一株高效同化氨氮酵母菌的筛选及发酵工艺优化

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

摘要:

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:

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