探討利用 3D 列印塗覆豆粕為載體固定化臺灣紫芝提升胞外多醣產量

Abstract

臺灣紫芝 (Ganoderma formosanum) 為臺灣特有種靈芝，其胞外多醣 (Exopolysaccharide, EPS) 在免疫調節與抗腫瘤方面展現出顯著的生物活性，具有極高的開發潛力。而 EPS 的大規模生產受限於液態發酵時微生物生長的穩定性與培養成本，因此需開發高效且經濟的發酵策略。本研究旨在w透過細胞固定化技術，結合 3D 列印載體與農業副產物豆粕 (Soybean meal, S)、酵母萃取物 (Yeast extract, Y)、蛋白腖 (Peptone, P) 及礦物質 (Minerals, M) 等營養物質，建立 3D 列印物件塗覆豆粕的固定化系統，以提升臺灣紫芝 EPS 的產量與產率，並降低生產成本。實驗中將載體分為 6 組，其中一組為未塗覆 (non)，以及五組塗覆不同營養組合之組別，依序為 S、SY、SP、SYP、SYPM。搖瓶培養結果顯示，在 60 rpm 下，臺灣紫芝菌絲體能夠成功附著於載體表面。在重複批次發酵中，當 3D 列印載體塗覆為豆粕與蛋白腖 (SP) 組時，相較於懸浮培養，EPS 產量在第三批次顯著提升 60%，整體產量提升 34.5%，並以 1 個載體/100 mL 的載體添加比例時有最高的平均單位生產效率 16.88 mg/L/day/carrier。5 L 生物反應器擴大培養的結果顯示，以 SP 組別進行固定化培養 (b-SP)，EPS 產量會於第 7 天達 289.29 ± 19.94 mg/L，生產率為 41.33 ± 2.85 mg/L/day，分別較搖瓶發酵 (f-SP) 提升 38.3% 與 27.3%。在生物反應器中進行重複批次發酵時，至第四批次仍能維持高生產率，介於 41.1 至 50.6 mg/L/day 之間。在生物活性分析中，於生物反應器中固定化培養的 b-SP 組 之 EPS，其 ABTS 與 DPPH 試驗之 IC₅₀ 分別為 2.91 ± 0.04 與 1.79 ± 0.34 mg/mL，抗氧化能力顯著優於其他組別。此外，SP 組別的 β-glucan 含量亦高於未塗覆固定化組，顯示此固定化系統有助於提升 EPS 之功能性與品質穩定性。本研究成功開發了一個能夠降低生產成本、提升產量、達成環保與永續發展目標的固定化平台，此技術能應用在商業發酵槽中進行商業規模生產，並為其提供更具效率與穩定的發酵策略。

Ganoderma formosanum, a Taiwan native species, produces exopolysaccharides (EPS) with significant bioactivities in immune modulation and anti-tumor effects, offering great potential for development. However, large-scale production of EPS is limited by the stability of microbial growth and the cost of cultivation during liquid fermentation, highlighting the need for efficient and cost-effective fermentation strategies. This study aimed to establish an immobilization system using 3D-printed carriers coated with agricultural by-products—soybean meal (S), yeast extract (Y), peptone (P), and minerals (M), to enhance the yield and productivity of G. formosanum EPS while reducing production costs. Six carrier groups were designed: one uncoated control group (non) and five coated groups (S, SY, SP, SYP, SYPM). In shake flask fermentation at 60 rpm, G. formosanum mycelia successfully adhered to the carrier surfaces. Aditionally, the SP-coated 3D-printed carrier group achieved a 60% increase in EPS production during the third batch and a 34.5% overall improvement in total yield compared to suspension culture. In 5L bioreactor scale-up experiments, the b-SP group reached an EPS concentration of 289.29 ± 19.94 mg/L on day 7 of fermentation, with a productivity of 41.33 ± 2.85 mg/L/day—representing 38.3% and 27.3% improvements in yield and productivity, respectively, compared to the shake flask culture (f-SP). The best specific productivity (16.88 mg/L/day/carrier) was obtained when 1 carrier was added per 100 mL. During repeated-batch fermentation in the bioreactor, high EPS productivity was maintained through the fourth batch, with EPS productivity ranging from 41.1 to 50.6 mg/L/day. Antioxidant analysis showed that EPS produced in the bioreactor using SP-coated 3D- printed carriers (b-SP) had IC₅₀ values of 2.91 ± 0.04 mg/mL in the ABTS assay and 1.79 ± 0.34 mg/mL in the DPPH assay, demonstrating superior antioxidant activity. The SP group also exhibited higher β-glucan content than uncoated carriers, indicating enhanced functionality and stability of EPS. This study successfully developed an immobilization platform that reduces costs, increases yield, and supports sustainable development, offering an efficient and stable strategy for commercial-scale fermentation.