**4. Discussion**

The purpose of the present study was to continue exploring the beneficial effects of treatment with a multispecies probiotic in patients diagnosed with FMS. For this, a group of patients with a mean time of 8 and a half years since diagnosis and a mean age of 52 years were treated for 8 weeks with a multispecies probiotic or with a placebo substance and evaluated immediately for its effects on attention and memory.

To our knowledge, the only study evaluating the role of probiotics in cognition in FMS patients is our previous study, which showed a reduction in impulsivity after treatment [31]. In the current research, we found no significant differences in memory after treatment. Although no other studies have used probiotics to improve memory in FMS, a recent systematic review and meta-analysis of preclinical and clinical studies indicates that probiotics could be a useful strategy to improve dementia and cognitive decline [35] in both healthy [36] and elderly populations [40]. Similarly, a probiotic-treated Alzheimer's experimental model demonstrated an improvement in learning [41] and memory [42]. In clinical studies of elderly people with mild cognitive impairment, an improvement in cognitive function (memory and attention) and an increase in brain-derived neurotrophic factor (BDNF) were reported after treatment with *Lactobacillus plantarum C29*-fermented soybean (DW2009) for 12 weeks [43]. Similar data were collected after the administration of *Bifidobacterium* A1 for 12 weeks in older adults with memory deficits, although the data are not conclusive and further research is required in this regard [44]. According to these studies, one possible explanation for the lack of positive results in our study could be the short length of treatment; studies demonstrating memory benefits were of significantly longer duration.

Regarding the attentional tasks, no differences in the Stroop effect or the negative priming effect (Stroop Task with Negative Priming) were observed among the participants after the treatment, implying that the probiotic treatment used did not affect the inhibitory mechanisms of attention. However, patients with FMS treated with the probiotic showed a tendency towards reduced errors of omission (Go trials) during the Go/No-Go Task and the group that received the placebo presented a number of errors that was slightly higher than those registered in the pre-treatment phase. This type of error occurs when there is an absence of response to a relevant stimulus, and it is assumed that it reflects symptoms of inattention [45]. Therefore, FMS patients treated and not treated with the

probiotic showed similar levels of inhibitory motor control and similar ability to inhibit information irrelevant to the task objective, but they differed in their ability to maintain attention for an extended period with the objective of responding to specific stimuli. This difference could be attributed to the effect that probiotics produced in these patients, which improved their ability to maintain attention, as evidenced by the results obtained in the Go/No-Go Task in the post-treatment phase.

Despite studies finding that the effects of probiotics on attention are reduced, similar results have been observed in other populations. In this regard, after 8 weeks of treatment with *Lactobacillus plantarum* 299v, patients with major depression showed an improvement in attention and work speed on the attention and perceptivity test, but no significant effects on the Stroop test [46]. Similarly, *Lactobacillus plantarum* DR7 treatment for 12 weeks improved basic attention and memory in healthy adults, as measured by the computerized CogState Brief Battery [47].

A recent systematic review and meta-analysis showed a positive effect of probiotics on cognition in both humans and animals [48]. Human studies showed an improvement in attention and memory in patients with Alzheimer's, in the healthy elderly individuals or those with depression. The only FMS study included in this analysis was the first part of our current research [31]. Most included studies used *Lactobacillus* and *Bifidobacterium* probiotic strains, but it is worth noting that the meta-analysis found that using just one probiotic was more effective than using a combination. In the same manner, the 12-week treatment was more effective than the 8-week treatment, implying that our findings on FM cognition could be significant after additional weeks of treatment.

The putative mechanisms of action of probiotics in cognitive function, as suggested by Lv and collaborators [48], are related to neuroinflammation. In this regard, the decline in cognitive function associated with aging is related to changes in brain immunoregulation, including decreases in IL-4 [49]. Several studies sugges<sup>t</sup> a decrease in the diversity of microbiota with cognition and inflammatory markers [50], in which changes in the intestinal metagenome appear to be associated with cognitive function and brain iron deposition [51]. In this context, factors associated with aging, such as oxidative stress and inflammation, are related to the intestinal microbiota [52], which influences the different sequences of cognitive impairment [53], and probiotic treatment could reverse this cognitive impairment via cytokine systems.

Interestingly, elevations of proinflammatory chemokines/cytokines could negatively impact symptoms of FMS. Proinflammatory cytokines have been shown to have an important modulatory role in pain transmission and perception. It is not surprising that high levels of them have been found, specifically of interleukins 1, 2, 6 and 8, in patients with FMS [54]. Therefore, probiotic administration could be an effective approach to treat cognitive deficits in FMS, as can be seen in our results. In other words, a multispecies probiotic treatment can improve some cognitive functions in FMS patients, such as impulse control, sustained attention and the ability to maintain attentional control in a context of change. The clinical relevance of microbiota modulation in FMS patients should be considered as an adjuvant treatment.

However, these results must be taken with caution, given that this study had several limitations. First of all, we had a limited number of subjects, since this was a pilot randomized controlled trial. Secondly, the nutritional habits of the participants should have been registered because they could influence or interfere with the results—for example, the effect of the consumption of other fermented foods. Finally, measuring the gu<sup>t</sup> microbiota would have given us more information about probiotic modulation. In this manner, future studies should be designed with a large sample size while keeping these limitations in mind.
