基于代谢组学解析白酒糟双菌发酵单细胞蛋白的原料品质

doi:10.13560/j.cnki.biotech.bull.1985.2026-0567

摘要/Abstract

摘要：

目的研究产朊假丝酵母与酿酒酵母双菌固态发酵白酒糟制备单细胞蛋白（single-cell protein, SCP）的工艺条件，基于代谢组学分析发酵产物的代谢物谱变化，及其与蛋白品质、风味特性、安全性的关联。方法以浓香型白酒糟为底物，采用单因素与Box-Behnken试验设计优化发酵工艺，利用代谢组学分析双菌发酵与空白原料中挥发性及非挥发性代谢物差异，并结合营养成分测定、体外消化试验及霉菌毒素检测进行系统品质评价。结果最优发酵工艺为麸皮15%、温度30 ℃、接种量15%、水分58%、pH 5.5和发酵时间3 d，发酵后酸溶蛋白（acid-soluble protein, ASP）明显提升35.03%，粗蛋白（crude protein, CP）和真蛋白（true protein, TP）分别提升16.64%、29.32%。代谢组学共鉴定出9类92种挥发性风味物质，其中酯类含量大幅增加，已醛等不良风味物质显著下降；差异代谢物共筛选出296个，其中脂类及萜类衍生物显著上调，芳香族等氨基酸类及蒽醌类显著下调；KEGG富集显示谷氨酸等氨基酸生物合成、2-氧代羧酸代谢等4条通路被激活。体外消化率显著提升，霉菌毒素未检出，符合国标饲料卫生标准。结论产朊假丝酵母与酿酒酵母双菌固态发酵白酒糟的工艺能显著改善蛋白品质与风味特性，为白酒糟高值化利用提供了基于代谢组学的数据支撑与品质评价新视角。

关键词: 白酒糟, 单细胞蛋白, 双菌协同发酵, 代谢组学, 品质评价

Abstract:

Objective This study aimed to investigate the process conditions for producing single-cell protein (SCP) from Baijiu distiller’s grains through solid-state fermentation with Candida utilis and Saccharomyces cerevisiae, and to analyze the changes in metabolite profiles of the fermented products based on metabolomics, as well as their associations with protein quality, flavor characteristics, and safety. Method Nongxiang-type Baijiu distiller’s grain served as the substrate. Single-factor and Box-Behnken experimental designs were used to optimize the fermentation process. Metabolomics analysis identified differences in volatile and non‑volatile metabolites between dual‑strain fermented samples and the blank control. Nutrient composition determination, in vitro digestion tests, and mycotoxin detection were performed to complete the systematic quality evaluation. Result The optimal fermentation parameters were determined as 15% wheat bran supplementation, 30 ℃, 15% inoculum size, 58% moisture content, pH 5.5, and 3-day fermentation duration. After fermentation, acid-soluble protein (ASP) increased markedly by 35.03%, while crude protein (CP) and true protein (TP) increased by 16.64% and 29.32%, respectively. Metabolomics analysis identified 92 volatile flavor compounds across 9 chemical classes. Ester contents increased substantially, while undesirable flavor substances such as hexanal decreased significantly. A total of 296 differential metabolites were screened, among which lipids and terpenoid derivatives were significantly up‑regulated, while aromatic amino acids and anthraquinones were significantly down‑regulated. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis revealed the activation of four metabolic pathways, including glutamic acid biosynthesis and 2-oxocarboxylic acid metabolism. In vitro digestibility improved significantly, mycotoxin tests showed negative results, and the product met national feed hygiene standards. Conclusion The solid-state fermentation of liquor residue using both Candida utilis and Saccharomyces cerevisiae can significantly improves protein quality and flavor characteristics, and providing metabolomics-based data support and a novel quality evaluation perspective for the high-value utilization of Baijiu distiller’s grains.

Key words: Baijiu distiller’s grains, single-cell protein, dual-bacteria synergistic fermentation, metabolomics, quality evaluation

桂元, 杨婷妃, 王风青, 刘军, 刘根侨, 李仲玄, 廖钰婷, 王川, 李丽, 周正富. 基于代谢组学解析白酒糟双菌发酵单细胞蛋白的原料品质[J]. 生物技术通报, 2026, 42(11): 283-296.

GUI Yuan, YANG Ting-fei, WANG Feng-qing, LIU Jun, LIU Gen-qiao, LI Zhong-xuan, LIAO Yu-ting, WANG Chuan, LI Li, ZHOU Zheng-fu. Raw Material Quality Analysis of Single‑cell Protein Production from Baijiu Distiller’s Grains via Dual‑strain Fermentation Based on Metabolomics[J]. Biotechnology Bulletin, 2026, 42(11): 283-296.

图/表 12

图1 不同因素对酒糟固态发酵蛋白含量的影响A：麸皮添加量；B：发酵温度；C：接种量；D：初始pH；E：发酵时间；F：水分含量。CP：粗蛋白；ASP：酸溶蛋白；TP：真蛋白。不同小写字母表示差异显著（P<0.05），下同

Fig. 1 Effect of different factors on the protein content in solid-state fermentation of distiller's grainsA: Addition amount of wheat bran. B: Fermentation temperature. C: Inoculation amount. D: Initial pH. E: Fermentation time. F: Moisture content. CP: Crude protein; ASP: acid soluble protein; TP: true protein. Different lowercase letters indicate significant difference (P<0.05). The same below

图2 各因素交互作用对ASP/CP含量的影响

Fig. 2 Effect of interaction among various factors on ASP/CP content

表1 响应面试验因素水平

Table 1 Factors and levels of response surface experiments

水平

Level

发酵温度（A）

Fermentation temperature （℃）

接种量（B）

Inoculation amount（%）

发酵时间（C）

Fermentation time （d）

-1

表2 响应面试验结果

Table 2 Results of response surface methodology experiment

试验编号 Test number	发酵温度（A） Fermentation temperature （℃）	接种量（B） Inoculation amount （%）	发酵时间（C） Fermentation time （d）	酸溶蛋白/粗蛋白 ASP/CP （%）	真蛋白 TP （g/100 g）
1	27	10	3	30.09	10.70
2	33	10	3	30.93	9.81
3	27	18	3	31.19	10.55
4	33	18	3	31.86	9.90
5	27	14	2	31.77	10.59
6	33	14	2	31.97	9.22
7	27	14	4	30.47	10.00
8	33	14	4	31.62	9.32
9	30	10	2	32.65	10.58
10	30	18	2	32.79	10.61
11	30	10	4	31.09	10.40
12	30	18	4	33.05	10.06
13	30	14	3	33.95	11.06
14	30	14	3	34.26	11.20
15	30	14	3	34.27	10.98
16	30	14	3	34.02	11.00
17	30	14	3	34.16	11.05

表3 ASP/CP的回归模型方差分析结果

Table 3 Results of variance analysis of the regression model of ASP/CP

方差来源 Variance source	平方和 Sum of squares	自由度 Degrees of freedom	均方 Mean square	F值 F-value	P值 P-value	显著性 Significance
模型	31.42	9	3.49	245.78	<0.000 1	**
A-温度（℃）	1.02	1	1.02	72.15	<0.000 1	**
B-接种量（%）	2.13	1	2.13	149.62	<0.000 1	**
C-时间（d）	1.10	1	1.10	77.12	<0.000 1	**
AB	0.007 9	1	0.007 9	0.553 1	0.481 3	ns
AC	0.228 2	1	0.228 2	16.07	0.005 1	**
BC	0.832 6	1	0.832 6	58.62	0.000 1	**
A²	17.24	1	17.24	1 213.61	<0.000 1	**
B²	4.98	1	4.98	350.89	<0.000 1	**
C²	1.76	1	1.76	123.91	<0.000 1	**
残差	0.099 4	7	0.014 2
失拟误差	0.017 8	3	0.005 9	0.290 7	0.831 0	ns
纯误差	0.081 6	4	0.020 4
总误差	31.52	16
R²：0.996 8， R²_Adj：0.970 6， R²_Pre：0.986 8

表4 TP含量的回归模型方差分析结果

Table 4 ANOVA results of the regression model for TP content

方差来源 Variance source	平方和 Sum of squares	自由度 Degrees of freedom	均方 Mean square	F值 F-value	P值 P-value	显著性 Significance
模型	5.78	9	0.642 6	59.75	<0.000 1	**
A-温度（℃）	1.61	1	1.61	150.13	<0.000 1	**
B-接种量（%）	0.016 0	1	0.016 0	1.49	0.261 9	ns
C-时间（d）	0.187 9	1	0.187 9	17.47	0.004 1	**
AB	0.014 1	1	0.014 1	1.31	0.289 4	ns
AC	0.116 6	1	0.116 6	10.84	0.013 2	*
BC	0.033 3	1	0.033 3	3.10	0.121 7	ns
A²	2.21	1	2.21	205.59	<0.000 1	**
B²	0.036 9	1	0.036 9	3.43	0.106 4	ns
C²	1.29	1	1.29	119.70	<0.000 1	**
残差	0.075 3	7	0.010 8
失拟误差	0.047 0	3	0.015 7	2.22	0.228 4	ns
纯误差	0.028 3	4	0.007 1
总误差	5.86	16
R²：0.987 2， R²_Adj：0.970 6， R²_Pre：0.864 0

图3 各因素交互作用对TP含量的影响

Fig. 3 Effect of interaction among various factors on TP content

表5 酒糟发酵前后营养指标变化

Table 5 Changes in nutritional indicators of distiller's grains before and after fermentation

组别

Group

粗蛋白

CP（g/100 g）

酸溶蛋白

ASP（g/100 g）

酸溶蛋白/粗蛋白

ASP/CP（%）

真蛋白

TP（g/100 g）

总酸

Total acid （g/kg）

粗脂肪

Ether extract（%）

含水量

Water content（%）

图4 空白原料和发酵酒糟的差异风味物质筛选火山图（A）与环形层次聚类热图（B）K：空白原料；Y：发酵酒槽。下同

Fig. 4 Volcanic map (A) and circular hierarchical clustering heat map (B) for screening differential flavor substances between blank raw materials and fermented distiller's grainsK: Blank raw materials; Y: fermented distiller’s grains. The same below

图5 空白原料和发酵酒糟的差异代谢物火山图（A）、火柴杆图（B）和层次聚类热图（C）

Fig. 5 Volcano plot (A), matchstick plot (B) and hierarchical clustering heat map (C) of differential metabolites between blank raw material and fermented distiller's grainsNote: Compound-X: (1S,6S,9S,10R,14R,17S,19S)-12-ethyl-9,19-dihydroxy-17-methoxy-14-methyl-5-oxa-12-azahexacyclo [8.7.2.1~2,6~.0~1,11~.0~3,9~.0~14,18~] icosan-4-one

图6 空白原料和发酵酒糟的差异代谢物KEGG富集图

Fig. 6 KEGG enrichment plots of differential metabolites between blank raw materials and fermented distiller's grains

图7 空白原料与发酵酒糟体外模拟消化后各蛋白成分消化率

Fig. 7 In vitro digestibility of individual protein fractions in blank raw material and fermented distiller's grains after simulated digestion

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