Effects of storage methods on total bacterial count and microbial composition of bovine colostrum

Neonatal calves need to acquire passive immunity through maternal colostrum, as they are immunologically naive and the structure of the bovine placenta does not allow passage of maternal antibodies during pregnancy. Milked colostrum is not initially sterile and may even contain high bacterial counts. Minimizing total bacterial counts in colostrum is generally advised, however bacterial quality of colostrum comprises more than just bacterial quantities, but also depends on the specific bacteria present. While duration and temperature of colostrum storage are known to affect total plate counts (TPC), less is known about the effects of storage on the actual bacterial composition of the TPC. We speculated that, depending on the storage conditions, colostrum is a substrate in which certain bacterial species can thrive affecting the quality of colostrum.We therefore aimed to characterize the effects of different colostrum storage methods on the composition of the viable, aerobic, microbial community. Colostrum samples were stored at different temperatures and for different durations. Next, bacterial growth was assessed using the aerobe plate count culture method, followed by 16S rRNA gene amplicon sequencing. Differences in the TPC bacterial compositions of the stored colostrum samples, as determined by 16S rRNA gene amplicon sequencing, were mostly explained by the variation in bacterial composition of the colostrum sample directly after milking. In line with earlier studies, the results from our study show that the TPC increased when colostrum was stored for 24 hours at room temperature, but not when stored in a refrigerator for the same duration. Community structure of the TPC of colostrum stored at room temperature for 24 hours and stored in a refrigerator for a week was significantly different from the baseline samples. The 16S rRNA sequencing results indicate this is because of increased numbers of Enterobacteriaceae. Enterobacteriaceae abundance in refrigerated samples seemed to remain stable for the first 24 hours, but increased drastically after one week. The results indicate that microbial composition of stored colostrum is mostly influenced by the composition of the colostrum sample directly after milking, which is most probably the result of contamination or other environmental influences during the milking process. This study provides a deeper insight in the changes in the microbial composition of colostrum TPC during practical storage conditions and provides a primer for more detailed research into the determinants of bacterial composition of colostrum and the linked health effects.Lisa Robbers, Hannes Bijkerk, Lars Ravesloot, Alex Bossers, Mirjam Nielen, Ruurd Jorritsma, Ad Koets and Lindert Benedictus

新生犊牛因免疫功能尚未成熟，且牛胎盘结构无法在孕期传递母源抗体，因此需通过摄入母源初乳（colostrum）获得被动免疫。采集得到的初乳初始并非无菌，甚至可能携带大量细菌。通常建议尽量降低初乳中的总细菌数，但初乳的细菌品质不仅取决于细菌总量，还与所存在的特定菌种相关。尽管已知初乳储存时长与温度会影响总平板计数（Total Plate Counts, TPC），但目前学界对储存如何影响TPC对应的实际细菌群落组成的了解仍较为有限。我们推测，根据储存条件的不同，初乳可作为特定细菌物种的增殖基质，进而影响初乳品质。
因此本研究旨在阐明不同初乳储存方式对需氧活微生物群落组成的影响。研究人员将初乳样本置于不同温度、储存不同时长，随后采用需氧平板计数培养法评估细菌生长情况，再通过16S rRNA基因扩增子测序进行分析。经16S rRNA基因扩增子测序分析可知，储存后初乳样本的TPC细菌组成差异，主要源于采集后即刻的初乳样本本身的细菌组成变异。
与既往研究结果一致，本研究显示初乳在室温下储存24小时后TPC会升高，但在冰箱中储存相同时长则无此变化。室温储存24小时与冰箱储存1周的初乳TPC群落结构，与基线样本相比存在显著差异。16S rRNA测序结果显示，这一差异源于肠杆菌科（Enterobacteriaceae）的数量增加。冷藏样本中的肠杆菌科丰度在最初24小时内似乎保持稳定，但在储存1周后会大幅上升。
研究结果表明，储存后初乳的微生物组成主要受采集后即刻的初乳样本组成影响，这大概率源于挤奶过程中的污染或其他环境因素干扰。本研究深入解析了实际储存条件下初乳TPC微生物组成的变化规律，为后续深入研究初乳细菌组成的影响因素及其相关健康效应提供了研究基础。
作者：Lisa Robbers, Hannes Bijkerk, Lars Ravesloot, Alex Bossers, Mirjam Nielen, Ruurd Jorritsma, Ad Koets 和 Lindert Benedictus