The Effect of the Combination of Two Postbiotics on Anxiety-like Behavior in Animal Models

Abstract

With increasing evidence showing the connections between the microbiome, neurophysiology, and behavior, our research endeavors to investigate whether the consumption of a combination of two postbiotics with antioxidant effects can affect behavior regulation in model species. Here, we worked with a combination (1:1 ratio) of heat-treated Bifidobacterium longum subsp. longum ES1 (CECT7347) and Lacticaseibacillus rhamnosus BPL15 (CECT8361) as a dietary supplement. To examine the potential benefit of using this formulation to alleviate anxiety-like behavior, we employed two model species, Caenorhabditis elegans and adult Danio rerio. In C. elegans, the postbiotic supplementation reduced the anxiety-related behavior analyzed by means of the octanol avoidance test. In zebrafish, the novel tank test indicated a different swimming pattern 2 and 4 months after the animals were fed with the postbiotic combination. While fish did not exhibit any variance in their locomotion parameters such as pace and speed, they showed a statistically significant preference to spend more time in the upper zone of the water tank, a behavior that is correlated with a lower anxiety-like behavior in these species. Our aim with this study is to present evidence that can be used to develop whole-cell postbiotic-based novel and innovative dietary supplements for anxiety-related conditions.

Keywords: Bifidobacterium; Lacticaseibacillus; anxiety-like; postbiotics.

Figure 1

The experimental design including the animal model species used in this project, the experimental groups, the conditions for each trial, and the basis of the chosen evaluation tests for anxiety-like behavior assessment.

Figure 2

The modulation of the time of response in avoidance of octanol in C. elegans. (A) The frequency of distribution of the times recorded for each nematode in each of the groups (bin size = 1). (B) Mean values of the response time in each experimental group. “Control” refers to nematodes grown under standard conditions; “Starved” refers to worms subjected to starvation (anxiety-like condition); and “Starved + Postbiotics” refers to individuals supplemented with the ES1 HT + BPL15 HT postbiotic blend and subjected to starvation (anxiety-like condition). Asterisks show significant differences (**** p < 0.0001; *** p < 0.0010).

Figure 3

Biometrical mean values. (A) At the beginning of the experiment, experimental groups were homogenously set up in terms of fish weight. (B) Weight mean values at 2 and 4 months of experimentation. (C) Length mean values at 2 and 4 months of experimentation. “Control” refers to zebrafish diet-supplemented with maltodextrin and “Postbiotics” refers to zebrafish diet-supplemented with the ES1 HT + BPL15 HT postbiotic blend (ns—no significant changes). Dots represent values from individual animals within each experimental group.

Figure 4

Analysis of the zebrafish swimming pattern at 2 and 4 months. (A) Fish velocity during the NTT. (B) Total distance swam by the fish during the NTT. (C) Time spent in the upper zone of the novel tank during the NTT. (D) Merge heatmaps of all the trajectories of each experimental group. “Control” refers to zebrafish diet-supplemented with maltodextrin and “Postbiotics” refers to zebrafish diet-supplemented with the ES1 HT + BPL15 HT postbiotic combination. (* p < 0.0500; ** p < 0.0100; ns—no significant changes). Dots represent values from individual animals within each experimental group.

Figure 5

Detailed study on the zebrafish swimming pattern in the upper zone of the tank. (A) Pie chart representing the percentage of fish spending less than 30 s (10% of the total NTT; stripped black) in the upper zone during the behavior assay at 2 months and 4 months after the beginning of the experiment in each experimental group. (B) Latency to the first entry in the upper zone. “Placebo” refers to zebrafish diet-supplemented with maltodextrin and “Postbiotics” refers to zebrafish diet-supplemented with the ES1 HT + BPL15 HT postbiotic combination. White dots represent fish swimming in the upper zone at the beginning of the NTT and black dots represent fish swimming throughout the NTT (300 s) in the lower zone.