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氨化预处理促进醋糟酶解产糖及生产酵母单细胞蛋白

Production of yeast single cell protein from vinegar residue through enzymatic saccharification and fermentation enhanced by ammoniation pretreatment

  • 摘要: 针对醋糟木质纤维素含量高、资源化利用率低的问题,该研究构建了“氨化预处理-酶解糖化-微生物转化”的醋糟资源化利用体系,旨在通过氨化预处理促进醋糟酶解产糖以生产酵母单细胞蛋白(single-cell protein, SCP)。首先,采用单因素试验筛选氨化预处理关键参数(氨水添加质量分数、温度、时间、含水率)的适宜范围,随后通过Box-Behnken响应面法(RSM)设计17组试验,以醋糟酶解还原糖产率为响应指标,优化醋糟氨化产糖的预处理工艺参数。基于对VRH中还原糖利用能力筛选出用于单细胞蛋白生产的酵母菌。以VRH中的还原糖为主要碳源,通过优化氮源复配、生长因子和碳氮比等参数,建立高产SCP的培养基配方,并通过摇瓶发酵和高密度发酵进行验证。结果表明:在醋糟中添加质量分数为21%的氨水,调整醋糟含水率为30%,64 ℃条件下氨化62 h,醋糟酶解还原糖产率达252.89 ± 15.12 mg/g,较未处理组(113.98 ± 3.10 mg/g)提高123.60%(P<0.05)。热带假丝酵母Candida tropicalis GB3能够较好的利用VRH中的还原糖,利用率达到60.68%。对C. tropicalisGB3的发酵培养基进一步优化为:在1 L VRH(含还原糖31.97 g/L、粗蛋白12.45 g/L)中加入酵母粉8.3 g/L、葡萄糖10.4g/L、MgSO4 2 g/L,调节初始pH值 为6.5。摇瓶发酵48 h后菌体干质量和蛋白质产量分别为17.39 ± 0.22 g/L和7.83 ± 0.10 g/L,5 L发酵罐高密度发酵24 h后,菌体干质量和蛋白质产量分别为24.64 和11.40 g/L。研究表明,氨化预处理可有效提高醋糟酶解效率,VRH可替代部分传统碳源实现酵母SCP的高效生产,为工业糟渣资源化利用提供了思路。

     

    Abstract: Vinegar residue (VR), the primary byproduct of traditional Chinese vinegar production, is rich in lignocellulosic resources. However, its efficient resource utilization was hampered by the recalcitrant structure of the lignin-carbohydrate complex. This study established a resource utilization system for VR based on ammonia pretreatment, enzymatic saccharification, and microbial conversion, aiming to enhance VR reducing sugar yield and utilize the hydrolysate for single-cell protein (SCP) production. Single-factor experiments were first conducted to screen key parameters for ammonia pretreatment (ammonia loading, temperature, time, moisture content), determining suitable ranges. Subsequently, a Box-Behnken response surface methodology (RSM) design comprising 17 trials was employed to optimize the pretreatment parameters for maximizing enzymatic reducing sugar yield. Pretreated samples were hydrolyzed using Cellic CTec3 HS cellulase. Three yeast strains (Saccharomyces cerevisiae 2, Candida tropicalis GB3, Meyerozyma caribbica GD1) were evaluated for their capacity to utilize reducing sugars in the VR hydrolysate (VRH) to identify the optimal SCP producer. Using VRH as the basal medium, the effects of nitrogen source combination (aqueous ammonia and yeast extract), growth factors (MgSO4, ZnSO4, MnSO4, biotin, pantothenic acid, thiamine), and carbon-to-nitrogen ratio (C/N 5:1-20:1) on biomass production were systematically investigated to establish high-yield SCP medium parameters. These parameters were then validated in shake-flask and high-cell-density fermentations. Results indicated that ammonia water dosage, temperature, time, and moisture content all significantly impacted saccharification efficiency (P<0.05). The optimal ammonia pretreatment conditions were 21% w/w NH3·H2O, 64 ℃, 62 h, and 30% moisture content. Under these conditions, the reducing sugar yield reached 252.89 ± 15.12 mg/g VR, representing a 123.6% increase compared to the untreated control (113.98 ± 3.10 mg/g) (P<0.05). Among the three yeast strains, C. tropicalis GB3 demonstrated superior performance, utilizing 60.68% of the VRH reducing sugars and achieving an initial dry cell weight (DCW) of 5.36 ± 0.51 g/L, significantly higher than the others (P<0.05). Biomass production was significantly enhanced by employing a 3:2 ammonia-yeast extract nitrogen source combination, adding MgSO4, and optimizing the C/N ratio to 10:1. The optimized high-yield SCP process comprised: VRH (containing 31.97 g/L reducing sugars, 12.45 g/L crude protein) 1 L, supplemented with 8.3 g/L yeast extract, 10.4 g/L glucose, and 2 g/L MgSO4, with an initial pH of 6.5. After 48 h of shake-flask fermentation, the DCW and dry protein weight reached 17.39 ± 0.22 g/L and 7.83 ± 0.10 g/L, respectively. This represented increases of 131.00% and 151.61% compared to the control using unoptimized VRH medium (7.53 ± 0.14 g/L DCW and 3.11 ± 0.06 g/L protein) (P<0.05). Subsequent high-cell-density fermentation in a 5-L bioreactor for 24 h further elevated DCW and protein yield to 24.64 ± 0.51 g/L and 11.25 ± 0.33 g/L, respectively, corresponding to increases of 61.79% and 38.35% compared to growth in YPD medium (P<0.05). While VRH fermentation reduced reliance on conventional carbon sources compared to YPD, the functional composition of the resulting biomass was reduced. Crude protein content decreased by 14.50%, mannan content by 18.78%, nucleotide content by 49.53%, essential amino acid content by 16.06%, and total amino acid content by 9.99% (P<0.05). This reduction was likely attributable to VRH lacking a complete amino acid profile, nucleic acid precursors, and B vitamins, coupled with the high C/N ratio of the optimized medium, which may have directed metabolic resources preferentially towards biomass synthesis over functional component accumulation. The inorganic nitrogen provided by residual ammonia in VRH contributed to cost reduction. Ammonia pretreatment effectively improved cellulose accessibility, enabling efficient enzymatic saccharification and microbial conversion. This optimized system confirmed VRH as a viable low-cost substrate for SCP production, with C. tropicalis GB3 achieving high biomass and protein yields. The study provides a novel strategy for the industrial-scale valorization of lignocellulosic waste, emphasizing the synergy between resource recovery and sustainable protein production. Future research should focus on enhancing functional protein quality through precursor supplementation and metabolic engineering to address nutritional limitations.

     

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