枯草芽孢杆菌固态发酵豆渣生产表面活性素及其应用前景

Solid-state fermentation of soybean residue by Bacillus subtilis for surfactin production and its application prospects

摘要:

背景 表面活性素(surfactin)是具有卓越表面/界面活性的生物表面活性剂。Surfactin的生产面临着碳源成本高昂及发酵过程中起泡严重等问题。豆渣(okara)的不合理利用也会导致资源浪费和环境污染。目的 鉴于对surfactin的可持续生产及豆渣的资源化利用，本研究探索了利用枯草芽孢杆菌(Bacillus subtilis)固态发酵豆渣生产surfactin生物表面活性剂，并评价了其应用前景。方法 通过液态发酵和固态发酵方法评估并比较枯草芽孢杆菌利用豆渣合成surfactin的可行性；利用弱碱水提取法提取固态发酵物中的表面活性产物，运用HPLC-MS法分析产物组成，评价产物理化活性；通过响应面法优化豆渣的固态发酵培养基，通过平板菌落计数发酵残渣中的枯草芽孢杆菌数。结果 在液态发酵和固态发酵中，豆渣转化为surfactin的转化率分别为0.6%-0.8%和1.2%-1.5%。枯草芽孢杆菌固态发酵豆渣生产surfactin既具有高产率，又实现了中无泡沫产生。产物中检测到了5种surfactin，主要是surfactin-C₁₃ (34.16%)、surfactin-C₁₄ (23.95%)和surfactin-C₁₅ (35.14%)。Surfactin产物的临界胶束浓度为35.0 mg/L，可将水表面张力降低至(26.0±0.1) mN/m，其乳化原油的乳化活性指数(emulsifying activity index, EI24)值为(73.1±3.2)%。产物可在4-121 ℃、pH 5.0-11.0及低于150 g/L NaCl条件下保持稳定。豆渣、NH₄Cl和CaCl₂·2H₂O是固态培养基中的关键组分。固态培养基优化使得surfactin产量提升52.1%。添加适量麦秸可能由于固态培养基通气性的改善，而进一步提高产量。B. subtilis AnPL-1在含有14.8 g豆渣和1.5 g麦秸的优化后固态培养基中产生了(263.2±7.8) mg surfactin。豆渣转化为表面素的转化率提升至1.8%。此外，固态发酵残渣中枯草芽孢杆菌含量达4.27×10¹⁰ CFU/g，有望开发为微生物肥料。结论 本研究开发了一条无泡沫发酵生产surfactin生物表面活性剂的可行途径，并实现了豆渣的资源化利用。

Abstract:

Background Surfactin is a biosurfactant with remarkable surface/interfacial activity. Surfactin production suffers from high costs of carbon sources and severe foaming problem during fermentation. Unreasonable utilization of soybean residue (okara) can cause resource waste and environmental pollution.Objective To achieve sustainable production of surfactin and value-added conversion of okara, we explored foam-free production of surfactin by Bacillus subtilis using okara as a low-cost substrate and evaluated its application prospects.Methods We evaluated and compared the feasibility of B. subtilis utilizing okara to synthesize surfactin through liquid and solid-state fermentation methods. Biosurfactants were extracted from solid-state culture via a weak alkaline water extraction method. The products were identified by HPLC-MS, and the physicochemical properties of the produced surfactin were analyzed. The solid-state medium for fermentation of okara was optimized by the response surface method. The viable count of B. subtilis in solid-state fermentation residue was determined by the plate colony counting method.Results The conversion rates of okara to surfactin were 0.6%-0.8% and 1.2%-1.5% in liquid and solid-state fermentation, respectively. Interestingly, solid-state fermentation of okara by B. subtilis achieved both high-yield and foam-free production of surfactin. Five surfactin homologues were produced from okara, mainly including surfactin-C₁₃ (34.16%), surfactin-C₁₄ (23.95%), and surfactin-C₁₅ (35.14%). The produced surfactin, with a critical micelle concentration of 35.0 mg/L, decreased water surface tension to (26.0±0.1) mN/m and emulsified crude oil with emulsifying activity index (EI24)(73.1±3.2)%. It was stable at 4-121 ℃, pH 5.0-11.0, and NaCl<150 g/L. Okara, NH₄Cl, and CaCl₂·2H₂O were significant components in the solid-state medium. The surfactin yield was increased by 52.1% through solid-state medium optimization. Adding wheat straw further enhanced surfactin production by improving aeration in the solid-state medium. B. subtilis AnPL-1 produced (263.2±7.8) mg surfactin in the optimized solid-state medium containing 14.8 g okara and 1.5 g wheat straw. The conversion rate of okara to surfactin was enhanced to 1.8%. In addition, the residue of solid-state fermentation was expected to be microbial fertilizer since it contained 4.27×10¹⁰ CFU/g of B. subtilis.Conclusion This study established a promising way for foam-free production of surfactin and value-added conversion of okara.