg/g、0.29 mg/g與 0.98 mg/g。另從樟芝A. cinnamomea AC0623菌株醱酵菌絲體中萃取多醣體，分析其對人類全血細胞激素的誘導產生量，藉此驗證樟芝多醣的免疫調控功能。醱酵液固液分離後，菌絲體再分別以熱水萃取，萃取液經酒精沉澱，獲得不同的區分，其中區分Ⅰ∼Ⅳ菌絲體熱水可溶區分，明顯誘導全血中IL-6，TNF-α 和IFN-γ等細胞激素的釋放。醱酵液亦獲致同樣的結果，顯示樟芝醱酵不論胞内之菌體或胞外醱酵液均可誘導IL-6 與TNF-α 的釋放。而進一步分析這些區分的多醣，並利用GPC 管柱測定分子量的分佈，得知本試驗經醱酵槽培養之樟芝菌體多醣體含有8.52 × 104 Da。樟芝醱酵菌絲體樣品測定β-D-glucan 含量約0.71%(w/w)。本菌株醱酵菌絲體進行總抗氧化力分析，發現樣品經萃取後使用DMSO 溶劑溶解具有相當高的抗氧化作用。樟芝醱酵液離心之上清液，經試驗證明有抑制tyrosinase 活性的功效，亦即可抑制黑色素的形成，因此在美白產品的應用上具無限的潛力與前景。
本研究並同時對樟芝BCRC 35396 菌株進行醱酵，生理活性與抗發炎之探討，另置於附錄中；BCRC 35396 菌株於醱酵過程中所分泌出的醱酵液代謝物中，含有極為顯著的抗發炎物質，於樟芝醱酵液的劃分過程中，我們得到一具有細胞毒性的成份，經分離純化後再以質譜儀測定其分子量，判斷此具細胞毒性的分子為1-hydroxy-3-isobuty1-4-[4- (3-methy1-2-butenyloxy)- pheny1]-pyrrole-2, 5-dione。

關鍵字

樟芝 ； 核醣體RNA基因 ； 三萜類 ； 子實體 ； 多醣體 ； 反應曲面法 ； 液體浸沉培養

並列摘要

The partial sequences of ribosomal RNA genes, including the internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA gene, internal transcribed spacer 2 (ITS2), and 18S ribosomal RNA gene, was sequenced and applied to identify the two strains, ACTS1 and AC0623, which were isolated from the fruiting bodies of Antrodia cinnamomea. Comparison of partial nucleotide sequences (ITS1, 5.8S, and ITS2) with those of the strains from different genus (Antrodiella semisupina, Antrodiella romellii and Trametes versicolor) was made. These DNA sequences data demonstrated that there was no difference among ACTS1, AC0623, BCRC 35396 and BCRC 35398. The results suggested that the two strains, ACTS1 and AC0623, isolated from fruiting bodies, and BCRC 35396 and 35398 strains, the original A. cinnamomea strain obtained from Food Industry Research and Development Institute (FIRDI), were the same species.
Further study using Antrodia cinnamomea AC0623 for large-scale fermentation was employed. Response surface methodology was used to optimize medium composition for the increment of biomass and triterpenoids production. The results indicated that when a submerged culture in shaking flasks was operated at 28℃, initial pH 5.5 and rotation speed 105 rpm, the biomass and triterpenoids content could be increased to 3.20 % (dry cell weight, w/w) and 31.8 mg/g, respectively. The experiments were further scale-up to 100-L and 700-L fermentors. Higher content of triterpenoids (63 mg/g) and total polysaccharide (5.59%, w/w) were obtained in 700-L fermentation by means of the control of cultural conditions and the modification of medium composition based on the RSM. It is profitable if the biomass reaches 2.0% (w/w), as a result, current study demonstrated a high possibility for commercialization.
The application of high-performance liquid chromatography (HPLC) method for quantitation of adenosine and cordycepin in 5-L and 100-L fermentation from A. cinnamomea AC0623 was also described. The strain A. cinnamomea AC0623 was cultivated in a 700-L fermentor. The contents of adenosine, cordycepin and ergosterol in the whole cell of spray-dried powder were 15.70 mg/g、0.29 mg/g and 0.98 mg/g, respectively. The immunomodulatory effect of the functional polysaccharides of A. cinnamomea AC0623 strain was investigated. The intracellular polysaccharide fraction, Fractions Ⅰ∼Ⅳ, which was prepared by hot water extraction of mycelium followed by ethanol precipitation, induced the release of IL-6, TNF-α and IFN-γ in human whole blood dose-dependently. Same results were obtained by using culture filtrate. The molecular mass distribution of polysaccharide was estimated 8.52 × 104 Da in Fraction Ⅰ, as determined by gel permeation chromatography (GPC) with refractive index detector. The β-D-glucan content was estimated about 0.71% (w/w) in the mycelium. Higher antioxidative activity was also found in DMSO solvent extract of A. cinnamomea AC0623. Cultural filtrate also inhibited the tyrosinase activity, it implied the potential in whitening cosmetic application.
The effects of A. cinnamomea BCRC 35396 on the inhibition of nitric oxide synthesis and cytotoxicity in murine macrophage RAW264.7, was investigated. The result indicated that the extract of. A. cinnamomea BCRC 35396 culture filtrate inhibited lipopolysaccharide-induced nitric oxide production. The cytotoxic compound was isolated and identified by NMR and LC-MS as 1-hydroxy-3-isobuty1-4-[4-(3-methy1-2-butenyloxy)-pheny1]-pyrrole-2, 5-dione.

並列關鍵字

Antrodia cinnamomea ； ribosomal RNA genes ； triterpenoid ； fruiting bodies ； polysaccharide ； response surface methodology ； submerged culture

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