Establishment and Preliminary Application of Determination Methods for Three Small Molecule Metabolites in Complex Yeast Cultures
YANG Lan1,*, ZHANG Xiao-Jie1,*, LIAN Hai-Fei2, LIU Da-Cheng1,**
1 College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; 2 Inner Mongolia Agricultural and Animal Husbandry Quality and Safety and Testing Research Institute/Inner Mongolia Agricultural and Animal Husbandry Science and Technology Achievement Transfer and Transformation Center, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Hohhot 010018, China
Abstract：Accurate quantification of active components in complex yeast culture (CYC) is essential for evaluating its quality and functionality. This study established quantitative analytical methods for 3 key small molecule metabolites—N-acetylglutamic acid (NAG), arachidonic acid (ARA), and taurine (Tau)—which were previously identified through non-targeted metabolomics by this research group. To address the complex matrix of CYC, specific methods were developed: Solid-phase extraction combined with high-performance liquid chromatography was used for the highly polar NAG; dansyl chloride pre-column derivatization followed by high-performance liquid chromatography was applied for the zwitterionic Tau; and methylation derivatization coupled with gas chromatography-flame ionization detection was employed for the lipid-soluble ARA. The results demonstrated good linearity (R2>0.999) for all 3 methods, with precision (relative standard deviation<2%), repeatability (relative standard deviation<5%), and spike recovery rates (81.87%~106.95%) all meeting analytical requirements. The developed methods were applied to analyze a laboratory-prepared CYC sample, revealing contents of NAG, ARA, and Tau as (253.80±8.41), (19.72±0.26), and (230.51±4.69) mg/kg (relative standard deviation≤3.31%), respectively. Significant variations were observed in commercially available yeast culture products, with concentration ranges of 38.28~502.31 mg/kg for NAG, 2.60~73.36 mg/kg for ARA, and 28.11~146.44 mg/kg for Tau. The established methods were accurate, reproducible, and suitable for routine quality monitoring of NAG, ARA, and Tau in CYC, providing a reliable analytical foundation for standardizing applications and optimizing production processes.
杨岚, 张小洁, 连海飞, 刘大程. 复合酵母菌培养物中三种小分子代谢物测定方法的建立与初步应用[J]. 农业生物技术学报, 2026, 34(3): 578-597.
YANG Lan, ZHANG Xiao-Jie, LIAN Hai-Fei, LIU Da-Cheng. Establishment and Preliminary Application of Determination Methods for Three Small Molecule Metabolites in Complex Yeast Cultures. 农业生物技术学报, 2026, 34(3): 578-597.
[1] 陆壮, 吕武兴, 舒剑成, 等. 2021. 牛磺酸的生物学功能及其在动物生产中的应用[J]. 饲料工业, 42(06): 15-20. (Lu Z, Lv W X, Shu J C, et al.The biological functions of taurine and its application in animal production[J]. Feed Industry, 42(06): 15-20. [2] Aubert E, Zhao M, Ferreira L, et al.2025. Development and validation of a pure methanol extraction method at room-temperature for glucosinolate quantification in white cabbage using UHPLC-Q-TOF-MS[J]. Journal of Food Composition and Analysis, 146: 107876. [3] Bao G, Niu J, Li S, et al.2022. Effects of ultrasound pretreatment on the quality, nutrients and volatile compounds of dry-cured yak meat[J]. Ultrasonics Sonochemistry, 82: 105864. [4] Belal S A, Kang D R, Cho E S R, et al.2018. Taurine reduces heat stress by regulating the expression of heat shock proteins in broilers exposed to chronic heat[J]. Brazilian Journal of Poultry Science, 20(3), 479-486. [5] Franke A A, Morrison C M, Custer L J, et al.2013. Simultaneous analysis of circulating 25-hydroxy-vitamin D3, 25-hydroxy-vitamin D2, retinol, tocopherols, carotenoids, and oxidized and reduced coenzyme Q10 by high performance liquid chromatography with photo diode-array detection using C18 and C30 columns alone or in combination[J]. Journal of Chromatography A, 1301: 1-9. [6] Gu F, Jiang L, Xie L, et al.2021. Supplementing N-carbamoylglutamate in late gestation increases newborn calf weight by enhanced placental expression of mTOR and angiogenesis factor genes in dairy cows[J]. Animal Nutrition, 7(04): 981-988. [7] Guo Z, Ge X, Yang L, et al.2021. Ultrasound-assisted thawing of frozen white yak meat: Effects on thawing rate, meat quality, nutrients, and microstructure[J]. Ultrasonics Sonochemistry, 70: 105345. [8] Hu B, Liu T, Xia B, et al.2024. Precise evaluation of the nutritional value of yeast culture and its effect on pigs fed low-protein diets[J]. Animal Nutrition, 19: 325-338. [9] Imam M S, Batubara A S, Gamal M, et al.2023. Adjusted green HPLC determination of nirmatrelvir and ritonavir in the new FDA approved co-packaged pharmaceutical dosage using supported computational calculations[J]. Scientific Reports, 13(1): 137. [10] Iseh A.2024. Optimization of heat exchanger cleaning in the candy industry: Comparative analysis of ultrasonic, heat treatment, and chemical methods[J]. Journal of Research in Science and Engineering, 6(12): 1-7. [11] Jeannot C, Macorps N, Bizec B L, et al.2025. Advancing PFASs monitoring in food: From targeted SPE-LC-MS/MS to non-targeted QuEChERS-LC-HRMS approaches[J]. Food Chemistry, 29: 102674. [12] Kojima A, Nadai M, Yamazaki H, et al.2025. Substrate inhibition potential of arachidonic acid on 14,15-epoxidation, a biological drug developmental target, mediated by recombinant human cytochrome P450 2J2 and 2C8 enzymes[J]. Drug Metabolism and Pharmacokinetics, 61: 101052-101052. [13] Li C, Zou Y, Liao G, et al.2024. Identification of characteristic flavor compounds and small molecule metabolites during the ripening process of Nuodeng ham by GC-IMS, GC-MS combined with metabolomics[J]. Food Chemistry, 15(440): 138188. [14] Lysholm S, Chaters G L, Bari C D, et al.2025. A framework for quantifying the multisectoral burden of animal disease to support decision making[J]. Frontiers in Veterinary Science, 12, 1476505-1476505. [15] Mandal A, Banerjee S, Ghosh S, et al.2025. Taurine alleviates colitis by regulating oxidative stress, inflammatory responses, ER stress, and apoptotic pathways[J]. Naunyn Schmiedebergs Arch Pharmacol, 14: 1-17. [16] Maphari P W, Simelane M B C, Madala N E, et al.2024. Comparative metabolites profiling of different solvent extracts of Asparagus species cladodes using liquid chromatography-mass spectrometry-based metabolomics and molecular networking[J]. Phytochemical Analysis: PCA, 36(3): 506-519. [17] Trindade V B, Teixeira R R, Vieira C P, et al.2023. N-Acetylglutamate and N-acetylmethionine compromise mitochondrial bioenergetics homeostasis and glutamate oxidation in brain of developing rats: Potential implications for the pathogenesis of ACY1 deficiency[J]. Biochemical and Biophysical Research Communications, 684: 149123-149123. [18] Vanessa B, Vieira C P, Teixeira R R, et al.2023. Experimental evidence that N-acetylglutamate and N-acetylmethionine compromises mitochondrial functions besides inhibiting citric acid cycle enzyme and respiratory chain activities in brain of young rats[J]. Free Radical Biology and Medicine, 208(S1): S63-S63. [19] Wang D, Wang X, Cheng H, et al.2025. Effect of yeast culture supplementation on rumen microbiota, regulation pathways, and milk production in dairy cows of different parities[J]. Animal Feed Science and Technology, 321: 116244-116244. [20] Wang H, Su M, Wang C, et al.2023. Yeast culture repairs rumen epithelial injury by regulating microbial communities and metabolites in sheep[J]. Frontiers in Microbiology, 14: 1305772-1305772. [21] Wang J, Yu Y, Guo Y, et al.2024. Metabolomics-based study of the effect of dietary N-carbamoylglutamic acid addition to heifers in late pregnancy on newborn calves[J]. Frontiers in Veterinary Science, 11: 1335897-1335897. [22] Xiao C, Xiao J, Wu Y, et al.2024. Determination of amino acids of novel food in food by HPLC coupled with pre-column derivatization[J]. Foods, 13(24), 4012. [23] Xu J, Li X, Fan Q, et al.2025. Effects of yeast culture on lamb growth performance, rumen microbiota, and metabolites[J]. Animals, 15(5): 738-738. [24] Xu Z, Ying C, Bai P, et al.2025. Microbial dynamics, metabolite profiles, and chemical composition in Saccharomyces cerevisiae and Kluyveromyces marxianus co-culture during solid-state fermentation[J]. Food Bioscience, 64, 105849. [25] Yoshinaga K, Iida Y, Inoue M, et al.2025. Analysis of fatty acid composition by gas chromatography using hydrogen or nitrogen as an alternative carrier gas to helium: A JOCS collaborative study[J]. Journal of Oleo Science, 74(4), 349-360. [26] Zhang H, Cui N, Ma X, et al.2025. Structural basis of augmenting taurine uptake by the taurine transporter in alleviating cellular senescence[J]. Cell Research, 35(5): 385-388. [27] Zhang S, You M, Shen Y, et al.2025. Improving fatty liver hemorrhagic syndrome in laying hens through gut microbiota and oxylipin metabolism by Bacteroides fragilis: A potential involvement of arachidonic acid[J]. Animal Nutrition, 20: 182-199. [28] Zhong Y, Jia Z, Zhou H, et al.2023. Comparative analysis of volatile compounds from four radish microgreen cultivars based on ultrasonic cell disruption and HS-SPME/GC-MS[J]. International Journal of Molecular Sciences, 24(19). [29] Zou Y, Kang D, Liu R, et al.2018. Effects of ultrasonic assisted cooking on the chemical profiles of taste and flavor of spiced beef[J]. Ultrasonics-Sonochemistry, 46: 36-45.
