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Page 8 of 12 Horie et al. Microbiome Res Rep 2024;3:35 https://dx.doi.org/10.20517/mrr.2024.08

DISCUSSION
The lactic acid bacteria species isolated by the culture method tended to differ depending on the feeding
habits of the host animals. Furthermore, 16S rRNA gene amplicon sequencing of the feces showed that the
composition of the intestinal flora depends on the feeding habits of the host animals. Different lactic acid
bacteria were isolated from carnivores and herbivores in this study. L. salivarius and L. saerimneri were
isolated from carnivores, and L. equi and Limosilactobacillus gorillae were isolated from the herbivores.
L. saerimneri was also isolated from the feces of the carnivorous polar bear. However, these species were not
found in the feces of the herbivorous Asian black bear or sun bear. Instead, L. animalis and Ligilactobacillus
murinus (L. murinus) were isolated from these herbivorous bears. The diet of the polar bear consisted of
60% chicken and 12% sausage, whereas that of the sun bear consisted of 60% vegetables, such as steamed
potato, apple, and banana. Asian black bears in zoos also tended to be herbivorous, with apples and Chinese
cabbage accounting for 40% of their diet. These results suggest that the type of lactic acid bacteria occupying
the intestine is more dependent on diet than host species.

The trends in the bacterial composition of the intestinal flora depended more on host feeding habits than on
differences in host species. Similar results have been reported in previous studies. A study comparing the
gut microbiota of bats in zoos found that while there were significant differences in microbial diversity
between the insectivorous species N. noctula and V. murinus and the frugivorous species C. perspicillata, the
distinctions between N. noctula and V. murinus were not statistically significant . Among insectivorous
[40]
bats, the phylum Bacillota was predominant, accounting for approximately 55% of the gut microbiota. In
the gut microbiota of frugivorous bats, the phylum Bacillota accounted for 35.7% and Bacteroidota
accounted for 30.4%. These results were similar to those of carnivores and herbivores in the present study.
On the other hand, at the order level, Lactobacillales were the most dominant among insectivorous bats,
which was different from the results of this study. Bacteroidales were predominant (29.9%) in the
frugivorous bat, consistent with our results for herbivores.

The presence of species belonging to the former genus Lactobacillus in the carnivores was also confirmed
through culture method. However, 16S rRNA gene amplicon sequencing did not detect Lactobacillus in
tiger, lion, horse, polar bear, or Asian black bear. The DNA extraction method and primer design used to
detect Lactobacillus species through 16S rRNA gene amplicon sequencing may need to be improved.

The microorganisms that make up the intestinal flora do not exist independently, but rather exist in mutual
interference. For example, the metabolites of one microorganism are used by other bacteria, or the
production of antibacterial substances such as bacteriocins eliminates similar related species. The microbial
environment in the intestine can be considered an ecosystem. When the intestinal flora, which is an
ecosystem, is disturbed, it may be effective to use lactic acid bacteria strains that are naturally present there
as probiotics in order to restore the original intestinal flora composition.

How are the microorganisms that compose the intestinal flora selected? The specific factors that determine
the constituent microorganisms of the intestinal flora have not yet been determined. The most likely
potential factor would be the energy source of the bacteria. In general, meat is richer in protein than plants,
and plants are richer in dietary fiber. The genus Paeniclostridium, found in the fecal flora of carnivores,
increased during fermentation using protein and peptone as substrates, and its growth was strongly
[53]
correlated with the production of ammonium, indole, and p-cresol . Analysis of the V4 region of the 16S
rRNA gene of healthy domestic ferrets and cats revealed that the fecal microbiota of ferrets exhibited a
higher representation of Bacillota and Proteobacteria, including Clostridium sensu stricto, Streptococcus,
Romboutsia, Paeniclostridium, Lactobacillus, Enterococcus, and Lactococcus . On the other hand,
[54]