固態發酵處理提升中草藥渣功能性之研究

温譽鈴

doi:10.6845/NCHU.2014.00651

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固態發酵處理提升中草藥渣功能性之研究

Study of Solid State Fermentation Treatment on Enhancing Functionality of Chinese Herbal Medicinal Residues

温譽鈴 , Ph.D　　Advisor：陳錦樹　　

繁體中文 DOI： 10.6845/NCHU.2014.00651

米麴菌；固態發酵；生物轉化；中藥；藥渣； Aspergillus oryzae ； solid state fermentation ； biotransformations ； Chinese herbal medicines ； residues

Abstract │ Reference (202) │ Altmetrics

Abstract 〈TOP〉

相關研究指出微生物可利用中藥中的纖維、醣類與蛋白質等物質，具有轉化藥性、提高藥物療效、降低藥物毒性等作用，存有物理或化學萃取方法無可比擬之優勢。故本研究利用微生物所具有生物轉化 (biotransformation) 之特性，以自行篩選分離出；可經誘導分泌高單寧酶活性之Aspergillus oryzae NCH 42為試驗菌株，對中藥之藥渣進行固態發酵，探討不同之發酵條件對中藥藥渣活性成分之影響；此外，評估藥渣發酵前後單寧含量之變化，期能降低單寧含量、提升機能性成分萃取率、減少中藥飲品製劑之澀味、增加國人對中藥飲品之接受度以及改善其適口性，並以體外細胞毒性試驗評估此萃取方法之安全性。期望能提高中藥有效成分之萃取率以及增加中藥藥渣之利用率，並為發酵中藥研究提供新的製備方法，亦使發酵中藥於醫療業及食品工業中的發展中能開闢出新的研究領域。
本研究之第一部份中以晉耆 (Hedysarum polybotrys Hand.-Mazz)、合歡皮 (Albizziae Cortex)、刺五加 (Eleutherococcus senticosus)、木賊 (Equisetum hiemale L.)、夜交藤 (Polygonum multiflorum Thunb.)、竹茹 (Bambusae Caulis in Taeniis.)、柴胡 (Bupleurum falcatum L.)、厚朴 (Magnolia officinalis)、穿心蓮 (Andrographis paniculata)、金銀花 (Lonicera japonica Thunb.)、一條根 (Glycine tomentella Hayata) 及括樓仁 (Trichosanthes kirilowii Maxim) 共12種經熱水萃取中藥渣為固態發酵基質，分別接種A. niger BCRC 32073或A. oryzae NCH 42，並分別以4種不同培養基組成，接種試驗菌株後於30℃下培養10天。結果顯示經Asp. niger BCRC 32073發酵後之合歡皮與穿心蓮藥渣之原兒茶酸 (protocatechuic acid) 含量明顯提升，刺五加和括樓仁則出現發酵前未含之成分；而經Asp. oryzae NCH 42發酵後之金銀花與括樓仁藥渣則產生紫丁香酸 (syringicacid) 及表兒茶素沒食子酸酯 (epicatechin gallate, ECG)；此外，部份中藥渣之抑菌範圍增加，其中以木賊、夜交藤、金銀花以及括樓仁之抑菌效果較為顯著。以上結果顯示經A. niger BCRC 32073及A. oryzae NCH 42固態發酵處理可提升中藥渣抑菌活性，增進中藥渣之附加價值。
接著以不同處理之金銀花接種A. oryzae NCH 42進行發酵，結果顯示發酵後之抗氧化能力 (亞鐵離子螯合能力、還原力以及DPPH自由基清除能力) 皆較傳統100℃水萃法為佳，且可維持一定之抗菌能力；而金銀花添加水後接種經A. oryzae NCH 42發酵1-7天，結果顯示金銀花經A. oryzae NCH 42固態發酵至第7天，對Bacillus cereus BCRC 10603, Staphylococcus aureus BCRC 15211, Staphylococcus aureus BCRC 12154之抑菌能力皆較未接種A. oryzae NCH 42之對照組為佳。另，由抗氧化能力分析之結果得知，在發酵至第3天時即可提升還原力、總抗氧化能力以及DPPH自由基清除能力，而由高效能液相層析儀 (HPLC)得知，發酵至第7天時可得最高量之咖啡酸，達425.04 μg/g。
而在第三部分試驗中，將A. oryzae NCH 42接種於四種中草藥藥渣 (栝樓仁、丹參、厚朴及甘草) 進一步探討機能性成分變化及藥渣再利用之可能性。結果顯示，發酵可強化機能性成份之釋放，發酵組之總酚含量及抗氧化活性之表現均優於未發酵者，此外可降低單寧含量，並有較廣泛之抑菌能力，且括樓仁發酵後之甲醇萃取物 (0.1%，w/v) 有助於RAW 264.7細胞存活。
綜合以上研究結果顯示，透過微生物生物轉化之效果可降低中草藥渣中殘存之單寧含量，其抗氧化能力如還原力、亞鐵離子螯合能力、DPPH自由基清除能力及總抗氧化能力等均可獲得不同程度的提升或改善；而於抗菌能力方面，可得知中草藥渣經由微生物固態發酵後對受測病原菌仍可保有不同程度之抑菌能力，甚具有開發抑制革蘭氏陰性菌之潛力；另由細胞試驗初步評估，發酵後之中草藥渣對細胞具有較低之刺激性。此外，以HPLC分析經固態發酵後之中草藥渣萃取物發現藥渣中殘存之酚類化合物可經由微生物轉化分解，產生新的成分。

Parallel Abstract 〈TOP〉

Relevant research point out such materials as the fibre, candies and protein in the usable traditional Chinese medicine of microorganism, etc. Transform nature, improve medicine curative effect, reduce such function as medicine toxicity, etc. Superior to physics or the chemistry and extract the method. This research utilizes the characteristic of microbial biotransformations, an extracvellularly tannase-producing fungal isolate, Aspergillus oryzae NCH 42 was used. Carry on the solid state and ferment to the dregs of a decoction of the traditional Chinese medicine. Probe into different influence on the active composition of dregs of a decoction of traditional Chinese medicine of fermented condition, In addition assess single peaceful change of content in front and at the back of dregs of a decoction ferment.
The feasibility of using solid state fermentation (SSF) treatment for increasing the utilization of extraction residues of Chinese herbal medicines (CHMs) was evaluated in part I of this study. Twelve kinds of CHMs, including Hedysarum polybotrys Hand.-Mazz., Albizziae Cortex, Eleutherococcus senticosus, Equisetum hiemale L., Polygonum multiflorum Thunb., Bambusae Caulis in Taeniis., Bupleurum falcatum L., Magnolia officinalis, Andrographis paniculata, Lonicera japonica Thunb., Glycine tomentella Hayata and Trichosanthes kirilowii Maxim were used. 
Their aqueous extraction residues (CHMRs) were inoculated with fungal strain, either Aspergillus orgzae NCH 42, and SSF was perfurmed at 37℃ for 10 days. The antimicrobial activity and phenolic substances after SSF treatment were determined. SSF was performed by dividing CHMRs into four groups based upon with or without nutrient supplement and inoculation.
 Results showed that fermentation treatment enhanced the release of functional ingredients from CHMRs. The protocatechuic acid contents of Albizziae Cortex and Andrographis paniculata increased after fermented with Asp. niger BCRC 32073, while presence of unknow compounds was observed for Eleutherococcus senticosus and Trichosanthes kirilowii Maxim. after fermentation. In addition, Syringic acid and Epicatechin gallate (ECG) were found for Lonicera japonica Thunb. and Trichosanthes kirilowii Maxim. after SSF with Asp. oryzae NCH 42. Moreover, the antimicrobial activity against some pathogenic bacteria increased for some fermented CHMRs, especially for those of Equisetum hiemale L., Bambusae Caulis in Taeniis., Lonicera japonica Thunb. and Trichosanthes kirilowii Maxim.
These results revealed that solid-state fermentation using either Asp. niger BCRC 32073 or A. oryzae NCH 42 increased the antimicrobial activity and changed phenolic substance content of aqueous extraction residues of some CHMRs, it therefore improves the utilization of extraction residues of CHMRs.
Followed by different treatment of Lonicera japonica Thunb. inoculated A. oryzae NCH 42 for SSF, the antioxidant capacity (ferrous ion chelating ability, reducing power and DPPH radical scavenging capacity) and the antimicrobial activity were better than the traditional 100 ℃ water extraction method. Furthermore, Lonicera japonica Thunb. inoculated with A. oryzae NCH 42, and SSF was performed for 1-7 days at 30 °C, the results showed that have better antimicrobial activity and antioxidant capacity of the seventh day. The results calculated by the HPLC analysis that the fermentation to 7 days when the highest amounts of caffeic acid, up to 425.04 mg/g.
The feasibility of using Aspergillus oryzae NCH 42, an extracellular tannase-producing fungus, for increasing the utilization of extraction residues of Chinese herbal medicines (CHMs) was evaluated in Part III. Four types of CHMs, including Trichosanthes kirilowii Maxim, Salvia miltiorrhiza Bge, Magnolia officinalis and Glycyrrhizae radix, were used. Their aqueous extraction residues were inoculated with A. oryzae NCH 42, and solid-state fermentation was performed for 5 days at 30 °C with moisture content of 86% (ratio of solid to water 1:6, w/v). The methanolic extracts of fermentation products (F&M) were examined for their functional components and properties, such as total phenolic content, antioxidant activity and tannin content. All experiments were performed using 0.1% (w/v) extracts, and antibacterial activities of the 10% (w/v) extracts were tested against seven gram-positive and gram-negative bacteria, including Bacillus cereus BCRC 10603, Listeria monocytogenes BCRC 14848, Staphylococcus aureus BCRC 15211, Staphylococcus aureus BCRC 12154, Escherichia coli BCRC 10675, Salmonella enterica BCRC 10747, and Salmonella enterica BCRC 12948. Results showed that fermentation enhanced the release of functional ingredients from extracts. The F&M products had higher total phenolic content and antioxidant activity than the unfermented ones, but the tannin content was lower. Moreover, F&M products of each residue showed a wide spectrum of antibacterial activities against the seven pathogenic bacteria tested. These results revealed that solid-state fermentation using A. oryzae NCH 42 improves the utilization of extraction residues of CHMs.
These results suggested that the effect of biotransformation can reduce the tannin content of Chinese herbal medicines residues and had higher total phenolic content and antioxidant activity, such as reducing power, ferrous ion chelating ability, DPPH radical scavenging and total antioxidant capacity etc. than the unfermented ones, but the cell irritant was lower. Moreover, that showed a wide spectrum of antibacterial activities against the seven pathogenic bacteria tested, even with the development of the potential of inhibiting Gram-negative bacteria. In addition, high-performance liquid chromatography analysis by solid state fermentation residue of herbal extracts found in the remaining dregs of phenolic compounds can be decomposed by microbial transformation to produce new ingredients, it can be developed in the food industry and related new applications.

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