Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou Universitygrid.268415.c, Yangzhou, People's Republic of China.
College of Animal Science and Technology, Yangzhou Universitygrid.268415.c, Yangzhou, People's Republic of China.
Microbiol Spectr. 2022 Apr 27;10(2):e0228021. doi: 10.1128/spectrum.02280-21. Epub 2022 Mar 28.
Lactic acid bacteria such as Streptococcus thermophilus are known to produce extracellular polysaccharide (EPS) in fermented foods that enhance the creaminess and mouthfeel of the product, such as yogurt. Strains producing larger amounts of EPS are highly sought-after, and therefore, robust and accurate quantification methodologies are important. This study found that two commonly used methodologies significantly underestimated the amount of EPS produced as measured using a milk matrix. To this end, a proteolytic step was implemented prior to EPS extraction (Method C). An initial proteolytic step using xanthan gum-spiked milk significantly increased recovery yield to 64%, compared to 27.8% for Method A and 34.3% for Method B. Method C showed no improvement when assessed using a chemically defined medium. Method C was further validated using three strains of S. thermophilus with varying EPS-production capabilities (ST, ST, ST). Overall, Method C demonstrated significant improvements in the EPS extraction yield for all three S. thermophilus strains in fermented milk. On average, Method C improved isolation yield by ∼3- to 6-fold compared with Method A and by ∼2- to 3-fold compared with method B. There were no significant differences between samples when they were grown in a chemically defined medium, highlighting the importance of a proteolytic step specifically for fermented milk samples. In commercial applications, accurate quantification of EPS-production is an important aspect when finding new strains. Extracellular polysaccharide (EPS) production by milk-fermenting microorganisms is a highly sought-after trait in improving the perceived thickness, creaminess, and mouthfeel of yogurt. Streptococcus thermophilus are commonly isolated and their EPS production is quantified in the search for higher-producing strains. In this study, we demonstrated that two commonly used methods for isolating EPS from milk samples significantly underestimated the true amount of EPS present. We demonstrated that the addition of a proteolytic step prior to EPS extraction isolated over 2-fold more EPS than identical samples processed using the traditional protocols. We further validated this method in fermented milk samples from three strains of S. thermophilus that included a low-, mid-, and high-EPS producing strain. Again, we showed significant improvements in EPS isolation using a proteolytic step. In the search for new S. thermophilus strains with enhanced EPS production, accurate quantification in an optimal medium is essential.
嗜热链球菌等乳酸菌在发酵食品中产生胞外多糖 (EPS)，可提高产品的奶油感和口感，如酸奶。因此，人们高度追求产生更多 EPS 的菌株，而强大且准确的定量方法则非常重要。本研究发现，两种常用方法在测量牛奶基质中的 EPS 产量时，会严重低估 EPS 的产量。为此，在 EPS 提取前进行了蛋白水解步骤（方法 C）。使用 xanthan 胶加牛奶进行初始蛋白水解步骤，与方法 A 的 27.8%和方法 B 的 34.3%相比，回收率提高到 64%。当使用化学定义培养基进行评估时，方法 C 没有改善。方法 C 进一步使用三种具有不同 EPS 生产能力的嗜热链球菌菌株 (ST、ST 和 ST) 进行验证。总体而言，方法 C 显著提高了所有三种嗜热链球菌菌株在发酵乳中的 EPS 提取产率。平均而言，与方法 A 相比，方法 C 使分离产率提高了约 3 至 6 倍，与方法 B 相比提高了约 2 至 3 倍。当在化学定义培养基中生长时，样品之间没有显著差异，这突出表明该蛋白水解步骤对于发酵乳样品非常重要。在商业应用中，准确量化 EPS 的产生是寻找新菌株的一个重要方面。 牛奶发酵微生物产生胞外多糖 (EPS) 是提高酸奶口感、奶油感和口感的高度追求的特性。嗜热链球菌通常被分离出来，并在寻找高产菌株时对其 EPS 产量进行定量。在这项研究中，我们证明了从牛奶样品中分离 EPS 的两种常用方法严重低估了实际存在的 EPS 量。我们证明，在 EPS 提取前添加蛋白水解步骤可分离出比使用传统方案处理的相同样品多 2 倍以上的 EPS。我们进一步在三种嗜热链球菌菌株的发酵乳样品中验证了这种方法，其中包括低、中、高 EPS 产生菌株。同样，我们使用蛋白水解步骤显示出在 EPS 分离方面的显著改进。在寻找具有增强 EPS 产生能力的新嗜热链球菌菌株时，在最佳培养基中进行准确的定量是必不可少的。
