新疆传统奶酒专用酵母菌株筛选及其生物学特性与风味贡献分析

Screening, biological characterization, and flavor contribution analysis of specialized yeast strains for Xinjiang traditional milk wine

摘要:

目的 挖掘新疆传统奶酒发酵体系中的优良酵母菌种资源，筛选适配奶酒发酵的专用菌株，为奶酒专用发酵剂的开发提供理论依据和菌种储备。方法 以产醇、产酯能力、乳糖利用能力以及耐酸(pH)、耐糖、耐乙醇性能为筛选指标，采用梯度稀释分离法结合选择性培养基分离酵母菌株。通过形态观察和分子生物学鉴定明确菌株的分类地位，研究其生长特性、碳源利用能力及生物安全性，结合感官评价和电子鼻技术评价菌株对发酵风味的贡献。结果 从新疆传统奶酒引子及传统发酵乳制品中分离纯化获得40株酵母菌株，经多轮筛选得到2株功能优良的菌株(J17和J23)。这2株菌可耐受pH 2.5、350 g/L葡萄糖及47.34 g/L乙醇的环境，同时具备优良的产醇、产酯能力和乳糖利用能力，鉴定为马克思克鲁维酵母(Kluyveromyces marxianus)和库德里阿兹威氏毕赤酵母(Pichia kudriavzevii)。两菌株的生长曲线显示，对数期始于4 h，稳定期分别始于12 h和18 h；对峙试验证实菌株之间无拮抗作用可共生；碳源利用试验表明二者可利用葡萄糖、乳糖、蔗糖等7种碳源，适应性强。生物安全性检测显示，两菌株均无溶血性，对康唑类抗真菌药物敏感，符合食品发酵菌株的安全标准。电子鼻分析显示，两菌株以1:1混菌(HJ)发酵奶酒基质的烷烃、硫化物、醇类、醛酮类等风味物质的响应值显著高于单菌发酵组及对照组，主成分分析累计贡献率与线性判别分析累计判别率均达99.97%。感官评价实验显示，混菌(HJ)发酵的奶酒发酵彻底，且在外观、香气、口感和风格4个维度的评分均高于其他组合。结论 马克思克鲁维酵母J17与库德里阿兹威氏毕赤酵母J23兼具高耐受性、广谱碳源利用能力、良好的生物安全性及协同增香能力，具备奶酒专用发酵剂的开发潜力，可为传统奶酒发酵提供优质菌种资源。

Abstract:

Objective To identify high-quality yeast strains in the Xinjiang traditional milk wine fermentation system and screen specialized strains suitable for the milk wine fermentation, thereby providing a theoretical basis and strain reserves for the development of dedicated fermentation agents.Methods With alcohol production, ester production, lactose utilization ability, as well as acid (pH), glucose, and ethanol tolerance as screening criteria, the gradient dilution separation method combined with selective media was employed to isolate yeast strains. Morphological observation and molecular biological identification were conducted to determine the taxonomic status of the strains. The growth characteristics, carbon source utilization ability, and biosafety of the strains were investigated. The fermentation flavor contributions of the strains were evaluated through sensory assessment and electronic nose technology.Results Forty yeast strains were isolated and purified from Xinjiang traditional milk wine starter and traditional fermented dairy products. After multiple rounds of screening, two strains (J17 and J23) with excellent functions were obtained, tolerating pH 2.5, 350 g/L glucose, and 47.34 g/L ethanol. They were identified as Kluyveromyces marxianus and Pichia kudriavzevii, respectively. The growth curves of the two strains showed that the logarithmic phase began at the time point of 4 h and the stationary phase started at 12 h and 18 h, respectively. The co-culture test confirmed no antagonistic effects and demonstrated symbiotic relationships between the two strains. Carbon source utilization tests indicated that both strains efficiently utilized seven carbon sources, including glucose, lactose, and sucrose, demonstrating strong metabolic adaptability. Biosafety testing revealed that neither strain exhibited hemolytic activity and was sensitive to antifungal drugs such as ketoconazole, meeting the safety standards for food fermentation strains. Electronic nose analysis revealed that the 1:1 mixed strain (HJ) fermentation of milk wine substrate exhibited significantly higher response values for flavor compounds such as alkanes, sulfides, alcohols, and aldehydes/ketones than the single-strain fermentation groups and controls. Both the cumulative contribution rate of PCA and the cumulative discriminant rate of LDA reached 99.97%. Sensory assessment demonstrated that the HJ fermented milk wine scored higher than that of other strain combinations in four dimensions: appearance, aroma, taste, and style.Conclusion K. marxianus J17 and P. kudriavzevii J23 demonstrate high tolerance, broad-spectrum carbon source utilization ability, excellent biosafety, and synergistic aroma enhancement, showing the potential as specialized fermentation agents for milk wine production and providing high-quality microbial resources for the fermentation of traditional milk wine.