基于复合菌的玉米芯发酵工艺构建与优化

doi:10.13560/j.cnki.biotech.bull.1985.2025-1369

摘要/Abstract

摘要：

目的高木质素秸秆因酸性洗涤木质素（acid detergent lignin, ADL）含量高导致饲料化利用率低。构建白腐真菌-枯草芽胞杆菌-毕赤酵母的复合菌发酵工艺，探究其协同降解玉米芯效果。方法以分离自香菇的白腐真菌BF-1为核心功能菌，搭配枯草芽胞杆菌、毕赤酵母构建三菌复合发酵体系（菌体量1∶1∶1），设BF-1单菌发酵为对照，均以玉米芯为基质。通过单因素试验（温度28-32 ℃、含水量40%-50%、接种量1%-5%）比较ADL降解效果，再用响应面法优化三菌体系发酵条件；结合扫描电镜观察玉米芯形态变化，测定粗蛋白（crude protein, CP）、中性洗涤纤维（neutral detergent fiber, NDF）、酸性洗涤纤维（acid detergent fiber, ADF）等指标，评价相对饲用价值（relative feed value, RFV）和相对饲用品质（relative feed quality, RFQ）。结果单因素试验表明，两种体系均在温度30 ℃、含水量45%、接种量3%时ADL降解效果最优，单菌发酵ADL降解率为42.29%，三菌发酵ADL降解率为58.83%，且三菌发酵降解效果显著优于单菌发酵（P<0.01）。响应面法优化三菌发酵最佳条件为温度29.8 ℃、含水量45.2%、接种量3.1%，此时ADL降解率达63.42%。发酵过程中，其Lac、MnP和LiP活性随时间持续升高。扫描电镜结果显示，三菌发酵后玉米芯表面结构破坏更明显，呈松散多孔特征；发酵21 d后，三菌体系玉米芯CP含量显著提升1.43%（P<0.01），NDF和ADF含量显著降低（P<0.01），其降解率分别为22.12%，33.97%，RFQ和RFV分别提高82.80%、17.21%。结论三菌协同发酵可高效降解玉米芯木质素，ADL降解率达63.42%，粗蛋白提升1.43%，纤维显著降解，相对饲用品质提高82.80%，饲用价值大幅提升，为玉米芯饲料化利用提供技术支撑。

关键词: 白腐真菌, 玉米芯, 酸性洗涤木质素, 营养品质, 饲用价值

Abstract:

Objective High-lignin straw exhibits low feed utilization efficiency due to its high content of acid detergent lignin (ADL). We developed a composite fermentation process using white-rot fungus, Bacillus subtilis, and Pichia pastoris, and evaluated its synergistic degradation effect in corncob. Method We constructed a three-strain composite fermentation system consisting of white-rot fungus BF-1 (isolated from Lentinula edodes) as the core functional strain, together with Bacillus subtilis and Pichia pastoris at a cell mass ratio of 1∶1∶1. Corncob served as the fermentation substrate, and single-strain fermentation of BF-1 was used as the control. The degradation efficiency of ADL was compared using single-factor experiments (temperature: 28–32 ℃, moisture content: 40%–50%, inoculum size: 1%–5%). Response surface methodology was then employed to optimize the fermentation conditions for the three-strain system. Morphological changes in corncobs were examined by scanning electron microscopy (SEM). Crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were measured to evaluate the relative feed value (RFV) and relative feed quality (RFQ). Result Single-factor experiments showed that both fermentation systems achieved optimal ADL degradation at 30 ℃, 45% moisture content, and 3% inoculum size. Under these conditions, the ADL degradation rate of single-strain fermentation was 42.29%, while that of the three-strain fermentation was 58.83%. The degradation efficiency of the three-strain system was significantly higher than that of the single-strain system (P<0.01). Response surface methodology identified the optimal conditions for three-strain fermentation as 29.8 ℃, 45.2% moisture, and 3.1% inoculum, achieving an ADL degradation rate of 63.42%. During fermentation, the activities of laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) increased continuously over time. Scanning electron microscopy revealed that after three-strain fermentation, the surface structure of the corncob was more significantly disrupted, exhibiting a loose and porous morphology. After 21 days of fermentation, the CP content in the three-strain system increased by 1.43% (P<0.01), while NDF and ADF contents decreased significantly (P<0.01), with degradation rates of 22.12% and 33.97%, respectively. RFQ and RFV increased by 82.80% and 17.21%, respectively. Conclusion The three-strain synergistic fermentation effectively degraded corncob lignin, achieving an ADL degradation rate of 63.42%, a 1.43% increase in CP content, and significant fiber degradation. RFQ was increased by 82.80%, and RFV was substantially enhanced. These findings provide a technical support for the utilization of corncobs as animal feed.

Key words: white-rot fungi, corncob, acid detergent lignin, nutritional quality, feeding value

陈文燕, 周瑞, 吴慧昊, 李耀东, 杨婕, 臧荣鑫, 徐红伟. 基于复合菌的玉米芯发酵工艺构建与优化[J]. 生物技术通报, 2026, 42(10): 37-46.

CHEN Wen-yan, ZHOU Rui, WU Hui-hao, LI Yao-dong, YANG Jie, ZANG Rong-xin, XU Hong-wei. Construction and Optimization of Corncob Fermentation Process Using Composite Microbial Inoculum[J]. Biotechnology Bulletin, 2026, 42(10): 37-46.

图/表 9

参考文献 35

[1]	Zeko-Pivač A, Bošnjaković A, Planinić M, et al. Improvement of the nutraceutical profile of brewer's spent grain after treatment with Trametes versicolor [J]. Microorganisms, 2022, 10(11): 2295.
[2]	李森, 罗霄天, 秦心儿, 等. 高粱秸秆栽培大球盖菇的品质、产量与优势分析 [J]. 生物资源, 2024, 46(1): 32-38.
	Li S, Luo XT, Qin XE, et al. Analysis of quality, yield and advantages of Stropharia rugosoannulata cultivated with sorghum straw [J]. Biotic Resour, 2024, 46(1): 32-38.
[3]	Nazar M, Tian JP, Wang X, et al. Effect of biological lignin depolymerization on rice straw enzymatic hydrolysis, anerobic fermentation characteristics and in vitro ruminal digestibility [J]. Int J Biol Macromol, 2025, 305: 141664.
[4]	Liu WX, Zhao MY, Li MX, et al. Laccase activities from three white-rot fungal species isolated from their native habitat in North China using solid-state fermentation with lignocellulosic biomass [J]. BioResources, 2022, 17(1): 1533-1550.
[5]	Ahmad Khan N, Khan M, Sufyan A, et al. Biotechnological processing of sugarcane bagasse through solid-state fermentation with white rot fungi into nutritionally rich and digestible ruminant feed [J]. Fermentation, 2024, 10(4): 181.
[6]	杨莉, 杨义, 郑碧君, 等. 木质素降解菌的筛选及其对酒糟的预处理研究 [J]. 生物质化学工程, 2024, 58(5): 45-52.
	Yang L, Yang Y, Zheng BJ, et al. Screening of lignin-degrading fungal strain and its pretreatment of distillers' grains [J]. Biomass Chem Eng, 2024, 58(5): 45-52.
[7]	Singh J, Mandal A, Nandabalan YK. Synergistic effect on lignolytic enzyme production through co-culturing of white rot fungi during solid state fermentation of agricultural residues [J]. Bull Natl Res Cent, 2025, 49: 3.
[8]	Campos AO, Asevedo EA, Souza Filho PF, et al. Extraction of cellulases produced through solid-state fermentation by Trichoderma reesei CCT-2768 using green coconut fibers pretreated by steam explosion combined with alkali [J]. Biomass, 2024, 4(1): 92-106.
[9]	Zhu DC, Zhang PP, Xie CX, et al. Biodegradation of alkaline lignin by Bacillus ligniniphilus L1 [J]. Biotechnol Biofuels, 2017, 10: 44.
[10]	Naranjo CA, Jivan AD, Vo MN, et al. Role of BGS13 in the secretory mechanism of Pichia pastoris [J]. Appl Environ Microbiol, 2019, 85(24): e01615-e01619.
[11]	Arora D, Sharma A, Singh S, et al. The Bacterial degradation of lignin—a Review [J]. Crit Rev Biotechnol, 2010, 30(4): 326-341.
[12]	Hardianto D, Safarrida A, Prayoga GS, et al. Increasing the production of insulin glargine in Pichia pastoris through medium modification [J]. BIO Web Conf, 2024, 127: 06001.
[13]	李强, 吴晓青, 张新建. 微生物降解秸秆木质素的研究进展 [J]. 微生物学报, 2023, 63(11): 4118-4132.
	Li Q, Wu XQ, Zhang XJ. Research progress in microbial degradation of straw lignin [J]. Acta Microbiol Sin, 2023, 63(11): 4118-4132.
[14]	Cui TW, Yuan B, Guo HW, et al. Enhanced lignin biodegradation by consortium of white rot fungi: microbial synergistic effects and product mapping [J]. Biotechnol Biofuels, 2021, 14: 162.
[15]	Jiménez-Barrera D, Chan-Cupul W, Fan ZL, et al. Fungal co-culture increases ligninolytic enzyme activities: statistical optimization using response surface methodology [J]. Prep Biochem Biotechnol, 2018, 48(9): 787-798.
[16]	司欣雨, 李佳琪, 周梦含, 等. 多功能复合菌剂的构建及盐碱胁迫下促生效果 [J]. 农业工程学报, 2024, 40(21): 82-90.
	Si XY, Li JQ, Zhou MH, et al. Constructing multifunctional composite microbial agents for promoting crop growth under saline-alkali stress [J]. Trans Chin Soc Agric Eng, 2024, 40(21): 82-90.
[17]	杨斌, 殷引, 张浩博, 等. 洗涤剂法测定烟草及烟草制品中中性洗涤纤维、酸性洗涤纤维、酸性洗涤木质素的研究 [J]. 中国烟草学报, 2012, 18(3): 10-15.
	Yang B, Yin Y, Zhang HB, et al. Determination of NDF, ADF and ADL in tobacco and tobacco products with detergent method [J]. Acta Tabacaria Sin, 2012, 18(3): 10-15.
[18]	王光荣, 高世霞, 于晓锋, 等. 改性玉米芯对Zn²⁺和Cu²⁺的吸附性能研究 [J]. 轻工学报, 2019, 34(2): 56-62.
	Wang GR, Gao SX, Yu XF, et al. Research of modified corn cob on the adsorption properties of Zn²⁺, Cu²⁺ [J]. J Light Ind, 2019, 34(2): 56-62.
[19]	刘宽庆, 张以恒. 木质素的生物降解和生物利用 [J]. 合成生物学, 2024, 5(6): 1264-1278.
	Liu KQ, Zhang YH. Biological degradation and utilization of lignin [J]. Synth Biol J, 2024, 5(6): 1264-1278.
[20]	杨航, 宋福超, 王诗涛, 等. 固态发酵平菇-青稞秸秆菌糠饲料化利用研究 [J]. 草地学报, 2025, 33(4): 1289-1298.
	Yang H, Song FC, Wang ST, et al. Research on the utilization of solid-state fermented oyster mushroom-highland barley straw fungus-bran as feed [J]. Acta Agrestia Sin, 2025, 33(4): 1289-1298.
[21]	罗俊杰, 肖燕子, 侯美玲, 等. 不同添加剂对苜蓿与羊草混合青贮品质及维生素含量的影响 [J]. 草地学报, 2025, 33(3): 992-1000.
	Luo JJ, Xiao YZ, Hou ML, et al. Effects of different additives on quality and vitamin content of mixed silage of alfalfa and Leymus chinensis [J]. Acta Agrestia Sin, 2025, 33(3): 992-1000.
[22]	郝迎辰, 黄钰, 张洪瑞, 等. 品种和添加剂对三江源区燕麦青贮品质和CNCPS蛋白组分的影响 [J]. 草地学报, 2025, 33(4): 1274-1280.
	Hao YC, Huang Y, Zhang HR, et al. Effects of varieties and additives on silage quality and CNCPS protein fractions of oats in the Sanjiangyuan area [J]. Acta Agrestia Sin, 2025, 33(4): 1274-1280.
[23]	梁文斌, 尹国丽, 陈三冬, 等. 全株玉米青贮过程中品质及真菌群落组成特征 [J]. 草地学报, 2024, 32(12): 3722-3732.
	Liang WB, Yin GL, Chen SD, et al. Characteristics of quality and fungal community composition during whole-plant corn silage [J]. Acta Agrestia Sin, 2024, 32(12): 3722-3732.
[24]	Sun Y, Liu WC, Shi X, et al. Inducing secondary metabolite production of Aspergillus sydowii through microbial co-culture with Bacillus subtilis [J]. Microb Cell Fact, 2021, 20: 42.
[25]	Datsomor O, Yan Q, Opoku-Mensah L, et al. Effect of different inducer sources on cellulase enzyme production by white-rot basidiomycetes Pleurotus ostreatus and Phanerochaete chrysosporium under submerged fermentation [J]. Fermentation, 2022, 8(10): 561.
[26]	Yang J, Zhao J, Jiang JC, et al. Isolation and characterization of Bacillus sp. capable of degradating alkali lignin [J]. Front Energy Res, 2021, 9: 807286.
[27]	Chen KJ, Tang JC, Xu BH, et al. Degradation enhancement of rice straw by co-culture of Phanerochaete chrysosporium and Trichoderma viride [J]. Sci Rep, 2019, 9: 19708.
[28]	Niu C, Sun LN, Tang R. Preparation of a bacterial consortium for straw degradation and optimization of conditions for its return to the field [J]. Agronomy, 2025, 15(8): 1947.
[29]	Zhang SZ, Duan Y, Teng CC, et al. Fast and selective degradation of biomass for xylose, glucose and lignin under mild conditions [J]. Molecules, 2023, 28(8): 3306.
[30]	蔡吉, 陈文聪, 刘军, 等. 基于两步法制备生物发酵蛋白工艺的研究 [J]. 中国酿造, 2022, 41(10): 207-212.
	Cai J, Chen WC, Liu J, et al. Preparation technology of bio-fermented protein based on two-step method [J]. China Brew, 2022, 41(10): 207-212.
[31]	夏欣欣, 林海, 杨谷良, 等. 混菌固态发酵米糠产谷维素的工艺及抗氧化活性评估 [J]. 食品与机械, 2023, 39(9): 154-161.
	Xia XX, Lin H, Yang GL, et al. Process study on mixed bacterial solid-state fermentation of rice bran for the preparation of oryzanol and evaluation of its antioxidant activity [J]. Food Mach, 2023, 39(9): 154-161.
[32]	Xu XQ, Xu ZQ, Shi S, et al. Lignocellulose degradation patterns, structural changes, and enzyme secretion by Inonotus obliquus on straw biomass under submerged fermentation [J]. Bioresour Technol, 2017, 241: 415-423.
[33]	Xu QQ, Zhao MJ, Yu ZZ, et al. Enhancing enzymatic hydrolysis of corn cob, corn stover and sorghum stalk by dilute aqueous ammonia combined with ultrasonic pretreatment [J]. Ind Crops Prod, 2017, 109: 220-226.
[34]	Tuyen DV, Phuong HN, Cone JW, et al. Effect of fungal treatments of fibrous agricultural by-products on chemical composition and in vitro rumen fermentation and methane production [J]. Bioresour Technol, 2013, 129: 256-263.
[35]	杜云龙, 潘金石, 冯鑫, 等. 基于体外产气法的吉林省食用菌菌糠的营养价值评定 [J]. 中国畜牧兽医, 2024, 51(4): 1520-1529.
	Du YL, Pan JS, Feng X, et al. Evaluation of nutritional value of fungus bran in Jilin Province based on in vitro gas production method [J]. China Anim Husb Vet Med, 2024, 51(4): 1520-1529.

因素 Factor	水平 Level	对照组 Control group	单菌处理组 Single-bacteria treatment group		三菌处理组 Three-bacteria treatment group		P-value
因素 Factor	水平 Level	ADL含量	ADL含量	ADL降解率	ADL含量	ADL降解率	P-value
温度	28 ℃	5.77±0.41A	4.9±0.22B	15.02±3.86b	4.65±0.1B	19.35±1.8a	<0.01
	30 ℃	5.77±0.41A	3.33±0.11B	42.29±1.93b	2.69±0.1B	53.4±1.72a	<0.01
	32 ℃	5.77±0.41A	5.14±0.09B	10.98±1.57b	4.31±0.1B	25.31±1.69a	<0.01
含水量	40%	5.77±0.41A	5.34±0.12BC	7.45±2b	3.74±0.6B	32.29±1.15a	<0.01
	45%	5.77±0.41A	4.42±0.2B	23.34±3.49b	2.38±0.07B	58.83±1.24a	<0.01
	50%	5.77±0.41A	5.27±0.14B	8.61±2.47b	4.14±0.67B	36.24±1.78a	<0.01
接种量	1%	5.77±0.41A	5.16±0.17B	10.63±2.86b	4.7±0.17B	18.48±2.91a	<0.01
	3%	5.77±0.41A	3.36±0.16B	41.83±2.8b	2.57±0.09C	55.42±1.54a	<0.01
	5%	5.77±0.41A	4.46±0.23B	22.76±3.95b	3.88±0.07C	32.78±1.27a	<0.01

因素 Factor	水平 Level	对照组 Control group	单菌处理组 Single-bacteria treatment group		三菌处理组 Three-bacteria treatment group		P-value
因素 Factor	水平 Level	ADL含量	ADL含量	ADL降解率	ADL含量	ADL降解率	P-value
温度	28 ℃	5.77±0.41A	4.9±0.22B	15.02±3.86b	4.65±0.1B	19.35±1.8a	<0.01
	30 ℃	5.77±0.41A	3.33±0.11B	42.29±1.93b	2.69±0.1B	53.4±1.72a	<0.01
	32 ℃	5.77±0.41A	5.14±0.09B	10.98±1.57b	4.31±0.1B	25.31±1.69a	<0.01
含水量	40%	5.77±0.41A	5.34±0.12BC	7.45±2b	3.74±0.6B	32.29±1.15a	<0.01
	45%	5.77±0.41A	4.42±0.2B	23.34±3.49b	2.38±0.07B	58.83±1.24a	<0.01
	50%	5.77±0.41A	5.27±0.14B	8.61±2.47b	4.14±0.67B	36.24±1.78a	<0.01
接种量	1%	5.77±0.41A	5.16±0.17B	10.63±2.86b	4.7±0.17B	18.48±2.91a	<0.01
	3%	5.77±0.41A	3.36±0.16B	41.83±2.8b	2.57±0.09C	55.42±1.54a	<0.01
	5%	5.77±0.41A	4.46±0.23B	22.76±3.95b	3.88±0.07C	32.78±1.27a	<0.01

方差来源 Variance source	平方和 Sum of squares	自由度 Degree of freedom	均方差 Mean square	F值 F value	P-value	显著性 Significance
模型Model	4 614.81	9	512.76	49.08	<0.000 1	**
A-温度	124.81	1	124.81	11.95	0.010 6	*
B-含水量	90.19	1	90.19	8.63	0.021 8	*
C-接种量	155.13	1	155.13	14.85	0.006 3	**
AB	0.005 6	1	0.005 6	0.000 5	0.982 1
AC	8.01	1	8.01	0.767 0	0.410 2
BC	43.45	1	43.45	4.16	0.080 8
A²	1 073.31	1	1 073.31	102.73	<0.000 1	**
B²	141.00	1	141.00	13.50	0.007 9	**
C²	1 731.93	1	1 731.93	165.78	<0.000 1	**
残差	73.13	7	10.45
失拟项	12.09	3	4.03	0.264 2	0.848 5	no significance
纯误差	61.04	4	15.26
总变异	4 687.94	16
R²					0.984 4
R²_Adj					0.964 3
R²_Pre					0.938 4
Adeq precision					21.201 5
CV （%）					7.84

方差来源 Variance source	平方和 Sum of squares	自由度 Degree of freedom	均方差 Mean square	F值 F value	P-value	显著性 Significance
模型Model	4 614.81	9	512.76	49.08	<0.000 1	**
A-温度	124.81	1	124.81	11.95	0.010 6	*
B-含水量	90.19	1	90.19	8.63	0.021 8	*
C-接种量	155.13	1	155.13	14.85	0.006 3	**
AB	0.005 6	1	0.005 6	0.000 5	0.982 1
AC	8.01	1	8.01	0.767 0	0.410 2
BC	43.45	1	43.45	4.16	0.080 8
A²	1 073.31	1	1 073.31	102.73	<0.000 1	**
B²	141.00	1	141.00	13.50	0.007 9	**
C²	1 731.93	1	1 731.93	165.78	<0.000 1	**
残差	73.13	7	10.45
失拟项	12.09	3	4.03	0.264 2	0.848 5	no significance
纯误差	61.04	4	15.26
总变异	4 687.94	16
R²					0.984 4
R²_Adj					0.964 3
R²_Pre					0.938 4
Adeq precision					21.201 5
CV （%）					7.84

组别 Group	对照组 Control group	发酵时间 Fermentation time （d）			P-value
组别 Group	对照组 Control group	7	14	21	P-value
粗蛋白（CP）	2.8±1.14C	4.69±0.62B	5.95±0.29A	6.79±0.44A	<0.001
CP增长率		0.68±0.22C	1.13±0.1B	1.43±0.16A	<0.001
酸性洗涤纤维（ADF）	43.57±1.16A	38.19±0.4B	35.67±1.98C	28.77±1.08D	<0.001
ADF降解率		12.36±0.92C	18.13±4.55B	33.97±2.47A	<0.001
中性洗涤粗纤维（NDF）	78.31±1.37A	68.13±1.36B	66.56±0.51B	60.99±1.83C	<0.001
NDF降解率		13.01±1.73C	15.01±0.65B	22.12±2.33A	<0.001
相对饲用品质（RFQ）	56.22±2.7D	74.55±0.76C	81.07±4.06B	102.77±3.5A	<0.001
相对饲用价值（RFV）	41.07±0.73B	40.7±1.44B	42.79±2.38B	48.14±2.04A	<0.001