复合菌剂QY12-A4对玉米秸秆的降解及其产物中抗氧化木质素的分离与纯化

doi:10.13560/j.cnki.biotech.bull.1985.2026-0026

摘要/Abstract

摘要：

目的为实现玉米秸秆可再生高效利用及其木质素增值化应用，探索玉米秸秆有效利用策略，挖掘具有抗氧化活性的木质素。方法采用固态发酵法高温传代培养，构建玉米秸秆降解的复合菌剂；采用16S rRNA高通量测序技术分析复合菌剂的稳定性及其微生物菌群的多样性；通过固态发酵（SSF）的发酵失重率测定和成分分析评价复合菌剂的玉米秸秆降解能力；采用碱提酸沉法提取SSF中的木质素（SSF-A）；采用D101大孔吸附树脂柱层析和Sephadex G-50凝胶过滤柱层析对SSF-A进行分离纯化。结果以玉米秸秆为底物，通过高温传代驯化培养，获得耐高温复合菌剂QY12-A4。QY12-A4在属水平趋于稳定，优势菌属为野野村氏菌属（Nonomuraea）、热杆菌属（Thermobacillus）、热芽胞杆菌属（Caldibacillus）和科恩氏菌属（Cohnella）。QY12-A4的玉米秸秆固态发酵的发酵失重率为25.26%±1.49%（w/w）。SSF-A的提取率为12.56%±0.90%（w/w），SSF-A中总酚含量为18.94%±0.41%（w/w），ABTS阳离子自由基清除能力的IC₅₀值为（158.00±4.24） μg/mL。SSF-A经柱层析分离，得到分子量分布依次减小的组分GL2-1、GL2-2和GL2-3。其中GL2-3的总酚含量最高，为19.65%±0.16%（w/w），在200-320 nm区域紫外线透射率<5.0%，抗氧化活性最大，即ABTS阳离子自由基清除能力IC₅₀值为（90.50±3.54） μg/mL；总还原力IC₅₀值为（1.38±0.02） mg/mL；相对氧自由基清除能力（ORAC）值为（3.14±0.03） mmol TE/g。结论复合菌剂QY12-A4可有效降解玉米秸秆木质纤维素，柱层析技术是一种获得高抗氧化活性的低分子量木质素类化合物的有效方法，为玉米秸秆木质素增值化应用提供有价值的参考。

关键词: 玉米秸秆, 复合菌剂, 木质素, 柱层析, 抗氧化活性

Abstract:

Objective Corn stover is a rich renewable biomass resource, with its lignin attracting significant attention for value-added applications. The effective utilisation strategies for corn stover are explored to develop the antioxidant lignin. Method A microbial consortiun for corn stover degradation was constructed by the solid-state fermentation (SSF) with serial subculture at high temperature. The stability of the microbial consortium and the diversity of its microbial community were analyzed using 16S rRNA high-throughput sequencing technology. The capacity of the microbial consortium to degrade corn stover was evaluated by measuring the fermentation weight loss rate and analyzing the components after SSF. Lignin (SSF-A) was extracted from SSF using the alkali extraction and acid precipitation method. SSF-A was separated and purified using macroporous adsorption resin D101 column chromatography and Sephadex G-50 gel filtration column chromatography. Result Using corn stover as the substrate, a high-temperature-resistant microbial consortium QY12-A4 was obtained through high-temperature subculturing and domestication. At the genus level, QY12-A4 tended to be stable, and the dominant genera were Nonomuraea, Thermobacillus, Caldibacillus, and Cohnella. The weight loss rate of corn stover fermented by QY12-A4 was 25.26%±1.49% (w/w). The extraction rate of SSF-A was 12.56%±0.90% (w/w), with a total phenolic content of 18.94%±0.41% (w/w) and IC₅₀ for ABTS cationic radical scavenging activity of (158.00±4.24) μg/mL. GL2-1, GL2-2, and GL2-3 from SSF-A with progressively decreasing molecular weight were obtained by column chromatography technology. Among them, GL2-3 exhibited the highest total phenolic content of 19.65%±0.16% (w/w), with the UV transmittance <5.0% in the 200–320 nm wavelength range, and the greatest antioxidant activity, manifested as an ABTS cation radical scavenging activity with an IC₅₀ of (90.50±3.54) μg/mL, reducing power with an IC₅₀ of (1.38±0.02) mg/mL, and relative ORAC value of (3.14±0.03) mmol TE/g. Conclusion The microbial consortium QY12-A4 can effectively degrade the lignocellulose in corn stover. Column chromatography technology is an effective method for obtaining low-molecular-weight lignin with high antioxidant activity. This provides a valuable reference for the value-added utilisation of lignin from corn stover.

Key words: corn stover, microbial consortia, lignin, column chromatography, antioxidant activity

李博宇, 蹇春晨, 侯佳俊, 钱明岩, 杨文钦, 章圣龙, 藏传刚, 马福开, 郑龙, 关宏. 复合菌剂QY12-A4对玉米秸秆的降解及其产物中抗氧化木质素的分离与纯化[J]. 生物技术通报, 2026, 42(7): 292-303.

LI Bo-yu, JIAN Chun-chen, HOU Jia-jun, QIAN Ming-yan, YANG Wen-qin, ZHANG Sheng-long, ZANG Chuan-gang, MA Fu-kai, ZHENG Long, GUAN Hong. Degradation of Corn Stover by Composite Microbial Consortia QY12-A4 and Purification of Antioxidant Lignin from Its Products[J]. Biotechnology Bulletin, 2026, 42(7): 292-303.

图/表 10

图1 OTU分类水平的Rank-abundance曲线

Fig. 1 Rank-abundance curves at the OTU level

图2 驯化过程中微生物菌群分类学概况A：门水平物种相对丰度；B：属水平物种相对丰度

Fig. 2 Taxonomic overview of microbial communities during the domestication processA: Relative abundance of species at the phylum level. B: Relative abundance of species at the genus level

表1 种子培养物α多样性分析

Table 1 Analysis of the α-diversity of the microbial consortium

样品 Sample	覆盖率 Good’s coverage （%）	香农指数 Shannon index	辛普森指数 Simpson index
QY12-A1	100	2.620	0.132
QY12-A2	100	2.590	0.117
QY12-A3	100	2.730	0.142
QY12-A4	100	2.490	0.153

表2 SSF-A成分分析

Table 2 Analysis of components in SSF-A

样品Sample

提取率Extraction rate （%， w/w）

木质素 Lignin （%， w/w）

总酚

Total phenols （%，w/w）

总糖

Total sugars （%，w/w）

糖醛酸

Uronic acids （%，w/w）

酸可溶木质素

Acid-soluble lignin

酸不溶木质素

Acid-insoluble lignin

图3 SSF-A紫外可见光谱图

Fig. 3 UV-Vis spectra of SSF-A

图4 SSF-A的分离纯化谱图A：D101大孔吸附树脂柱层析图谱；B：D101大孔吸附树脂柱层析中总酚、总糖及糖醛酸分布曲线；C：Sephadex G-50凝胶过滤柱层析图谱；D：Sephadex G-50凝胶过滤柱层析中酚类化合物、总糖和糖醛酸分布曲线

Fig. 4 Separation and purification profile of SSF-AA: Chromatogram profile of D101 macroporous resin column. B: Distribution curves of total phenols, total sugars, and uronic acids on D101 macroporous resin column. C: Chromatogram profile of Sephadex G-50 gel filtration column. D: Distribution curves of total phenols, total sugars, and uronic acids on Sephadex G-50 gel filtration

表3 SSF-A经分离后不同组分的成分分析

Table 3 Component analysis of fractions separated from SSF-A

组分 Fraction	总酚 Total phenols （%， w/w）	总糖 Total sugars （%， w/w）	糖醛酸 Uronic acids （%， w/w）
L1	10.30±0.86e	19.30±1.10b	3.56±0.21a
L2	17.37±0.18b	15.09±0.20c	2.07±0.22c
GL2-1	12.75±0.02d	21.59±0.27a	2.73±0.06b
GL2-2	15.85±0.51c	14.63±0.81c	1.47±0.30c
GL2-3	19.65±0.16a	12.68±0.61d	2.32±0.08d

图5 SSF-A经分离后各个组分紫外线透射率光谱

Fig. 5 UV transmittance spectra of fractions separated from SSF-A

图6 SSF-A经分离后不同组分的抗氧化活性评价A：ABTS阳离子自由基清除率；B：总还原能力；C：荧光衰退曲线分析

Fig. 6 Evaluation of antioxidant activity in fractions separated from SSF-AA: ABTS⁺· radical scavenging rate. B: Total reducing capacity. C: Fluorescence decay curve analysis

表4 SSF-A经分离后不同组分的抗氧化活性评价

Table 4 Evaluation of antioxidant activity of different fractions separated from SSF-A

组分 Fraction	ABTS ABTS （IC₅₀ μg/mL）	总还原力 Reducing power （mg/mL）	相对ORAC值 Relative ORAC value （mmol TE/g）
L1	241.60±10.75a	3.38±0.04a	1.50±0.07d
L2	120.50±3.54c	1.33±0.01d	2.44±0.08c
GL2-1	218.00±11.31b	2.65±0.07b	1.95±0.07b
GL2-2	127.50±0.71c	1.51±0.04c	2.41±0.02b
GL2-3	90.50±3.54b	1.38±0.02d	3.14±0.03a
Trolox	58.00±5.66e	0.26±0.01e	-

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