Supplementing Glycerol to Inoculum Induces Changes in pH, SCFA Profiles, and Microbiota Composition in In-Vitro Batch Fermentation

Qingtao Gao,Lei Liu,Cheng Long,Kai Li,Liang Chen,Hongfu Zhang,Ruqing Zhong

doi:10.3390/fermentation8010018

Abstract

Glycerol was generally added to the inoculum as a cryoprotectant. However, it was also a suitable substrate for microbial fermentation, which may produce more SCFAs, thereby decreased pH of the fermentation broth. This study investigated the effect of supplementing glycerol to inoculum on in vitro fermentation and whether an enhanced buffer capacity of medium could maintain the pH stability during in vitro batch fermentation, subsequently improving the accuracy of short chain fatty acids (SCFAs) determination, especially propionate. Two ileal digesta were fermented by pig fecal inoculum with or without glycerol (served as anti-frozen inoculum or frozen inoculum) in standard buffer or enhanced buffer solution (served as normal or modified medium). Along with the fermentation, adding glycerol decreased the pH of fermentation broth (p < 0.05). However, modified medium could alleviate the pH decrement compared with normal medium (p < 0.05). The concentration of total propionic acid production was much higher than that of other SCFAs in anti-frozen inoculum fermentation at 24 and 36 h, thereby increasing the variation (SD) of net production of propionate. The α-diversity analysis showed that adding glycerol decreased Chao1 and Shannon index under normal medium fermentation (p < 0.05) compared to modified medium (p < 0.05) along with fermentation. PCoA showed that all groups were clustered differently (p < 0.01). Adding glycerol improved the relative abundances of Firmicutes, Anaerovibrio, unclassified_f_Selenomonadaceae, and decreased the relative abundance of Proteobacteria (p < 0.05). The relative abundances of Firmicutes, such as Lactobacillus, Blautia and Eubacterium_Ruminantium_group in modified medium with frozen inoculum fermentation were higher than (p < 0.05) those in normal medium at 36 h of incubation. These results showed that adding glycerol in inoculum changed the fermentation patterns, regardless of substrate and medium, and suggested fermentation using frozen inoculum with modified medium could maintain stability of pH, improve the accuracy of SCFA determination, as well as maintain a balanced microbial community.

Highlights

Fiber-rich ingredients contain high amounts of Dietary fiber (DF) resulting in a greater production of short-chain fatty acids (SCFA) [5], which depended on the action of cellulose-utilizing microorganisms colonized in hindgut whereby organization of the enzymes into cellulosomes [6,7]
Anti-frozen inoculum had a significant lower (p < 0.05) pH value of fermentation broth compared to frozen inoculum at 12 h (Figure 2A,B), 24 h (Figure 2C,D) and 36 h (Figure 2E,F) of fermentation for 2 substrates, regardless of media
The results indicated that glycerol has a distinct bacteria protection effects, but with the progress of fermentation, the decrement of pH caused by glycerol fermentation lead to microbial community disorder

Summary

Introduction

Dietary fiber (DF) bring benefits to the animals in term of well-being by improving satiety [1,2] and intestinal health by modulating microbiota and metabolites [3,4]. Fiber-rich ingredients contain high amounts of DF resulting in a greater production of short-chain fatty acids (SCFA) [5], which depended on the action of cellulose-utilizing microorganisms colonized in hindgut whereby organization of the enzymes into cellulosomes [6,7]. Recent studies have shown that dietary fiber shapes intestinal microbiota. Zhao et al [8] 4.0/).

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