Centro de Investigación Biomédica, Departamento de Nutrición y Bromatología, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Granada, 18071 Granada, Spain.
Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Nutrients. 2021 Jun 27;13(7):2207. doi: 10.3390/nu13072207.
The gut microbiota has a profound effect on human health and is modulated by food and bioactive compounds. To study such interaction, in vitro batch fermentations are performed with fecal material, and some experimental designs may require that such fermentations be performed with previously frozen stools. Although it is known that freezing fecal material does not alter the composition of the microbial community in 16S rRNA gene amplicon and metagenomic sequencing studies, it is not known whether the microbial community in frozen samples could still be used for in vitro fermentations. To explore this, we undertook a pilot study in which in vitro fermentations were performed with fecal material from celiac, cow's milk allergic, obese, or lean children that was frozen (or not) with 20% glycerol. Before fermentation, the fecal material was incubated in a nutritious medium for 6 days, with the aim of giving the microbial community time to recover from the effects of freezing. An aliquot was taken daily from the stabilization vessel and used for the in vitro batch fermentation of lentils. The microbial community structure was significantly different between fresh and frozen samples, but the variation introduced by freezing a sample was always smaller than the variation among individuals, both before and after fermentation. Moreover, the potential functionality (as determined in silico by a genome-scaled metabolic reconstruction) did not differ significantly, possibly due to functional redundancy. The most affected genus was , a fiber degrader. In conclusion, if frozen fecal material is to be used for in vitro fermentation purposes, our preliminary analyses indicate that the functionality of microbial communities can be preserved after stabilization.
肠道微生物群对人类健康有深远影响，并受到食物和生物活性化合物的调节。为了研究这种相互作用，采用粪便进行体外批量发酵，一些实验设计可能需要使用先前冷冻的粪便进行此类发酵。尽管已知在 16S rRNA 基因扩增子和宏基因组测序研究中，冷冻粪便不会改变微生物群落的组成，但尚不清楚冷冻样本中的微生物群落是否仍可用于体外发酵。为了探索这一点，我们进行了一项初步研究，在该研究中，我们使用患有乳糜泻、牛奶过敏、肥胖或瘦小孩子的粪便进行体外发酵，这些粪便用 20%的甘油冷冻（或不冷冻）。在发酵之前，粪便在营养培养基中孵育 6 天，以使微生物群落有时间从冷冻的影响中恢复。每天从稳定容器中取出一份样品，用于体外批量发酵扁豆。新鲜和冷冻样本之间的微生物群落结构有显著差异，但冷冻样本引入的变异始终小于发酵前和发酵后的个体间变异。此外，潜在功能（通过基因组规模的代谢重建进行计算确定）没有显著差异，可能是由于功能冗余。受影响最大的属是 ，一种纤维降解菌。总之，如果要将冷冻粪便用于体外发酵目的，我们的初步分析表明，在稳定化后可以保留微生物群落的功能。
