Gum Arabic changes the gut bacteria-brain system and influences overall health in zebrafish

Diet modulates the dynamic nature of the gut microbiome, influencing not only its composition but also its function and diversity. Different diets vary in their nutritional profile, whole-food composition, caloric value, and dietary fibre (DF) content, all of which can alter the microbial architecture, leading to either positive or negative health ramifications¹. A diet rich in DFs is essential for optimal health and well-being by preventing various non-communicable diseases due to its putative positive effects on cardiovascular, metabolic, and cognitive health in addition to weight management, appetite regulation, and inflammation^1–5. DFs constitute a wide range of heterogeneous compounds that resist digestion, and the gut microbiome plays a key role in breaking them down to expose their beneficial properties to the host organism^6,7. This bidirectional interaction between the gut microbiome and DFs not only influences intestinal health and metabolism but also mediates host behaviour and fitness via the gut-brain axis^8,9.

Gum Arabic (GA) is an edible tree gum exudate obtained by the incision of the trunks and the branches of Acacia senegal (L.) Willdenow, native to the arid and semi-arid regions of Africa. Also known by several names like gum Acacia, Senegal gum, and Sudan Arabic gum, GA is chemically composed of highly branched and high molecular weight glycoproteins and polysaccharide complexes of which 90% is arabinogalactan, a polymer of arabinose (17–34%) and galactose (32–50%) with prebiotic properties, in addition to some amino acids and minerals^10,11. It is a key additive (E-Number 414) in the food industry, mainly used as an emulsifier and a stabiliser, but its potential benefits as a DF have been largely overlooked¹². Traditional medicine has long suggested health benefits from its oral consumption, particularly with reports indicating positive effects on vital organ functions, such as the kidneys¹³, the brain¹⁴, the heart¹⁵, and the liver¹⁶ in both humans and animals. Although not primarily consumed in the Western world as a source of DF, people in sub-Saharan regions like Sudan have incorporated GA into their day-to-day life for both medicinal and alimentary purposes long before it was considered safe for human consumption by regulatory bodies like the Food and Drug Administration (FDA) and the Food and Agriculture Organization (FAO) in the 1970s^17–19.

GA displays a range of potential health benefits. A randomised clinical trial revealed that after 12 weeks of receiving 20 g/day of GA, participants showed a significant decrease in systolic and diastolic blood pressure and fasting blood glucose²⁰. Similarly, the administration of GA was associated with improved renal function in patients with progressive chronic kidney disease (CKD)¹³, and the oral administration of 30 g GA per day had antioxidant and anti-inflammatory effects in patients undergoing haemodialysis²¹. A recently published systematic review of 29 clinical trials utilising GA as a therapeutic agent for various human diseases and conditions indicated that GA improves oral health, gastrointestinal functions, and supports healthy weight management in addition to alleviating the adverse effects of sickle cell anaemia and rheumatoid arthritis²². Beneficial effects have also been identified in mice where the administration of GA mitigated the expression of key pro-inflammatory plasma cytokines: IL-6, IL-1β, and TNF-α; despite being fed an inflammatory high-fat diet²³. Furthermore, GA improved semen quality in rats²⁴ and ovarian function in mice²⁵ while exhibiting no teratogenic effects²⁶; and in rats, GA enhanced cognitive ability whilst demonstrating neuroprotective properties^14,27. Additionally, GA has prebiotic and modulatory effects on the gut microbiome^10,28–30 via its fermentation by bacteria into microbial-derived metabolites, mainly short-chain fatty acids (SCFAs)^29,31,32. This microbiota-gut-brain axis is directly influenced by the diet, which in turn can affect the cognitive behaviour and immunity of the organism^33–35. The broad range of health benefits associated with GA supplementation suggests that its effects are likely to occur at various levels along the gut-brain axis; however, the exact mechanisms are not yet fully understood.

Nutritional science using animal models recognises that no single animal model perfectly recapitulates human biology nor replicates every nuance of human nutrition. Zebrafish is a pertinent translational model for nutrition, offering valuable cross-species insight⁴⁸. Thus, it complements, but not replace, mammalian models such as mice and rats. The dosages chosen for this study were based on other animal models, including rats (Rattus sp.) and Nile tilapia (Oreochromis niloticus). In rats, the GA dosages ranged between 4000 mg/kg and 20,000 mg/kg⁴⁹, which is equivalent to a human equivalent dose (HED) based on body surface area (BSA) of 649—3243 mg/kg⁵⁰. This translates to 2 mg of GA/fish/day (i.e. 4000 mg/kg body weight when assuming fish weight = 0.0005 kg) in our 6% GA experiment. In Nile tilapia and other fish species, a ratio of GA ≤ 10% (total food weight) showed beneficial health effects^51–54, which translates into the HED of 6% GA in zebrafish (i.e. 4000 mg GA/kg body weight) being the equivalent of 280,000 mg GA per day in a 70 kg human adult. Similarly, 60% GA (i.e. 40,000 mg GA/kg) in zebrafish is the equivalent of 2,800,000 mg GA in a 70 kg human adult. However, when applying the BSA normalisation method, the HEDs are around 256 mg/kg in the 6% GA and 2564 mg/kg in the 60% GA experiments, respectively. The K_m factor of a 70 kg human adult with a BSA of 1.81 m² is 39⁵⁵. On the other hand, the K_m factor of zebrafish is around 2.5 (weight = 0.0005 kg and BSA = 2 × 10^–4 m²; Table S5 in the SI Appendix). In summary, the total HED of 256 mg/kg is around 18 g of GA (number of servings = 4 tsp) and around 180 g (number of servings = 36 tsp) of GA for 2564 mg/kg in a 70 kg human adult. Evidently, even the HED of 60% GA is impractical and not realistic. Therefore, results at the higher exposure (60% GA) are unlikely to generalise to typical human intakes, and extrapolation from zebrafish to humans should be made with caution.

Our results demonstrate that GA* supplementation affects female and male zebrafish at multiple levels, spanning from the gut microbiome to metabolic turnover in the gut and brain, and ultimately, overall fitness. The strength of the effects was dosage dependent, where 6% GA* supplementation had positive effects on the gut microbiome in both females and males, with no further implications on reproductive fitness, whereas the effects of 60% GA* supplementation went beyond the microbiome and affected the metabolic rates in the intestines and overall brain activity, potentially at the cost of reduced reproductive fitness in females. The microbiota-gut-brain axis is increasingly recognised to play a key role in organismal health. Changes in the gut microbiota are associated with a range of neurological conditions such as Alzheimer's, Parkinson's disease, autism spectrum disorders (ASD), and potentially even mood^56,57. GA has previously been shown to enhance cognitive abilities and, more recently, to be anti-epileptic in rats⁵⁸. Our results suggest that the substantial changes in gut microbiota in GA*-supplemented fish after just two weeks may have downstream effects and ramifications from gut health to brain function and behaviour. Due to the highly dynamic nature of the gut microbiome, dietary changes and the availability of nutritional substrates influence its composition and architecture. DFs have been thoroughly documented to exhibit gut modulatory effects in humans and animals, which contribute to their positive health benefits^59,60. In line with previous findings on other DFs, we demonstrated that GA* can favourably shift the Proteobacteria/Fusobacteria (P/F) ratio towards the more beneficial Fusobacteria, and consequently, Cetobacterium (Fig. 2A–C and Fig. S2). In zebrafish, a drop in Fusobacteria and a corresponding increase in the P/F ratio is directly linked with gut dysbiosis⁶¹. In our study, 6% GA* was sufficient to generate beneficial effects on the gut microbiome over just two weeks. Our findings suggest a possible difference in gut microbiome composition according to sex but primarily highlight the sex-specific response to GA* supplementation.

The role of sex in shaping the gut microbiome is contested and inconsistent^62–66. However, research has shown that sex-specific hormones can alter the gut microbiome composition by influencing the host-microbiome interactions. At the same time, the sex hormones differentially affect the physiology and behaviour of the two sexes, which in turn affects the microbiome^62,67,68. However, variation in diet has been consistently shown to exert sex-specific changes on the gut microbiome across different species, including the zebrafish^69–71. In line with the concept of a sex-specific microbiome, we observed that the increase in the relative abundance of Cetobacterium was more pronounced in females across both GA* treatments (Fig. S2). However, the higher dosage of 60% GA* only reduced the Observed α-richness in females (Fig. 3A). Since the zebrafish display sexual dimorphism in anatomical, physiological, and behavioural aspects^72–74. We speculate that this sex-specific effect of GA* on the microbiome is due to diet-host interactions. Cetobacterium, a prominent bacterium in the Fusobacteria phylum, is known to produce acetate⁴⁰ which can cross the blood–brain barrier to control appetite and glucose metabolism^40,75. Acetate, like all SCFAs, is primarily produced in the colon due to microbial fermentation of dietary fibres. In mammals, acetate can be endogenously produced by the liver, albeit to a lesser extent^76,77. In the zebrafish, the current data are suggestive but still scarce, with almost all papers highlighting and emphasising the role of the gut microbiome in generating acetate as the primary source.

Our results provide further evidence for a positive effect of GA on the microbiota-gut-brain axis. The observed increase in the relative abundance of Cetobacterium under 60% GA* in female zebrafish may be linked to the downregulation of the cart1 gene, an anorexigenic gene, in female brain tissues via an acetate-dependent pathway. The effects of acetate on the regulation of the cart1 gene remain inconsistent with some studies indicating no significant change⁸⁶ whilst others show an up-regulation⁸⁵ in gene expression. This discrepancy emphasises that the acetate-mediated effects on metabolism and appetite can differ depending on whether the source is microbial-derived or exogenous⁸³. From a mechanistic perspective, our study suggests that the microbial-derived acetate from the GA* fermentation can potentially counter the anorexigenic effects of the cart1 gene, particularly in females (Fig. 7). Similarly, the gut microbiota has a direct influence on the hypothalamic–pituitary–adrenal axis (HPA), which in turn regulates the organism's response to metabolic, physiological, and psychological stimuli⁸⁷. Interestingly, there is also a sex-specific link between the cart1 gene and the HPA axis, particularly in the hypothalamus, with potential downstream effects on the glucocorticoid hormone cortisol and subsequently, stress⁸⁸. In female zebrafish, the observed downregulation of cart1 may relate to activity in the hypothalamic-pituitary-interrenal (HPI) axis, the teleost homologue of the mammalian HPA axis⁸⁹, with possible ramifications on stress-related behaviours and appetite regulation⁹⁰. However, cortisol levels were not measured, and we can only propose, but not corroborate the exact mechanisms. Since the brain acetate levels were mostly abundant in the females of the 60% GA*-supplemented cohort (Fig. 4), the activation of the HPA axis via acetate might also be dosage-dependent. Furthermore, cortisol and insulin have antagonistic effects on metabolism⁹¹. We suggest that the stimulation of insulin via the parasympathetic nervous system in response to the microbial-derived acetate has a sex-specific impact on cortisol regulation potentially influencing the contrasting phenotypic data (Fig. 7). The mechanism of the sex-dependent effects of acetate on metabolism and hunger control involves a crosstalk between the microbiota-gut-brain axis and the endocrine system, which warrants further investigation. Additionally, de novo vitamin B₁₂ biosynthesis can be derived from Cetobacterium³⁸ which is essential for maintaining proper brain functioning and supporting the nervous system⁹². Thus, Cetobacterium-derived B₁₂ can potentially play a role in zebrafish development and influence its locomotion^92–94. Under 6% and 60% GA*, the raw abundance and relative abundance of Cetobacterium was higher in females; however, this apparent increase was only evident in males under 6% GA* supplementation (Fig. 2C and Supplementary Fig. S2). The impact of vitamin B₁₂ on reproductive fitness and embryo quality is scarce. From our data, any reproductive fitness under 60% GA* supplementation was primarily evident in male fish and not in females (Fig. 5). After conducting a differential abundance analysis, LEfSe (Linear Discriminant Analysis Effect Size) identified Cetobacterium enrichment in females under 60% GA* vs Control (p < 0.05); however, effect size was small (LDA score < 2.5). On the other hand, in males, Cetobacterium was slightly higher in the Control group compared to the 60% GA* group. However, neither the p nor the LDA effect size reached conventional thresholds⁹⁵. For this reason, any contribution of Cetobacterium-derived B12, if present, to reproduction or locomotion remains uncertain and warrants further investigation. Separately, DFs have been shown to exhibit immunomodulatory effects on the gut and to shape the homeostasis of the immune system^96,97. We detected the upregulation of the gene clec3ba in 60% GA*-supplemented fish compared to the Control fish (Table 1), which is in line with the idea that GA may have an immunomodulatory effect and increase the expression of C-type lectins in innate immune cells, namely, macrophages/microglia located in the zebrafish brain^98,99. Incorporating GA into the zebrafish diet can potentially activate defence mechanisms against infections.

Overall health is often directly linked with reproductive fitness^100,101. We found potentially beneficial effects of GA* supplementation on male reproductive fitness, particularly at high dosages, whereas the effects were detrimental in females. In animals, the implications of DFs on the reproductive parameters of both sexes have been promising. In boars, the incorporation of inulin and wheat bran during the pre-puberty period enhanced testosterone and semen production¹⁰². Also, in pigs, the incorporation of a variety of DFs primarily before mating, enhanced reproductive fitness and offspring survival by improving oocyte maturation¹⁰³. The health implications of DFs on human reproduction are currently understudied as most research has focused on metabolic health, but a high-fibre diet can reduce the reabsorption of oestradiol in the gut and subsequently be detrimental to the menstrual cycle in women due to the diet's influence on the hypothalamic-pituitary axis, supporting the potential role of the gut-brain axis in reproduction^104,105. Moreover, female gonadal hormones can have contrasting effects; therefore, a low intake of DF may negatively affect male fertility¹⁰⁶. A possible explanation for the positive impact of GA on male reproductive fitness in our study is that it improves sperm quality as previously reported in mammals^24,107,108. Also, GA can influence the steroid hormones governing gametogenesis^108–110 and exhibit antioxidant activity to protect the gametes from damage^{25,27,107,110} in both males and females. Despite potentially displaying some adverse effects under specific conditions, DFs play a crucial role in safeguarding metabolic health which in turn can have positive ramifications on fecundity and reproductive nutrition^111,112. The association between DFs and reproduction is not linear and likely dosage-dependent, which warrants further investigation. Our study highlights the importance of considering differences in female and male physiology when making dietary recommendations and the potential role of the microbiota-gut-brain axis has on reproductive fitness^67,113–115.

One possible reason for the associations detected between the 60% GA* dosage and reproductive fitness may be the behavioural changes we identified. We observed that fish supplemented with 60% GA* exhibited increased speed (mm/s) but without swimming a longer distance (mm) compared to Control fish (Fig. 6A and B). This is indicative of a burst swimming pattern rather than a sustained movement. We propose three different possible explanations for the increased speed in GA*-supplemented fish. The first theory is that this hyperactivity may benefit males during courtship and spawning activities, where the males rapidly chase females while nudging their flanks with their snouts and attempting to lead them to the spawning site¹¹⁶. The second theory relates to foraging and prey-capture behaviour^117,118, which could be linked to the downregulation of the cart1 gene (anti-anorexigenic effect) in the 60% GA*-supplemented females and may induce an increased desire for food or a change in feeding behaviour^119–121 (Table 1). The increase in activity under GA* supplementation could, in turn, explain the reduced reproductive fitness in females caused by their energy allocation strategy, which favours somatic growth over ovarian growth and reproduction^122–124. Thirdly, since the HPA axis might be involved, this increase in locomotion observed in the GA*-supplemented fish, specifically the rapid burst in swimming (speed) without increased exploration (distance) might be related to the role of cortisol in response to stress or anxiety¹²⁵. Additional studies investigating the possible behavioural and physiological phenotypes of stress and anxiety in zebrafish fed a high-dietary fibre diet are warranted. According to UK guidelines, human adults should aim to consume 30 g of dietary fibre daily for optimal health benefits¹²⁶. On average, most human clinical trials evaluating the effects of GA on different medical diseases provide around 25–30 g GA per day, which is the equivalent of 357—429 mg per kg body weight^22,49. Assuming an adult human weighs 70 kg, a typical human dosage would be approximately 350 mg/kg which is equivalent to around 5 teaspoons (1 tsp = approx. 5 g) of GA powder per day. Interestingly, the European Food Safety Authority (EFSA) suggests that a numerically Acceptable Daily Intake (ADI) for GA is not required as even oral intake of large amounts (430—760 mg/kg per day) is tolerated and nontoxic; albeit, with potential gastrointestinal discomfort¹²⁷.

A diet rich in DFs such as GA yields downstream health benefits on the gut microbiome, metabolic, and cognitive functions. Despite that, the optimal daily nutritional requirements are still highly debated^128–130 and we are just beginning to understand the effects of DFs on overall fitness^131,132. Additionally, sex differences are often not considered, and varying results of mixed-sex cohorts are typically reported^133,134. Our results suggest that GA* can positively affect the gut microbiome composition and subsequently influence brain gene expression and functions. However, we also found negative effects on female reproductive fitness at very high dosages. It is essential to understand when and how DFs exhibit optimal benefits and to enhance our understanding of the interplay between nutrition and health. We employed a holistic approach and demonstrated that a change in GA*, even over a short period, has substantial effects on the microbiota-gut-brain axis and even reproduction, but in a dosage-specific manner. However, further in-depth investigation and mechanistic studies are warranted to verify our findings. Finally, we anticipate follow-up studies in mammalian models, considering sex as a biological variable, to be carried out to help establish sex-specific guidelines for optimal dietary fibre intake.

Supplementary Information.