微生物学报

Levels	A: Amount of inoculation (%)	B: Amount of inorganic nitrogenadded (%)	C: Time of fermentation(h)
1	5	2	36
2	8	5	48
3	10	8	60

Levels	A: Amount of inoculation (%)	B: Amount of inorganic nitrogenadded (%)	C: Time of fermentation(h)
1	5	2	36
2	8	5	48
3	10	8	60

Groups	Substrate of fermentation
A	100% Wheat bran
B	98% Wheat bran+2% Enzymatic hydrolysis of soybean meal
C	95% Wheat bran+5% Enzymatic hydrolysis of soybean meal
D	97.8% Wheat bran+2% Soybean meal+0.1% Protease+0.1% Cellulase
E	94.8% Wheat bran+5% Soybean meal+0.1% Protease+0.1% Cellulase

Groups	Substrate of fermentation
A	100% Wheat bran
B	98% Wheat bran+2% Enzymatic hydrolysis of soybean meal
C	95% Wheat bran+5% Enzymatic hydrolysis of soybean meal
D	97.8% Wheat bran+2% Soybean meal+0.1% Protease+0.1% Cellulase
E	94.8% Wheat bran+5% Soybean meal+0.1% Protease+0.1% Cellulase

Strain No.	Colony number (×10⁸ CFU/mL)
NJ15	0.57±0.01b
CJ12	0.72±0.03a
NJ16	0.25±0.01d
YJ19	0.40±0.02c
P-value	<0.01

Strain No.	Colony number (×10⁸ CFU/mL)
NJ15	0.57±0.01b
CJ12	0.72±0.03a
NJ16	0.25±0.01d
YJ19	0.40±0.02c
P-value	<0.01

Strain No.	Ammonia nitrogen utilization (%)	GS activity (μmol/(h·g))
NJ15	46.23±0.39	0.18±0.03b
CJ12	55.39±0.24	0.29±0.04a
NJ16	48.19±0.21	0.16±0.02b
YJ19	50.02±1.42	0.06±0.03c
P-value	>0.05	0.04

Strain No.	Ammonia nitrogen utilization (%)	GS activity (μmol/(h·g))
NJ15	46.23±0.39	0.18±0.03b
CJ12	55.39±0.24	0.29±0.04a
NJ16	48.19±0.21	0.16±0.02b
YJ19	50.02±1.42	0.06±0.03c
P-value	>0.05	0.04

No.	KEGG metabolic pathway	Number of differential metabolites
1	Biosynthesis of phenylpropanoids	15
2	Tryptophan metabolism	11
3	Arginine and proline metabolism	10
4	ABC transporters	8
5	Purine metabolism	8
6	Biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid	7
7	Nicotinate and nicotinamide metabolism	7
8	Citrate cycle (TCA cycle)	7
9	Folate biosynthesis	7
10	Isoflavonoid biosynthesis	7
11	d-amino acid metabolism	7
12	Pyrimidine metabolism	7
13	Histidine metabolism	6
14	Phenylalanine metabolism	6
15	Methane metabolism	6
16	Biosynthesis of terpenoids and steroids	5
17	Biosynthesis of alkaloids derived from histidine and purine	5
18	Pantothenate and CoA biosynthesis	5
19	Two-component system	5
20	Tyrosine metabolism	5
21	Phosphotransferase system (PTS)	4
22	Cysteine and methionine metabolism	4
23	Lysine biosynthesis	4
24	Glycine, serine and threonine metabolism	4
25	Biosynthesis of alkaloids derived from terpenoid and polyketide	4
26	Biosynthesis of unsaturated fatty acids	4
27	Biosynthesis of various antibiotics	4
28	Alanine, aspartate and glutamate metabolism	3
29	Chlorocyclohexane and chlorobenzene degradation	3
30	Ubiquinone and other terpenoid-quinone biosynthesis	3
31	Bisphenol degradation	3
32	C5-branched dibasic acid metabolism	3
33	Biotin metabolism	2
34	Amino sugar and nucleotide sugar metabolism	2
35	Cell cycle-yeast	1

No.	KEGG metabolic pathway	Number of differential metabolites
1	Biosynthesis of phenylpropanoids	15
2	Tryptophan metabolism	11
3	Arginine and proline metabolism	10
4	ABC transporters	8
5	Purine metabolism	8
6	Biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid	7
7	Nicotinate and nicotinamide metabolism	7
8	Citrate cycle (TCA cycle)	7
9	Folate biosynthesis	7
10	Isoflavonoid biosynthesis	7
11	d-amino acid metabolism	7
12	Pyrimidine metabolism	7
13	Histidine metabolism	6
14	Phenylalanine metabolism	6
15	Methane metabolism	6
16	Biosynthesis of terpenoids and steroids	5
17	Biosynthesis of alkaloids derived from histidine and purine	5
18	Pantothenate and CoA biosynthesis	5
19	Two-component system	5
20	Tyrosine metabolism	5
21	Phosphotransferase system (PTS)	4
22	Cysteine and methionine metabolism	4
23	Lysine biosynthesis	4
24	Glycine, serine and threonine metabolism	4
25	Biosynthesis of alkaloids derived from terpenoid and polyketide	4
26	Biosynthesis of unsaturated fatty acids	4
27	Biosynthesis of various antibiotics	4
28	Alanine, aspartate and glutamate metabolism	3
29	Chlorocyclohexane and chlorobenzene degradation	3
30	Ubiquinone and other terpenoid-quinone biosynthesis	3
31	Bisphenol degradation	3
32	C5-branched dibasic acid metabolism	3
33	Biotin metabolism	2
34	Amino sugar and nucleotide sugar metabolism	2
35	Cell cycle-yeast	1

Strain No.	A	B	C	Ammonia nitrogen utilization (%)
1	1	1	1	61.23
2	1	2	3	26.51
3	1	3	2	49.48
4	2	1	3	72.08
5	2	2	2	44.18
6	2	3	1	48.26
7	3	1	2	50.67
8	3	2	1	47.40
9	3	3	3	49.88
K₁	45.74	61.33	52.30
K₂	54.84	39.36	48.11
K₃	49.32	39.36	49.49
R	9.10	21.97	4.19

Strain No.	A	B	C	Ammonia nitrogen utilization (%)
1	1	1	1	61.23
2	1	2	3	26.51
3	1	3	2	49.48
4	2	1	3	72.08
5	2	2	2	44.18
6	2	3	1	48.26
7	3	1	2	50.67
8	3	2	1	47.40
9	3	3	3	49.88
K₁	45.74	61.33	52.30
K₂	54.84	39.36	48.11
K₃	49.32	39.36	49.49
R	9.10	21.97	4.19

Groups	Colony number (×10¹⁰ CFU/mL)
A	0. 17±0.02c
B	0.22±0.05b
C	0.26±0.02a
D	0.24±0.01b
E	0.28±0.03a
P-value	0.04

Groups	Colony number (×10¹⁰ CFU/mL)
A	0. 17±0.02c
B	0.22±0.05b
C	0.26±0.02a
D	0.24±0.01b
E	0.28±0.03a
P-value	0.04

Strain No.	Control group	Experimental group	P-value
Dry matter (%)	50.58±1.16a	48.46±0.63b	0.04
Crude protein (%)	20.88±0.12b	24.21±0.47a	0.02
Small peptide (%)	2.30±0.34	2.61±0.32	0.58
Organic nitrogen (%)	2.82±0b	3.63±0.04a	0.00
Ash (%)	6.47±0.25	7.37±0.16	0.82
Calcium (%)	0.26±0.05	0.28±0.02	0.79
Total phosphorus (%)	1.22±0.04	1.37±0.04	0.57
Crude fat (%)	4.80±0.53a	4.21±0.98b	0.04
Crude fiber (%)	5.77±1.54a	4.65±1.36b	0.02
Phytic phosphoric acid (%)	0.37±0.01a	0.32±0.01b	0.04

Strain No.	Control group	Experimental group	P-value
Dry matter (%)	50.58±1.16a	48.46±0.63b	0.04
Crude protein (%)	20.88±0.12b	24.21±0.47a	0.02
Small peptide (%)	2.30±0.34	2.61±0.32	0.58
Organic nitrogen (%)	2.82±0b	3.63±0.04a	0.00
Ash (%)	6.47±0.25	7.37±0.16	0.82
Calcium (%)	0.26±0.05	0.28±0.02	0.79
Total phosphorus (%)	1.22±0.04	1.37±0.04	0.57
Crude fat (%)	4.80±0.53a	4.21±0.98b	0.04
Crude fiber (%)	5.77±1.54a	4.65±1.36b	0.02
Phytic phosphoric acid (%)	0.37±0.01a	0.32±0.01b	0.04

Amino acid	Control group	Experimental group	P-value
Tryptophan	5.50±0.36a	0.41±0.05b	<0.01
Phenylalanine	0.56±0.04a	0.39±0.07b	0.02
Methionine	0.09±0.00a	0.02±0.00	<0.01
Valine	1.76±0.11b	2.27±0.21a	0.02
4-aminobutyric acid	6.22±0.30a	1.64±0.19b	<0.01
Tyrosine	0.56±0.05b	1.64±0.30a	<0.01
Proline	2.51±0.11a	1.62±0.23b	<0.01
Beta-alanine	0.38±0.04b	4.51±0.44a	<0.01
Alanine	7.63±0.33b	15.92±0.43a	<0.01
Glycine	1.97±0.03a	1.59±0.13b	0.01
Glutamic acid	2.14±0.08b	6.47±1.01a	<0.01
Hydroxyproline	0.07±0.01	0.08±0.01	0.08
Threonine	0.87±0.03b	1.38±0.15a	<0.01
Aspartic acid	2.06±0.29a	1.23±0.08b	0.01
Glutamine	0.08±0.01b	8.91±0.87a	<0.01
Serine	0.89±0.01	0.86±0.04	0.29
Citrulline	0.03±0.00	0.03±0.00	0.96
Asparagine	8.68±0.13a	1.26±0.07b	<0.01
Arginine	1.05±0.02b	1.79±60.03a	<0.01
3-methyl-l-histidine	0.03±0.00a	0.02±0.00b	<0.01
Lysine	0.50±0.06b	1.50±0.04a	<0.01
Histidine	0.22±0.02b	1.17±0.08a	<0.01
Ornithine	0.07±0.01b	0.18±0.02a	<0.01
5-hydroxylysine	N.D.	N.D.
1-methyl-l-histidine	N.D.	N.D.
Total	43.84±1.90b	54.87±3.16a	<0.01

Amino acid	Control group	Experimental group	P-value
Tryptophan	5.50±0.36a	0.41±0.05b	<0.01
Phenylalanine	0.56±0.04a	0.39±0.07b	0.02
Methionine	0.09±0.00a	0.02±0.00	<0.01
Valine	1.76±0.11b	2.27±0.21a	0.02
4-aminobutyric acid	6.22±0.30a	1.64±0.19b	<0.01
Tyrosine	0.56±0.05b	1.64±0.30a	<0.01
Proline	2.51±0.11a	1.62±0.23b	<0.01
Beta-alanine	0.38±0.04b	4.51±0.44a	<0.01
Alanine	7.63±0.33b	15.92±0.43a	<0.01
Glycine	1.97±0.03a	1.59±0.13b	0.01
Glutamic acid	2.14±0.08b	6.47±1.01a	<0.01
Hydroxyproline	0.07±0.01	0.08±0.01	0.08
Threonine	0.87±0.03b	1.38±0.15a	<0.01
Aspartic acid	2.06±0.29a	1.23±0.08b	0.01
Glutamine	0.08±0.01b	8.91±0.87a	<0.01
Serine	0.89±0.01	0.86±0.04	0.29
Citrulline	0.03±0.00	0.03±0.00	0.96
Asparagine	8.68±0.13a	1.26±0.07b	<0.01
Arginine	1.05±0.02b	1.79±60.03a	<0.01
3-methyl-l-histidine	0.03±0.00a	0.02±0.00b	<0.01
Lysine	0.50±0.06b	1.50±0.04a	<0.01
Histidine	0.22±0.02b	1.17±0.08a	<0.01
Ornithine	0.07±0.01b	0.18±0.02a	<0.01
5-hydroxylysine	N.D.	N.D.
1-methyl-l-histidine	N.D.	N.D.
Total	43.84±1.90b	54.87±3.16a	<0.01

Type of free radical	Sample	Linear regression equation	IC₅₀ value (mg/mL)	Mass equivalent (mg)
DPPH free radical	Control group	y=2.55x+2.25	18.76±0.02a	1.00
Experimental group	y=5.67x+21.91	4.97±0.04b	3.78
ABTS free radical	Control group	y=7.38x-6.56	7.66±0.01a	1.00
Experimental group	y=7.36x-2.78	7.20±0.02b	1.06

Type of free radical	Sample	Linear regression equation	IC₅₀ value (mg/mL)	Mass equivalent (mg)
DPPH free radical	Control group	y=2.55x+2.25	18.76±0.02a	1.00
Experimental group	y=5.67x+21.91	4.97±0.04b	3.78
ABTS free radical	Control group	y=7.38x-6.56	7.66±0.01a	1.00
Experimental group	y=7.36x-2.78	7.20±0.02b	1.06

一株高效同化氨氮酵母菌的筛选及发酵工艺优化

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杨连弟 ¹ , 尹梦丽 ¹ , 陈煜 ¹ , 王乐 ¹ , 李军训 ² , 甘晓峰 ³ , 万堃 ¹ , 左福元 ¹ , 黄文明 ¹^,^*

微生物学报 | 微生物资源新技术新方法 2025,65(4): 1635-1649

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微生物学报 | 微生物资源新技术新方法 2025, 65(4): 1635-1649

一株高效同化氨氮酵母菌的筛选及发酵工艺优化

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杨连弟¹, 尹梦丽¹, 陈煜¹, 王乐¹, 李军训², 甘晓峰³, 万堃¹, 左福元¹, 黄文明¹^,^*

作者信息

^1.西南大学动物科学技术学院，重庆市肉牛工程技术研究中心，重庆

^2.山东泰山生力源集团股份有限公司，山东泰安

^3.成都奥盛康生物科技有限公司，四川成都

Screening and fermentation process optimization of an efficient ammonia nitrogen-assimilating yeast strain

Liandi YANG¹, Mengli YIN¹, Yu CHEN¹, Le WANG¹, Junxun LI², Xiaofeng GAN³, Kun WAN¹, Fuyuan ZUO¹, Wenming HUANG¹^,^*

Affiliations

^1.Chongqing Beef Cattle Engineering Technology Research Center, College of Animal Science and Technology, Southwest University, Chongqing, China

^2.Shandong Taishan Shengliyuan Group Co. , Ltd. , Tai’an, Shandong, China

^3.Chengdu Aoshengkang Biotechnology Co. , Ltd. , Chengdu, Sichuan, China

出版时间: 2025-04-04 doi: 10.13343/j.cnki.wsxb.20240853

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摘要

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【目的】筛选出一株能够高效同化氨氮的酵母菌，并进行固态发酵，通过检测发酵饲料的营养成分、抗氧化活性和氨基酸含量等指标，优化发酵工艺，为利用该酵母菌生产单细胞蛋白饲料提供科学依据。【方法】采用硫酸铵作为唯一氮源的培养基分别培养5株产朊假丝酵母(Candida utilis)、4株异常毕赤酵母(Pichia anomala)、5株酿酒酵母(Saccharomyces cerevisiae)和3株东方伊萨酵母(Issatchenkia orientalis)，筛选氨氮利用率和谷氨酰胺合成酶(glutamine synthetase, GS)活性均较高的酵母菌作为供试菌株，并对其发酵工艺参数和发酵底物进行优化。测定发酵饲料的常规营养成分、植酸磷、氨基酸含量，以及1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基和2,2′-联氮-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐[2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid), ABTS]自由基的清除率。【结果】筛选到的产朊假丝酵母CJ12氨氮利用率为55.39%，GS活性为0.29 μmol/(h·g)，高于其他酵母菌。代谢组学结果表明，氨基酸代谢是CJ12氨同化的关键代谢途径。产朊假丝酵母CJ12的最佳发酵工艺为：硫酸铵添加量2%，接种量8%，发酵时间36 h。组成最佳发酵底物为94.8%麦麸、5%豆粕、0.1%蛋白酶和0.1%纤维素酶。产朊假丝酵母CJ12发酵后，饲料的粗蛋白质和有机氮含量较发酵前分别提高了15.95%和28.46% (P<0.05)；干物质、粗纤维、粗脂肪和植酸磷含量分别降低了4.19%、19.41%、12.29%、13.51% (P<0.05)；总氨基酸含量显著提高(P<0.05)，其中谷氨酰胺和谷氨酸含量分别是对照组的111.38倍和3.02倍，而对照组的色氨酸、天冬酰胺、4-氨基丁酸含量分别是试验组的13.41倍、8.27倍和6.41倍。DPPH和ABTS自由基的IC₅₀值分别降低了73.51%和6.01% (P<0.05)，自由基清除能力显著提高。【结论】产朊假丝酵母CJ12具有高效的氨氮同化能力和谷氨酰胺合成能力，能够显著提高饲料的营养价值和抗氧化性能，具有生产功能性单细胞蛋白饲料的潜力。

关键词

氨氮同化 / 酵母菌 / 氨基酸 / 单细胞蛋白

Abstract

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[Objective] To screen out a yeast strain that can efficiently assimilate ammonia nitrogen, optimize the solid-state fermentation conditions based on the nutrient composition, antioxidant activity, and amino acid content of the feed, and provide a scientific basis for the production of single-cell protein feed with this yeast strain. [Methods] Five strains of Candida utilis, four strains of Pichia anomala, five strains of Saccharomyces cerevisiae, and three strains of Issatchenkia orientalis were cultured with (NH₄)₂SO₄ as the sole nitrogen source. The yeast strain with the highest ammonia utilization rate and the highest glutamine synthetase (GS) activity was selected as the test strain, and then the fermentation parameters and fermentation substrates were optimized for this strain. The routine nutrient composition, content of phytate phosphorusand amino acids, and scavenging rates against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline- 6-sulfonicacid) (ABTS) free radicals of the fermented feed were determined. [Results] C. utilis CJ121 showed an ammonia utilization rate of 55.39% and a GS activity of 0.29 μmol/(h·g), which were higher than those of other yeast strains. The optimal fermentation process for C. utilis CJ12 was fermentation with a (NH₄)₂SO₄ addition amount of 2% and an inoculation amount of 8% for 36 h. The optimal fermentation substrate was composed of 94.8% wheat bran, 5% soybean meal, 0.1% protease, and 0.1% cellulase. After fermentation with C. utilis CJ12, the content of crude protein and organic nitrogen increased by 15.95% and 28.46%, respectively (P<0.05), while that of dry matter, crude fiber, crude fat, and phytate phosphorus decreased by 4.19%, 19.41%, 12.29%, and 13.51%, respectively (P<0.05). The total amino acid content increased after fermentation (P<0.05), with glutamine and glutamic acid levels being 111.38 times and 3.02 times those of the control group, respectively. However, the control group exhibited higher levels of tryptophan, asparagine, and 4-aminobutyric acid, which were 13.41, 8.27, and 6.41 times those of the experimental group, respectively. In addition, the scavenging abilities against DPPH and ABTS free radicals increased after fermentation (P<0.05), with the IC₅₀ values decreasing by 73.51% and 6.01%, respectively (P<0.05). [Conclusion] C. utilis CJ12 has high capacities of utilizing ammonia nitrogen and synthesizing glutamine. This strain improves the nutritional value and antioxidant performance of feed after solid-state fermentation, thus demonstrating the potential for producing functional single-cell protein feed.

Key words

ammonia nitrogen assimilation / yeast / amino acids / single-cell protein

引用本文

杨连弟, 尹梦丽, 陈煜, 王乐, 李军训, 甘晓峰, 万堃, 左福元, 黄文明. 一株高效同化氨氮酵母菌的筛选及发酵工艺优化. 微生物学报, 2025 , 65 (4) : 1635 -1649 . DOI: 10.13343/j.cnki.wsxb.20240853

Liandi YANG, Mengli YIN, Yu CHEN, Le WANG, Junxun LI, Xiaofeng GAN, Kun WAN, Fuyuan ZUO, Wenming HUANG. Screening and fermentation process optimization of an efficient ammonia nitrogen-assimilating yeast strain[J]. Acta Microbiologica Sinica, 2025 , 65 (4) : 1635 -1649 . DOI: 10.13343/j.cnki.wsxb.20240853

正文

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单细胞蛋白(single cell protein, SCP)是由酵母、霉菌、细菌和藻类等各种微生物在适宜条件下产生的蛋白质^[1]，具有生产时间短、蛋白质含量高、营养物质丰富、生产成本低等优点。SCP生产是目前缓解我国蛋白质饲料资源短缺、实现畜禽饲料中豆粕减量替代以及畜禽养殖节本增效的主要途径。酵母菌自身粗蛋白质含量高达40%-55%^[2]，还含有几乎所有种类的氨基酸和大量的维生素^[3]，是目前生产SCP应用最多、最广的一类微生物。Yadav等^[4]使用酿酒酵母处理小麦秸秆生产SCP，发酵过程中产SCP生物量最高可达到22.3×10⁷ CFU/mL。Jalasutram等^[5]使用产朊假丝酵母发酵家禽粪便生产SCP，经优化后其SCP产量可达29%。乔传丽等^[6]研究表明，酵母菌的蛋白酶活力为55-82 U/mL，能够有效提高发酵饲料的氨基酸和粗蛋白质含量。吴海燕等^[7]使用热带假丝酵母液态发酵马铃薯渣，其蛋白质产率达到43.12%。这些结果表明，酵母菌是利用低成本底物生产SCP的优良菌株。然而，不同酵母菌生产SCP的效果主要受菌种、碳源、氮源和发酵时间等因素的影响，其中，碳源与氮源作为微生物生长和繁殖的基本营养要素，对微生物的生长和蛋白质产量具有显著影响^[8]。

氨氮同化是指微生物将无机氮(如氨氮)转化为有机氮(如氨基酸和蛋白质)的过程。酵母菌同化氨氮时，主要由谷氨酸脱氢酶(glutamate dehydrogenase, GDH)和谷氨酰胺合成酶(glutamine synthetase, GS)催化完成，氨氮经转运蛋白进入酵母细胞后由GDH催化生成谷氨酸，谷氨酸与NH₄⁺在GS催化下生成谷氨酰胺^[9-10]。酵母作为重要的工业微生物，其氨氮同化能力直接影响发酵产物的品质和产量。然而，不同酵母菌株对氨氮的同化效率存在显著差异。因此，筛选高效同化氨氮的酵母菌并优化其发酵工艺，对于提高单细胞蛋白的产量和品质具有重要意义。本研究筛选一株可以高效同化氨氮的酵母菌，优化酵母菌的发酵工艺，并对酵母菌发酵麦麸前后的理化性质进行分析比较，以期为利用该酵母菌生产单细胞蛋白饲料提供科学依据。

1 材料与方法

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1.1 材料

本研究所用的17株酵母菌均为山东泰山生力源集团股份有限公司保藏菌株，其中，产朊假丝酵母(Candida utilis) 5株，编号为CJ10-CJ14；异常毕赤酵母(Pichia anomala) 4株，编号为YJ17-YJ20；酿酒酵母(Saccharomyces cerevisiae) 5株，编号为NJ14-NJ18；东方伊萨酵母(Issatchenkia orientalis) 3株，编号为DJ10-DJ12。

PDA培养基：参照李孝辉等^[11]的方法进行配制。

无机氮液体培养基(g/L)：葡萄糖30.0，硫酸铵20.0，磷酸氢二钾2.0，磷酸二氢钾1.0。其固体培养基的配制额外添加15.0 g/L的琼脂即可。

1.2 高效同化氨氮酵母菌的筛选

1.2.1 初筛

将保藏菌株在PDA固体培养基中划线，于32 ℃培养过夜直至菌落形成。用同一规格和大小的接种环挑取单菌落接种到100 mL无机氮液体培养基中，每个菌株设3个重复，32 ℃、250 r/min培养48 h后计酵母菌活菌数，挑取菌落数量大于1×10⁶ CFU/mL的菌株到新的无机氮培养基平板上，32 ℃培养48 h，重复3次，保存生长稳定的菌株。

1.2.2 复筛

将初筛后的菌株接种于100 mL无机氮培养基中(每个菌株重复3次)，在32 ℃、250 r/min下培养48 h。参照行标HJ535—2009测定菌液的氨氮含量^[12]，利用GS试剂盒(南京建成生物工程研究所)测定GS活性，根据公式(1)计算氨氮利用率，选择氨氮利用率和GS活性较高的菌株为目的菌株。

氨氮利用率=(M₀-M₁)/M₀×100%(1)

式中：M₀为空白培养基的氨氮含量；M₁为菌液中剩余氨氮含量。

1.3 生长曲线的绘制

将目的菌株在PDA培养基中培养48 h作为种子液，以体积分数为2%的接种量分别接种于100 mL无机氮培养基和PDA培养基中，32 ℃、250 r/min培养。在0-60 h测量OD₆₀₀值后绘制生长曲线，每个时间点设3个重复。

1.4 供试菌株的代谢组分析

样品准备与提取参照黄文明等^[13]的方法。供试菌株分别接种于PDA与无机氮培养基(各设置3次重复)，32 ℃、250 r/min培养至对数生长期(OD₆₀₀值分别为9.70和10.94)，5 000 r/min离心15 min取上清液于-80 ℃保存。提取时，上清液与含内标提取液混匀，超声5 min后4 ℃、12 000 r/min离心15 min，干燥后复溶，取120 μL上清液进样，制备成质控样本(quality control, QC)检测。

1.5 发酵工艺优化

1.5.1 发酵工艺参数优化

采用三因素三水平正交试验，准确称量240 g麦麸，固定发酵温度为32 ℃，含水量为50%，以接种量(A)、硫酸铵添加量(B)和发酵时间(C)为考察因素，以氨氮利用率为评价指标优化酵母菌发酵工艺，正交试验因素与水平见表1。

1.5.2 发酵底物优化

基于发酵工艺参数对酵母菌发酵底物进行优化。将酵母菌发酵底物分成5个处理组(表2)，处理组A：100%麦麸；处理组B：98%麦麸+2%酶解豆粕；处理组C：95%麦麸+5%酶解豆粕；处理组D：97.8%麦麸+2%豆粕+0.1%蛋白酶+0.1%纤维素酶；处理组E：94.8%麦麸+5%豆粕+0.1%蛋白酶+0.1%纤维素酶。发酵底物的含水量为50%，搅拌均匀后分装80 g至1 L三角瓶中，于121 ℃灭菌30 min后分别添加2%的硫酸铵，接种8%的酵母菌，发酵36 h后取出并计酵母菌活菌数。每个处理组设3个重复，选择活菌数最高的处理组为最佳发酵底物组合。

1.6 供试菌株发酵饲料的指标测定与方法

1.6.1 酵母活菌计数

取7支灭菌试管，用移液枪以无菌操作法取4.5 mL无菌水于各编号试管中。充分混匀待测菌液，用移液枪取0.5 mL菌液样品至编号为10^-1的试管中，混匀，以同样的方式吸取0.5 mL菌液至编号为10^-2的试管中，如此稀释至10^-7为止。选取10^-6和10^-7两个稀释度，吸取0.1 mL菌液加入准备好的PDA平板中，均匀涂布至菌液吸收，每个稀释度取3个重复。将平板倒置放于32 ℃培养箱中培养48 h后计活菌数。

1.6.2 营养成分和抗营养因子测定

根据最优组合发酵饲料。对照组除未接种酵母菌外，其他处理与试验组相同。将试验组和对照组饲料在65 ℃烘干，制备成风干样品。参照国标GB/T 6435—2014、GB/T 6432—2018、GB/T 22492—2008、GB/T 6434—2022、GB/T 6433— 2006、GB/T 6438—2007、GB/T 6436—2018、GB/T 6437—2018及三氯乙酸法分别测定水分、粗蛋白质、小肽、粗纤维、粗脂肪、粗灰分、钙、总磷和植酸磷的含量^[14-22]。参照国标GB/T 6432—2018的方法^[15]测定总氮含量。

参照GB 5009.234—2016方法测定无机氮含量^[23]，按公式(2)计算有机氮含量。

样品有机氮含量=总氮含量-无机氮含量(2)

氨氮利用率：取待检样品10 g置于锥形瓶内，加入90 mL蒸馏水混匀制成10^-1的稀释液，取1 mL稀释液加入9 mL蒸馏水中制成10^-2的均匀稀释液，以此类推，倍比稀释至10^-4，利用纳氏分光光度计法测定氨氮利用率。

采用GS试剂盒(南京建成生物工程研究所)测定GS活性。

1.6.3 抗氧化能力测定

准确称取1 g粉碎后的饲料样品，加20 mL蒸馏水，在80 ℃水浴中浸提，冷却后5 000 r/min离心15 min，取上清液并稀释成1、2、3、4、5 mg/mL的样品提取液，用于后续测定。

向试管中加入0.2 mL样品提取液及4 mL 0.2 mmol/L 1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)工作液，样品管常温避光反应30 min后于517 nm测吸光度；以蒸馏水替代提取液为对照管。按公式(3)计算DPPH自由基清除率。

DPPH自由基清除率=(1-A_样品管/A_对照管)×100%(3)

向试管中加入0.1 mL样品提取液及6 mL 2,2′-联氮-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐[2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid), ABTS]工作液，样品管常温避光反应10 min后于734 nm测吸光度；以蒸馏水替代提取液为对照管。按公式(4)计算ABTS自由基清除率。

ABTS自由基清除率=(1-A_样品管/A_对照管)×100%(4)

1.6.4 氨基酸组学分析

参照黄文明等^[13]的方法。取20 mg饲料样本于EP管，加钢珠及1 mL预冷提取液(乙腈:甲醇:水=2:2:1，含同位素内标)，涡旋、研磨、冰水浴超声处理后离心取上清，稀释后取80 μL进样，用于UHPLC-MS/MS分析。

1.7 数据统计与分析

采用SPSS 26.0软件对菌株的菌落数量、氨氮利用率和GS活性进行单因素方差分析(one-way ANOVA)和Duncan氏法多重比较；对发酵工艺参数优化进行极差分析；对发酵饲料营养成分含量、自由基清除率和氨基酸含量进行独立样本t检验。结果以平均值±标准差表示，P<0.05表示差异显著，P≥0.05表示差异不显著。代谢组数据由MassLynx v4.2系统采集，经Progenesis QI处理，基于在线METLIN等数据库鉴定。基于OPLS-DA模型的变量重要性投影(variable importance in projection, VIP)值、P-value及差异倍数(fold change)筛选VIP≥1、P-value≤0.05且|fold change|≥1的差异代谢物，并基于KEGG数据库进行富集通路分析。采用Agilent MassHunter Work Station Software (B.08.00)对25种氨基酸进行定量分析。

2 结果与分析

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2.1 高效同化氨氮酵母菌的筛选

从17株酵母菌中，通过无机氮培养基筛选出4株菌落数量大于1×10⁶ CFU/mL的菌株(表3)，分别为NJ15、CJ12、NJ16和YJ19。如表4所示，4株菌的氨氮利用率无显著差异(P>0.05)，而CJ12号菌株的GS活性显著高于NJ15、NJ16和YJ19号菌株(P<0.05)。因此，选择CJ12号菌株为目的菌株。

2.2 生长曲线的测定

CJ12菌株的生长曲线如图1所示。在无机氮培养基中，CJ12菌株经历12 h的延迟生长期后进入对数生长期(12-36 h)，随后保持稳定；而在PDA培养基中，CJ12菌株经历6 h的延迟生长期后进入对数生长期(6-28 h)，并于36 h后进入衰退期。结果表明，CJ12菌株在无机氮培养基中生长良好。

2.3 菌株在不同培养基的代谢组分析

2.3.1 代谢组数据主成分分析

对无机氮培养基培养的菌株(JN)和PDA培养基培养的菌株(JP)的代谢物进行主成分分析，结果显示，第一主成分(PC1)和第二主成分(PC2)对样品差异的贡献占比分别为51.55%和20.60%。不同组间存在明显的分离现象，表明JN和JP培养基中的代谢物在主成分上存在显著的差异。

2.3.2 代谢组数据OPLS-DA分析

两组样品在空间点上明显分离，进一步表明无机氮培养基培养的菌株和PDA培养基培养的菌株的代谢物存在差异。OPLS-DA模型的预测参数R²X为0.707，R²Y为0.997，Q²Y为0.948，说明模型对样本的解释度和预测度较高。Q²Y拟合回归线斜率为正，表明模型有意义。蓝点普遍位于红点上方，说明建模训练集和测试集的独立性较好，可根据VIP值筛选JP与JN代谢组间的差异代谢物。

2.3.3 差异代谢物筛选结果

基于非靶向代谢技术共筛选出代谢物3 235个，其中1 038个代谢物差异显著，636个显著上调，402个显著下调。筛选出在无机氮培养基中相对含量较高的32种代谢物。与JP组相比，JN组上调和下调的前10种代谢物的log₂ fold change结果如图2所示。其中，累积量上调的代谢产物主要分为维生素类、抗生素类、生物碱类和酶类；累积量下调的代谢物主要分为氨基酸类、甾醇类和脂肪酸类。

2.3.4 差异代谢物的KEGG分析

对差异代谢物富集的通路进行KEGG注释，筛选出富集差异代谢物最多的35个通路(表5)。在默认模式下，有12个代谢通路与各种氨基酸代谢相关，其次是苯丙烷生物合成、磷酸转移酶系统、嘌呤和嘧啶代谢、三羧酸循环、异黄酮生物合成和双组分系统等。这些结果表明，氨基酸代谢可能是产朊假丝酵母CJ12氨同化的关键代谢通路。

2.4 发酵工艺优化

2.4.1 发酵工艺参数优化

对氨氮利用率影响最大的因素为无机氮添加量，其次为接种量和发酵时间。最佳发酵工艺组合为A₂B₁C₁，即无机氮添加量为2%，接种量为8%，发酵时间为36 h (表6)。

2.4.2 发酵底物优化

如表7所示，处理组C和处理组E的活菌数显著高于处理组A、B和D (P<0.05)，处理组E的活菌数在数值上高于处理组C (P>0.05)。因此，选择处理组E作为酵母菌CJ12的最佳发酵底物，底物组合为94.8%麦麸+5%豆粕+0.1%蛋白酶+0.1%纤维素酶。

2.5 酵母菌发酵饲料的营养成分变化和氨基酸定量分析

如表8所示，酵母菌发酵饲料的粗蛋白质和有机氮含量显著高于未发酵饲料(P<0.05)，而干物质、粗脂肪、粗纤维和植酸磷含量显著低于未发酵饲料(P<0.05)，其他营养成分含量无显著差异(P>0.05)。如表9所示，发酵饲料中缬氨酸、酪氨酸、丙氨酸、谷氨酸、谷氨酰胺、赖氨酸和鸟氨酸的含量以及氨基酸总量显著高于对照组(P<0.05)。其中，谷氨酸和谷氨酰胺含量分别由2.14 mmol/100 g和0.08 mmol/100 g增加到6.47 mmol/100 g和8.91 mmol/100 g。色氨酸、苯基丙氨酸、甲硫氨酸、4-氨基丁酸、脯氨酸、甘氨酸、天冬氨酸、天冬酰胺和3-甲基-l-组氨酸的含量显著低于对照组(P<0.05)，而羟基脯氨酸、丝氨酸和瓜氨酸的含量无显著差异(P≥0.05)。

2.6 酵母菌发酵饲料的抗氧化活性变化

如图3所示，DPPH和ABTS自由基清除率随样品浓度的增加而升高。试验组发酵饲料的DPPH和ABTS自由基清除率均显著高于未发酵饲料(P<0.05)；如表10所示，发酵饲料与未发酵饲料的IC₅₀值分别为4.97 mg/mL和18.76 mg/mL，即1 mg发酵饲料对DPPH自由基的清除能力相当于3.78 mg未发酵饲料；发酵饲料与未发酵饲料的IC₅₀值分别为7.20 mg/mL和7.66 mg/mL，即1 mg发酵饲料对ABTS自由基的清除能力相当于1.06 mg未发酵饲料。

3 讨论

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3.1 高效同化氨氮的酵母菌筛选

微生物将无机氮转运至胞内转化为NH₄⁺-N，随后NH₄⁺-N与碳骨架结合将无机氮转化为有机氮的代谢过程称为氨同化^[24]。Sims等^[25]用¹⁵N同位素法发现，在以氨氮为唯一氮源的培养基中，产朊假丝酵母中仅有谷氨酸和谷氨酰胺中的氮元素来自¹⁵N，并且谷氨酸和谷氨酰胺的合成决定着酵母菌体内其他氨基酸的合成。研究表明，酵母菌氨同化作用主要通过2个途径进行：第1个途径是通过激活GDH促使氨与α-酮戊二酸合成谷氨酸；第2个途径是氨与谷氨酸通过GS合成谷氨酰胺，随后谷氨酰胺在谷氨酸合成酶(GOGAT)催化下合成2分子谷氨酸，然而GDH对氨的亲和力较GS/GOGAT途径低^[16-27]。Sieg等^[26]通过构建酵母突变体发现，当以氨为唯一氮源时，酵母菌主要通过GS/GOGAT途径产生谷氨酸。凌晓等^[28]以硫酸铵为唯一氮源筛选出的酿酒酵母和戴尔有孢圆酵母，其氨氮利用率分别为45.93%和35.35%。本研究中筛选出的酵母菌氨氮利用率为55.39%，GS活性为0.29 μmol/(h·g)。上述结果表明，CJ12产朊假丝酵母具有较好的氨氮同化能力，可以作为生产SCP的备用菌株。

3.2 菌株在不同培养基中的代谢途径

通过非靶向代谢组学注释到的前10个上调差异代谢物主要分为维生素类、抗生素类、生物碱类和酶类。二氢叶酸由二氢蝶酸和谷氨酸结合而成，谷氨酸是酵母菌氨同化的产物，从而导致无机氮培养基中酵母菌代谢物的二氢叶酸含量显著上调。研究发现，在饲料中添加酵母可以提高动物的免疫力^[29-30]，这可能与酵母同化氨氮的代谢物中产生大量抗生素类物质有关。乌头酸酶是三羧酸循环中的酶，催化柠檬酸转变为异柠檬酸，异柠檬酸又可在异柠檬酸脱氢酶的催化作用下氧化脱羧生成α-酮戊二酸^[31]，参与酵母菌氨同化反应，因此顺乌头酸酶显著上调。前10个下调差异代谢物主要包括氨基酸类、甾醇类和脂肪酸类。下调的氨基酸类均为氨基酸衍生物，可能是合成氨基酸的前体物质，在氨基酸代谢反应中不断合成氨基酸，从而降低了氨基酸衍生物的含量。脂肪酸类物质可氧化生成乙酰辅酶A，进入三羧酸循环，通过脱氨基作用生成相应的氨基酸，因此脂肪酸类物质下调。

基于KEGG功能注释及富集分析，含有差异代谢物最多的通路是苯丙烷的生物合成，苯丙烷生物合成不仅会产生大量的黄酮类物质，还可为酵母菌氨同化提供大量的氨^[32]。维生素的代谢和合成主要包括叶酸的生物合成、泛酸和CoA的生物合成、烟酸酯和烟酰胺代谢、生物素代谢。其中，叶酸的生物活化形式是四氢叶酸，四氢叶酸是一个碳原子的供体，参与氨基酸和核苷酸的合成；泛酸和CoA是三羧酸循环中的物质，为酵母菌氨同化提供α-酮戊二酸；烟酸酯和烟酰胺是烟酰胺腺嘌呤二核苷酸(NADH)和烟酰胺腺嘌呤二核苷酸磷酸(NADPH)的组成成分，NADH和NADPH是参与氨同化GS/GOGAT和GDH途径的辅酶^[33]；生物素直接参与亮氨酸、异亮氨酸等氨基酸的脱氨基及核酸代谢^[34]，蛋氨酸、异亮氨酸、苏氨酸、缬氨酸因生物素酶的作用，经琥珀酰辅酶A进入三羧酸循环。氨基酸的代谢和合成主要包括酪氨酸、半胱氨酸、蛋氨酸、色氨酸、组氨酸、精氨酸、脯氨酸、d-氨基酸、苯丙氨酸、甘氨酸、丝氨酸、苏氨酸、丙氨酸、天冬氨酸、谷氨酸的代谢，以及组氨酸、鸟氨酸和赖氨酸的生物合成。组氨酸参与Amts转运铵的蛋白质形成，Amts蛋白的单体包含一个狭窄的疏水通道，其中一个高度保守的组氨酸残基和一个谷氨酸残基位于该通道的中间^[35]。酵母菌含有表达真核GOGAT蛋白的前序列，可以产生并加工GOGAT，半胱氨酸是GOGAT的N末端，是谷氨酰胺氨基转移酶活性的关键成分^[36]。谷氨酰胺是酵母菌氨同化的最终产物，其酰胺氮通过酰胺转移酶反应转移，参与嘌呤和嘧啶的生物合成^[37]。双组分系统是酵母菌最重要的信号转导途径之一，负责激活编码各种酶和转运蛋白的基因的转录，由组氨酸蛋白激酶引入信号，并通过一系列磷酸化物质传递，从而调节蛋白的活性^[38]。综上所述，三羧酸循环、苯丙烷生物合成、维生素代谢、氨基酸代谢以及双组分系统共同调节并促进了产朊假丝酵母CJ12的氨同化过程，其中氨基酸代谢可能是其氨同化的关键代谢途径。

3.3 发酵工艺优化及发酵效果评价

微生物的发酵作用可改变饲料原料的成分，提高饲料的营养价值。张玉诚等^[39]利用有益菌固态发酵白酒糟后，其中蛋白质和总氨基酸含量分别提高了57.85%和24.47%，粗纤维和粗脂肪含量分别降低了42.39%和21.48%。袁凯红等^[40]接种米曲霉发酵多种利用率较低的动植物蛋白原料生产优质蛋白饲料，发酵产物的可溶性蛋白质、氨基氮和粗蛋白质含量显著增加。本研究结果与上述研究一致，酵母菌CJ12发酵显著提高了饲料的粗蛋白质、有机氮和氨基酸含量，同时降低了干物质、粗脂肪和粗纤维含量。粗蛋白含量的显著增加可能是因为酵母菌体含有丰富的菌体蛋白，且发酵过程中酵母菌通过同化作用将底物中的硫酸铵转化为有机氮^[41]。粗纤维含量的显著降低可能是因为微生物发酵过程中产生了纤维素酶降解了纤维素。粗脂肪含量的显著降低可能是因为微生物生长过程中利用脂肪作为能量来源，支持菌体生长^[42]。

Higuera-Ciapara等^[43]研究表明，氧化分子能够与蛋白质、脂质、脱氧核糖核酸(DNA)和其他细胞成分发生链式反应，导致蛋白质氧化、脂质过氧化和DNA损伤等，从而引发多种疾病。因此，提高饲料的抗氧化能力对保证动物健康和增强免疫力具有重要意义。抗氧化能力是指清除DPPH自由基、ABTS自由基、超氧阴离子自由基等体外自由基的能力。自由基具有强氧化性，可引发机体内的大分子过氧化反应，测定自由基清除能力是评价抗氧化活性的有效方法。本研究选择DPPH自由基和ABTS自由基清除能力来评价酵母菌CJ12发酵饲料的抗氧化能力。结果表明，酵母菌CJ12发酵后的饲料对DPPH自由基和ABTS自由基的清除能力均高于未发酵饲料，且随着样品浓度的增加，抗氧化能力也随之升高，可能是因为微生物发酵过程中产生了多种非酶抗氧化剂，如抗坏血酸、生育酚、类黄酮、生物碱和类胡萝卜素，以及抗氧化酶，如过氧化物酶、超氧化物歧化酶和过氧化氢酶^[44]。因此，酵母菌CJ12可以通过发酵作用显著提高饲料的抗氧化活性。

本研究结果中，试验组的氨基酸总量从43.84 mmol/100 g增加到54.87 mmol/100 g，其中谷氨酰胺和谷氨酸含量分别为对照组的111.38倍和3.02倍。谷氨酰胺对合成三羧酸循环代谢物、氨基酸、核苷酸、抗氧化剂以及ATP至关重要^[45-46]。谷氨酰胺在细胞基质中可以作为合成核苷酸、己糖胺以及天冬酰胺的氮供体^[47]。然而，在通过谷氨酰胺转运体SLC1A5运输进入线粒体后，谷氨酰胺会被线粒体上的谷氨酸脱氢酶转变为谷氨酸^[48]。这些生成的谷氨酸随后经转运蛋白转出线粒体，用于合成谷胱甘肽、丙氨酸、精氨酸、赖氨酸、鸟氨酸等物质^[49]。相关研究发现色氨酸通过犬尿氨酸途径分解代谢产生烟酸，烟酸是NAD和NADP的前体，参与酵母菌的氨同化^[50-51]。脯氨酸由PUT1和PUT2基因编码的脯氨酸氧化酶和1-吡咯烷-5-羧酸脱氢酶生成谷氨酸^[26,52]。酵母菌天冬酰胺酶或水解酶将天冬酰胺水解为天冬氨酸和氨^[53]。天冬氨酸作为酵母菌生长的氮源优于氨或谷氨酸，酵母菌可通过谷草转氨酶将天冬氨酸的氮转移到其他氨基酸^[13,54]。天冬氨酸随后进入氨基酸代谢途径，通过谷草转氨酶等酶的催化作用，将其氮原子转移到其他氨基酸上，从而实现氮的同化利用。细胞外的天冬酰胺在天冬酰胺酶作用下裂解为氨和天冬氨酸，4-氨基丁酸在4-氨基丁酸转氨酶的催化下，与丙酮酸和α-酮戊二酸反应生成琥珀酸半醛，再形成琥珀酸进入三羧酸循环^[55]。因此，本研究中对照组的色氨酸、脯氨酸、天冬氨酸、天冬酰胺以及4-氨基丁酸含量分别是试验组的13.41倍、1.95倍、1.67倍、8.27倍和6.41倍，可能与谷氨酰胺和谷氨酸的合成有关。谷氨酰胺作为关键中间产物，其合成需求可能竞争性地消耗了色氨酸等前体物质，同时谷氨酰胺向谷氨酸的转化也促进了谷氨酸的积累，谷氨酸的进一步代谢利用加剧了这些氨基酸含量的降低。这说明氨基酸代谢途径对产朊假丝酵母CJ12的氨同化调控过程可能是通过合成谷氨酰胺来完成的。

4 结论

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本研究以硫酸铵为唯一氮源，筛选出具有高效氨氮同化能力的产朊假丝酵母CJ12，其氨氮利用率达55.39%，GS活性为0.29 μmol/(h·g)。确立的最佳发酵工艺参数为：硫酸铵添加量2%，接种量8%，发酵时间36 h，最佳发酵底物为94.8%麦麸、5%豆粕、0.1%蛋白酶和0.1%纤维素酶。代谢组学分析揭示，CJ12的氨同化过程与多种代谢途径紧密相关，其中氨基酸代谢尤为关键。经CJ12发酵后，麦麸饲料中的粗蛋白质、有机氮、小肽、总氨基酸含量及抗氧化能力显著提高。谷氨酸和谷氨酰胺含量分别是对照组的3.02倍和111.38倍，而对照组的色氨酸、天冬酰胺、4-氨基丁酸含量分别是试验组的13.41倍、8.27倍和6.41倍。综上所述，CJ12具有较高的氨氮同化能力和谷氨酰胺合成能力，其氨同化过程可能通过氨基酸代谢途径调控谷氨酰胺合成，具有生产功能性单细胞蛋白饲料的潜力。

基金

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国家重点研发计划(2023YFD1301604)

参考文献

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文献

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参考文献引证文献

排序方式：

[1]

寇慧, 文晓霞, 叶思廷, 邹伟. 微生物发酵生产饲用菌体蛋白的研究进展[J]. 饲料工业, 2021, 42(21): 26-33.

KOU

, WEN

, YE

, ZOU

. Research progress on microbial fermentation for production of feed bacterial proteins[J]. Feed Industry, 2021, 42(21): 26-33 (in Chinese).

[2]

AGBOOLA

, ØVERLAND

, SKREDE

, JØ

HANSEN

. Yeast as major protein-rich ingredient in aquafeeds: a review of the implications for aquaculture production[J]. Reviews in Aquaculture, 2021, 13(2): 949-970.

[3]

SHURSON

. Yeast and yeast derivatives in feed additives and ingredients: sources, characteristics, animal responses, and quantification methods[J]. Animal Feed Science and Technology, 2018, 235: 60-76.

[4]

YADAV

, KUMAR

, SHARMA

, KAUR

, GAT Y, KAUR

, MISHRA

. Process optimisation for saccharification and fermentation of wheat straw for the production of single cell protein[J]. International Journal of Environment and Waste Management, 2020, 25(2): 176.

[5]

JALASUTRAM

, KATARAM

, GANDU

, ANUPOJU

. Single cell protein production from digested and undigested poultry litter by Candida utilis: optimization of process parameters using response surface methodology[J]. Clean Technologies and Environmental Policy, 2013, 15(2): 265-273.

[6]

乔传丽, 金丹, 蒋艾廷, 薛林林, 卢士玲, 李宝坤. 传统发酵酸牛奶中产蛋白酶酵母菌的筛选及产酶条件优化[J]. 食品与生物技术学报, 2019, 38(5): 73-78.

QIAO

, JIN

, JIANG

, XUE

, LU

, LI

. Screening of yeast with high protease activities from traditional yogurt and optimization of enzyme conditions[J]. Journal of Food Science and Biotechnology, 2019, 38(5): 73-78 (in Chinese).

[7]

吴海燕, 钟振声, 盖春慧. 新鲜马铃薯渣的高效利用[J]. 食品与发酵工业, 2012, 38(1): 112-115.

, ZHONG

, GAI

. Efficient usage of fresh potato residue[J]. Food and Fermentation Industries, 2012, 38(1): 112-115 (in Chinese).

[8]

季小禹, 王永伟, 刘宽博, 李爱科. 发酵生产饲用单细胞蛋白的研究进展[J]. 动物营养学报, 2024, 36(6): 3525-3533.

, WANG

, LIU

, LI

. Progress on fermentation production of single-cell protein for animal feed[J]. Chinese Journal of Animal Nutrition, 2024, 36(6): 3525-3533 (in Chinese).

[9]

BRO C, REGENBERG

, NIELSEN

. Genome-wide transcriptional response of a Saccharomyces cerevisiae strain with an altered redox metabolism[J]. Biotechnology and Bioengineering, 2004, 85(3): 269-276.

[10]

SCHURE EG

TER

, van RIEL

, VERRIPS

. The role of ammonia metabolism in nitrogen catabolite repression in Saccharomyces cerevisiae[J]. FEMS Microbiology Reviews, 2000, 24(1): 67-83.

[11]

李孝辉, 付浩, 吴杰, 邓波. 一株耐高温酵母的分离鉴定及生物学特性[J]. 饲料工业, 2024, 45(16): 139-144.

, FU

, WU

, DENG

. Isolation and identification of a thermotolerant yeast and its biological characteristics[J]. Feed Industry, 2024, 45(16): 139-144 (in Chinese).

[12]

中华人民共和国环境保护部.水质. 氨氮的测定纳氏试剂分光光度法：HJ 535—2009[S].北京：中国环境科学出版社，2010.

Ministry of Environmental Protection of the People’s Republic of China. Water quality-Determination of ammonia nitrogen-Nessler’s reagent spectrophotometry: HJ 535—2009[S]. Beijing: China Environmental Science Press, 2010 (in Chinese).

[13]

黄文明, 尹梦丽, 陈煜, 李军训, 王常童, 张进, 左福元. 一株高效同化氨氮霉菌的筛选及其代谢组分析[J]. 微生物学报, 2024, 64(1): 161-173.

HUANG

, YIN

, CHEN

, LI

, WANG

, ZHANG

, ZUO

. Screening and metabolomic analysis of a fungal strain efficiently assimilating ammonia nitrogen[J]. Acta Microbiologica Sinica, 2024, 64(1): 161-173 (in Chinese).

[14]

国家质量监督检验检疫总局, 中国国家标准化管理委员会. 饲料中水分的测定：GB/T 6435—2014[S]. 北京: 中国标准出版社, 2015.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China. Determination of moisture in feedstuffs: GB/T 6435—2014[S]. Beijing: Standards Press of China, 2015 (in Chinese).

[15]

国家质量监督检验检疫总局, 中国国家标准化管理委员会. 饲料中粗蛋白的测定　凯氏定氮法：GB/T 6432—2018[S]. 北京: 中国标准出版社, 2019.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China. Determination of crude protein in feeds—Kieldahl method: GB/T 6432—2018[S]. Beijing: Standards Press of China, 2019 (in Chinese).

[16]

国家质量监督检验检疫总局. 大豆肽粉：GB/T 22492—2008[S]. 北京: 中国标准出版社, 2009.

General Administration of Quality Supervision. Soy peptides powder: GB/T 22492—2008[S]. Beijing: Standards Press of China, 2009 (in Chinese).

[17]

国家质量监督检验检疫总局, 中国国家标准化管理委员会. 饲料中粗纤维的含量测定：GB/T 6434—2022[S]. 北京: 中国标准出版社, 2023.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China. Determination of crude fiber content in feeds: GB/T 6434—2022[S]. Beijing: Standards Press of China, 2023 (in Chinese).

[18]

国家质量监督检验检疫总局, 中国国家标准化管理委员会. 饲料中粗脂肪的测定：GB/T 6433—2006[S]. 北京: 中国标准出版社, 2006.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China. Determination of crude fat in feeds: GB/T 6433—2006[S]. Beijing: Standards Press of China, 2006 (in Chinese).

[19]

国家质量监督检验检疫总局, 中国国家标准化管理委员会. 饲料中粗灰分的测定：GB/T 6438—2007[S]. 北京: 中国标准出版社, 2007.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China. Animal feeding stuffs-Determination of crude ash: GB/T 6438—2007[S]. Beijing: Standards Press of China, 2007 (in Chinese).

[20]

国家质量监督检验检疫总局, 中国国家标准化管理委员会. 饲料中钙的测定：GB/T 6436—2018[S]. 北京: 中国标准出版社, 2018.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China. Determination of calcium in feeds: GB/T 6436—2018[S]. Beijing: Standards Press of China, 2018 (in Chinese).

[21]

国家质量监督检验检疫总局, 中国国家标准化管理委员会. 饲料中总磷的测定分光光度法：GB/T 6437—2018[S]. 北京: 中国标准出版社, 2019.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China. Determination of phosphorus in feeds—Spectrophotometry: GB/T 6437—2018[S]. Beijing: Standards Press of China, 2019 (in Chinese).

[22]

高营. 不同来源与水平的植酸酶对鸡磷吸收利用的影响[D]. 泰安：山东农业大学硕士学位论文, 2022.

GAO

. Effects of phytase sources and supplemental levels on phosphorus utilization of chickens [D]. Tai’an: Master Thesis of Shandong Agricultural University, 2022 (in Chinese)

[23]

国家卫生和计划生育委员会. 食品安全国家标准　食品中铵盐的测定：GB 5009.234—2016[S]. 北京: 中国标准出版社, 2017.

National Health and Family Planning Commission. National Food Safety Standard—Determination of Ammonium Salts in Foods: GB 5009.234—2016[S]. Beijing: Standards Press of China, 2019 (in Chinese).

[24]

杨小龙, 刘莉华, 毕永红, 王洵, 董薇, 邓自发, 王寿兵. 蓝藻光合氮同化的特征与分子调控机理研究进展[J]. 湖泊科学, 2023, 35(3): 766-779.

YANG

, LIU

, BI

, WANG

, DONG

, DENG

, WANG

. Progress on photosynthetic nitrogen assimilation and its regulatory mechanisms in cyanobacteria[J]. Journal of Lake Sciences, 2023, 35(3): 766-779 (in Chinese).

[25]

SIMS

, FOLKES

. A kinetic study of the assimilation of (15N)-ammonia and the synthesis of amino acids in an exponentially growing culture of Candida utilis[J]. Proceedings of the Royal Society B: Biological Sciences, 1964, 159: 479-502.

[26]

SIEG

, TROTTER

. Differential contribution of the proline and glutamine pathways to glutamate biosynthesis and nitrogen assimilation in yeast lacking glutamate dehydrogenase[J]. Microbiological Research, 2014, 169(9/10): 709-716.

[27]

YAN

. Protection of the glutamate pool concentration in enteric bacteria[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(22): 9475-9480.

[28]

凌晓. 高氨氮利用酵母筛选及其固态发酵苜蓿粉的应用研究[D]. 晋中: 山西农业大学硕士学位论文, 2021.

LING

. Screening of high ammonia nitrogen utilizing yeast and its application in solid fermentation of alfalfa meal[D]. Jinzhong: Master Thesis of Shanxi Agricultural University, 2021 (in Chinese).

[29]

NASIRI

, TOWHIDI

, SHAKERI

, ZHANDI

, DEHGHAN

, POOYAN

, SEHATI

, ROSTAMI

, KARAMZADEH

, KHANI

, AHMADI

. Effects of Saccharomyces cerevisiae supplementation on milk production, insulin sensitivity and immune response in transition dairy cows during hot season[J]. Animal Feed Science and Technology, 2019, 251: 112-123.

[30]

KIM

, HYUN

, SOHN

, KIM

, WOO HJ, HAN

. Effects of mannanoligosaccharide and protein levels on growth performance and immune status in pigs weaned at 21 days of age[J]. Korean Journal of Animal Science, 2000, 42: 489-498.

[31]

张文利, 沈文飚, 叶茂炳, 徐朗莱. 植物顺乌头酸酶及其生理功能[J]. 植物生理学通讯, 2003, 39(4): 391-398.

ZHANG

, SHEN

, YE

, XU

. Aconitase and its physiological roles in plants[J]. Plant Physiology Communications, 2003, 39(4): 391-398 (in Chinese).

[32]

TEMPLE

, VANCE

, STEPHEN GANTT

. Glutamate synthase and nitrogen assimilation[J]. Trends in Plant Science, 1998, 3(2): 51-56.

[33]

MARA

, FRAGIADAKIS

, GKOUNTROMICHOS

, ALEXANDRAKI

. The pleiotropic effects of the glutamate dehydrogenase (GDH) pathway in Saccharomyces cerevisiae[J]. Microbial Cell Factories, 2018, 17(1): 170.

[34]

邓奇风, 高凤仙. 生物素的营养代谢调控及生理功能研究进展[J]. 饲料与畜牧, 2016(9): 33-35.

DENG

, GAO

. Advances in the regulation of nutrient metabolism and physiological functions of biotin[J]. Feed and animal husbandry, 2016(9): 33-35 (in Chinese).

[35]

BOECKSTAENS

, ANDRÉ

, MARINI

. Distinct transport mechanisms in yeast ammonium transport/sensor proteins of the Mep/Amt/Rh family and impact on filamentation[J]. Journal of Biological Chemistry, 2008, 283(31): 21362-21370.

[36]

VANONI

, CURTI

. Glutamate synthase: a complex iron-sulfur flavoprotein[J]. Cellular and Molecular Life Sciences, 1999, 55(4): 617-638.

[37]

WATFORD

. Glutamine and glutamate: nonessential or essential amino acids?[J]. Animal Nutrition, 2015, 1(3): 119-122.

[38]

LIAO

, YE

, CHEN

, ZHOU

, CHENG

, ZHOU

, REN

. The two-component signal transduction system and its regulation in Candida albicans[J]. Virulence, 2021, 12(1): 1884-1899.

[39]

张玉诚, 薛白, 达勒措, 李秋瑾, 何宇. 混菌固态发酵白酒糟开发为蛋白质饲料的条件优化及营养价值评定[J]. 动物营养学报, 2016, 28(11): 3711-3720.

ZHANG

, XUE

, DA LC, LI

, HE

. Distillers grains: optimization of mixed bacterial solid-state fermentation conditions to produce protein feed and nutrient value analysis[J]. Chinese Journal of Animal Nutrition, 2016, 28(11): 3711-3720 (in Chinese).

[40]

袁凯红, 李灵平, 张文强, 李焦魏. 米曲霉固态发酵动植物蛋白生产优质蛋白饲料的工艺研究[J]. 饲料广角, 2011(20): 14-18.

YUAN

, LI

, ZHANG

, LI

. Process study of solid-state fermentation of animal and plant proteins by Aspergillus oryzae for the production of high quality protein feeds[J]. Feed China, 2011(20): 14-18 (in Chinese).

[41]

汤江武, 薛智勇, 钱红, 陈志良, 吴逸飞, 吴传珍. 酵母固体发酵对物料营养组分及生物活性的影响[J]. 浙江农业科学, 2003(5): 274-276.

TANG

, XUE

, QIAN

, CHEN

, WU

. Effect of yeast solid-state fermentation on nutrient fractions and biological activity of materials[J]. Journal of Zhejiang Agricultural Sciences, 2003(5): 274-276 (in Chinese).

[42]

HONG

, TANG

, YAO

, WU

, WANG

, FENG

. Improvement of the nutritional quality of cottonseed meal by Bacillus subtilis and the addition of papain[J]. International Journal of Agriculture and Biology, 2012, 14(4): 563-568.

[43]

HIGUERA-CIAPARA

, FÉLIX-VALENZUELA

, GOYCOOLEA

. Astaxanthin: a review of its chemistry and applications[J]. Critical Reviews in Food Science and Nutrition, 2006, 46(2): 185-196.

[44]

HAN

, LI

, ZHONG

, XIE

, ZHANG

, LU

, LI

, XU

, CHEN

, LENG

, ZHOU

. Anti-oxidation properties and therapeutic potentials of spirulina[J]. Algal Research, 2021, 55: 102240.

[45]

SON J, LYSSIOTIS

, YING

, WANG

, HUA

, LIGORIO

, PERERA

, FERRONE

, MULLARKY

, SHYH-CHANG

, KANG

, FLEMING

, BARDEESY

, ASARA

, HAIGIS

, DePINHO

, CANTLEY

, KIMMELMAN

. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway[J]. Nature, 2013, 496(7443): 101-105.

[46]

FAN

, KAMPHORST

, MATHEW

, CHUNG

, WHITE

, SHLOMI

, RABINOWITZ

. Glutamine-driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia[J]. Molecular Systems Biology, 2013, 9: 712.

[47]

NGUYEN

, DURÁN

. Glutamine metabolism in cancer therapy[J]. Cancer Drug Resistance, 2018, 1(3): 126-138.

[48]

STINE

, DANG

. Glutamine skipping the Q into mitochondria[J]. Trends in Molecular Medicine, 2020, 26(1): 6-7.

[49]

XIAO

, ZENG

, YAO

, KONG

, WU

, YIN

. The glutamine-alpha-ketoglutarate (AKG) metabolism and its nutritional implications[J]. Amino Acids, 2016, 48(9): 2067-2080.

[50]

CASTRO-PORTUGUEZ

, SUTPHIN

. Kynurenine pathway, NAD⁺ synthesis, and mitochondrial function: targeting tryptophan metabolism to promote longevity and healthspan[J]. Experimental Gerontology, 2020, 132: 110841.

[51]

龙凡, 梅文亮, 许兰娇, 瞿明仁. 烟酸的生物学功能及其在畜禽生产中的应用[J]. 动物营养学报, 2022, 34(7): 4143-4154.

LONG

, MEI

, XU

, QU

. Biological functions of niacin and its application in livestock and poultry production[J]. Chinese Journal of Animal Nutrition, 2022, 34(7): 4143-4154 (in Chinese).

[52]

WANDURAGALA

, SANYAL

, LIANG

, BECKER

. Purification and characterization of Put1p from Saccharomyces cerevisiae[J]. Archives of Biochemistry and Biophysics, 2010, 498(2): 136-142.

[53]

CORAL-MEDINA

, FENTON

, VARELA

, BARANOV

, CAMARASA

, MORRISSEY

. The evolution and role of the periplasmic asparaginase Asp3 in yeast[J]. FEMS Yeast Research, 2022, 22(1): foac044.

[54]

THOMULKA

, MOAT

. Inorganic nitrogen assimilation in yeasts: alteration in enzyme activities associated with changes in cultural conditions and growth phase[J]. Journal of Bacteriology, 1972, 109(1): 25-33.

[55]

王凯凯, 孙朦, 宋佳敏, 王鸿飞, 邵兴锋, 李和生, 周文化, 许凤. γ-氨基丁酸(GABA)形成机理及富集方法的研究进展[J]. 食品工业科技, 2018, 39(14): 323-329.

WANG

, SUN

, SONG

, WANG

, SHAO

, LI

, ZHOU

, XU

. Research progress in the formation mechanism and accumulation methods of γ-aminobutyric acid (GABA)[J]. Science and Technology of Food Industry, 2018, 39(14): 323-329 (in Chinese).

2025年第65卷第4期

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doi: 10.13343/j.cnki.wsxb.20240853

接收时间：2024-12-30
首发时间：2026-02-06
出版时间：2025-04-04

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收稿日期：2024-12-30
录用日期：2025-02-16

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National Key Research and Development Program of China(2023YFD1301604)

国家重点研发计划(2023YFD1301604)

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^1.西南大学动物科学技术学院，重庆市肉牛工程技术研究中心，重庆

^2.山东泰山生力源集团股份有限公司，山东泰安

^3.成都奥盛康生物科技有限公司，四川成都

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Levels	A: Amount of inoculation (%)	B: Amount of inorganic nitrogenadded (%)	C: Time of fermentation(h)
1	5	2	36
2	8	5	48
3	10	8	60

Levels

A: Amount of inoculation (%)

B: Amount of inorganic nitrogenadded (%)

C: Time of fermentation(h)

Groups	Substrate of fermentation
A	100% Wheat bran
B	98% Wheat bran+2% Enzymatic hydrolysis of soybean meal
C	95% Wheat bran+5% Enzymatic hydrolysis of soybean meal
D	97.8% Wheat bran+2% Soybean meal+0.1% Protease+0.1% Cellulase
E	94.8% Wheat bran+5% Soybean meal+0.1% Protease+0.1% Cellulase

Groups

Substrate of fermentation

100% Wheat bran

98% Wheat bran+2% Enzymatic hydrolysis of soybean meal

95% Wheat bran+5% Enzymatic hydrolysis of soybean meal

97.8% Wheat bran+2% Soybean meal+0.1% Protease+0.1% Cellulase

94.8% Wheat bran+5% Soybean meal+0.1% Protease+0.1% Cellulase

Strain No.	Colony number (×10⁸ CFU/mL)
NJ15	0.57±0.01b
CJ12	0.72±0.03a
NJ16	0.25±0.01d
YJ19	0.40±0.02c
P-value	<0.01

Strain No.

Colony number (×10⁸ CFU/mL)

NJ15

0.57±0.01b

CJ12

0.72±0.03a

NJ16

0.25±0.01d

YJ19

0.40±0.02c

P-value

<0.01

Strain No.	Ammonia nitrogen utilization (%)	GS activity (μmol/(h·g))
NJ15	46.23±0.39	0.18±0.03b
CJ12	55.39±0.24	0.29±0.04a
NJ16	48.19±0.21	0.16±0.02b
YJ19	50.02±1.42	0.06±0.03c
P-value	>0.05	0.04

Strain No.

Ammonia nitrogen utilization (%)

GS activity (μmol/(h·g))

NJ15

46.23±0.39

0.18±0.03b

CJ12

55.39±0.24

0.29±0.04a

NJ16

48.19±0.21

0.16±0.02b

YJ19

50.02±1.42

0.06±0.03c

P-value

>0.05

0.04

No.	KEGG metabolic pathway	Number of differential metabolites
1	Biosynthesis of phenylpropanoids	15
2	Tryptophan metabolism	11
3	Arginine and proline metabolism	10
4	ABC transporters	8
5	Purine metabolism	8
6	Biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid	7
7	Nicotinate and nicotinamide metabolism	7
8	Citrate cycle (TCA cycle)	7
9	Folate biosynthesis	7
10	Isoflavonoid biosynthesis	7
11	d-amino acid metabolism	7
12	Pyrimidine metabolism	7
13	Histidine metabolism	6
14	Phenylalanine metabolism	6
15	Methane metabolism	6
16	Biosynthesis of terpenoids and steroids	5
17	Biosynthesis of alkaloids derived from histidine and purine	5
18	Pantothenate and CoA biosynthesis	5
19	Two-component system	5
20	Tyrosine metabolism	5
21	Phosphotransferase system (PTS)	4
22	Cysteine and methionine metabolism	4
23	Lysine biosynthesis	4
24	Glycine, serine and threonine metabolism	4
25	Biosynthesis of alkaloids derived from terpenoid and polyketide	4
26	Biosynthesis of unsaturated fatty acids	4
27	Biosynthesis of various antibiotics	4
28	Alanine, aspartate and glutamate metabolism	3
29	Chlorocyclohexane and chlorobenzene degradation	3
30	Ubiquinone and other terpenoid-quinone biosynthesis	3
31	Bisphenol degradation	3
32	C5-branched dibasic acid metabolism	3
33	Biotin metabolism	2
34	Amino sugar and nucleotide sugar metabolism	2
35	Cell cycle-yeast	1

No.

KEGG metabolic pathway

Number of differential metabolites

Biosynthesis of phenylpropanoids

Tryptophan metabolism

Arginine and proline metabolism

ABC transporters

Purine metabolism

Biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid

Nicotinate and nicotinamide metabolism

Citrate cycle (TCA cycle)

Folate biosynthesis

Isoflavonoid biosynthesis

d-amino acid metabolism

Pyrimidine metabolism

Histidine metabolism

Phenylalanine metabolism

Methane metabolism

Biosynthesis of terpenoids and steroids

Biosynthesis of alkaloids derived from histidine and purine

Pantothenate and CoA biosynthesis

Two-component system

Tyrosine metabolism

Phosphotransferase system (PTS)

Cysteine and methionine metabolism

Lysine biosynthesis

Glycine, serine and threonine metabolism

Biosynthesis of alkaloids derived from terpenoid and polyketide

Biosynthesis of unsaturated fatty acids

Biosynthesis of various antibiotics

Alanine, aspartate and glutamate metabolism

Chlorocyclohexane and chlorobenzene degradation

Ubiquinone and other terpenoid-quinone biosynthesis

Bisphenol degradation

C5-branched dibasic acid metabolism

Biotin metabolism

Amino sugar and nucleotide sugar metabolism

Cell cycle-yeast

Strain No.	A	B	C	Ammonia nitrogen utilization (%)
1	1	1	1	61.23
2	1	2	3	26.51
3	1	3	2	49.48
4	2	1	3	72.08
5	2	2	2	44.18
6	2	3	1	48.26
7	3	1	2	50.67
8	3	2	1	47.40
9	3	3	3	49.88
K₁	45.74	61.33	52.30
K₂	54.84	39.36	48.11
K₃	49.32	39.36	49.49
R	9.10	21.97	4.19

Strain No.

Ammonia nitrogen utilization (%)

61.23

26.51

49.48

72.08

44.18

48.26

50.67

47.40

49.88

K₁

45.74

61.33

52.30

K₂

54.84

39.36

48.11

K₃

49.32

39.36

49.49

9.10

21.97

4.19

Groups	Colony number (×10¹⁰ CFU/mL)
A	0. 17±0.02c
B	0.22±0.05b
C	0.26±0.02a
D	0.24±0.01b
E	0.28±0.03a
P-value	0.04

Groups

Colony number (×10¹⁰ CFU/mL)

0. 17±0.02c

0.22±0.05b

0.26±0.02a

0.24±0.01b

0.28±0.03a

P-value

0.04

Strain No.	Control group	Experimental group	P-value
Dry matter (%)	50.58±1.16a	48.46±0.63b	0.04
Crude protein (%)	20.88±0.12b	24.21±0.47a	0.02
Small peptide (%)	2.30±0.34	2.61±0.32	0.58
Organic nitrogen (%)	2.82±0b	3.63±0.04a	0.00
Ash (%)	6.47±0.25	7.37±0.16	0.82
Calcium (%)	0.26±0.05	0.28±0.02	0.79
Total phosphorus (%)	1.22±0.04	1.37±0.04	0.57
Crude fat (%)	4.80±0.53a	4.21±0.98b	0.04
Crude fiber (%)	5.77±1.54a	4.65±1.36b	0.02
Phytic phosphoric acid (%)	0.37±0.01a	0.32±0.01b	0.04

Strain No.

Control group

Experimental group

P-value

Dry matter (%)

50.58±1.16a

48.46±0.63b

0.04

Crude protein (%)

20.88±0.12b

24.21±0.47a

0.02

Small peptide (%)

2.30±0.34

2.61±0.32

0.58

Organic nitrogen (%)

2.82±0b

3.63±0.04a

0.00

Ash (%)

6.47±0.25

7.37±0.16

0.82

Calcium (%)

0.26±0.05

0.28±0.02

0.79

Total phosphorus (%)

1.22±0.04

1.37±0.04

0.57

Crude fat (%)

4.80±0.53a

4.21±0.98b

0.04

Crude fiber (%)

5.77±1.54a

4.65±1.36b

0.02

Phytic phosphoric acid (%)

0.37±0.01a

0.32±0.01b

0.04

Amino acid	Control group	Experimental group	P-value
Tryptophan	5.50±0.36a	0.41±0.05b	<0.01
Phenylalanine	0.56±0.04a	0.39±0.07b	0.02
Methionine	0.09±0.00a	0.02±0.00	<0.01
Valine	1.76±0.11b	2.27±0.21a	0.02
4-aminobutyric acid	6.22±0.30a	1.64±0.19b	<0.01
Tyrosine	0.56±0.05b	1.64±0.30a	<0.01
Proline	2.51±0.11a	1.62±0.23b	<0.01
Beta-alanine	0.38±0.04b	4.51±0.44a	<0.01
Alanine	7.63±0.33b	15.92±0.43a	<0.01
Glycine	1.97±0.03a	1.59±0.13b	0.01
Glutamic acid	2.14±0.08b	6.47±1.01a	<0.01
Hydroxyproline	0.07±0.01	0.08±0.01	0.08
Threonine	0.87±0.03b	1.38±0.15a	<0.01
Aspartic acid	2.06±0.29a	1.23±0.08b	0.01
Glutamine	0.08±0.01b	8.91±0.87a	<0.01
Serine	0.89±0.01	0.86±0.04	0.29
Citrulline	0.03±0.00	0.03±0.00	0.96
Asparagine	8.68±0.13a	1.26±0.07b	<0.01
Arginine	1.05±0.02b	1.79±60.03a	<0.01
3-methyl-l-histidine	0.03±0.00a	0.02±0.00b	<0.01
Lysine	0.50±0.06b	1.50±0.04a	<0.01
Histidine	0.22±0.02b	1.17±0.08a	<0.01
Ornithine	0.07±0.01b	0.18±0.02a	<0.01
5-hydroxylysine	N.D.	N.D.
1-methyl-l-histidine	N.D.	N.D.
Total	43.84±1.90b	54.87±3.16a	<0.01

Amino acid

Control group

Experimental group

P-value

Tryptophan

5.50±0.36a

0.41±0.05b

<0.01

Phenylalanine

0.56±0.04a

0.39±0.07b

0.02

Methionine

0.09±0.00a

0.02±0.00

<0.01

Valine

1.76±0.11b

2.27±0.21a

0.02

4-aminobutyric acid

6.22±0.30a

1.64±0.19b

<0.01

Tyrosine

0.56±0.05b

1.64±0.30a

<0.01

Proline

2.51±0.11a

1.62±0.23b

<0.01

Beta-alanine

0.38±0.04b

4.51±0.44a

<0.01

Alanine

7.63±0.33b

15.92±0.43a

<0.01

Glycine

1.97±0.03a

1.59±0.13b

0.01

Glutamic acid

2.14±0.08b

6.47±1.01a

<0.01

Hydroxyproline

0.07±0.01

0.08±0.01

0.08

Threonine

0.87±0.03b

1.38±0.15a

<0.01

Aspartic acid

2.06±0.29a

1.23±0.08b

0.01

Glutamine

0.08±0.01b

8.91±0.87a

<0.01

Serine

0.89±0.01

0.86±0.04

0.29

Citrulline

0.03±0.00

0.96

Asparagine

8.68±0.13a

1.26±0.07b

<0.01

Arginine

1.05±0.02b

1.79±60.03a

<0.01

3-methyl-l-histidine

0.03±0.00a

0.02±0.00b

<0.01

Lysine

0.50±0.06b

1.50±0.04a

<0.01

Histidine

0.22±0.02b

1.17±0.08a

<0.01

Ornithine

0.07±0.01b

0.18±0.02a

<0.01

5-hydroxylysine

N.D.

1-methyl-l-histidine

N.D.

Total

43.84±1.90b

54.87±3.16a

<0.01

Type of free radical	Sample	Linear regression equation	IC₅₀ value (mg/mL)	Mass equivalent (mg)
DPPH free radical	Control group	y=2.55x+2.25	18.76±0.02a	1.00
Experimental group	y=5.67x+21.91	4.97±0.04b	3.78
ABTS free radical	Control group	y=7.38x-6.56	7.66±0.01a	1.00
Experimental group	y=7.36x-2.78	7.20±0.02b	1.06

Type of free radical

Sample

Linear regression equation

IC₅₀ value (mg/mL)

Mass equivalent (mg)

DPPH free radical

Control group

y=2.55x+2.25

18.76±0.02a

1.00

Experimental group

y=5.67x+21.91

4.97±0.04b

3.78

ABTS free radical

Control group

y=7.38x-6.56

7.66±0.01a

1.00

Experimental group

y=7.36x-2.78

7.20±0.02b

1.06