Gamma-aminobutyric acid as a potential postbiotic mediator in the gut–brain axis

Gamma-aminobutyric acid (GABA) plays a crucial role in the central nervous system as an inhibitory neurotransmitter. Imbalances of this neurotransmitter are associated with neurological diseases, such as Alzheimer’s and Parkinson’s disease, and psychological disorders, including anxiety, depression, and stress. Since GABA has long been believed to not cross the blood–brain barrier, the effects of circulating GABA on the brain are neglected. However, emerging evidence has demonstrated that changes in both circulating and brain levels of GABA are associated with changes in gut microbiota composition and that changes in GABA levels and microbiota composition play a role in modulating mental health. This recent research has raised the possibility that GABA may be a potent mediator of the gut–brain axis. This review article will cover up-to-date information about GABA-producing microorganisms isolated from human gut and food sources, explanation why those microorganisms produce GABA, food factors inducing gut–GABA production, evidence suggesting GABA as a mediator linking between gut microbiota and mental health, including anxiety, depression, stress, epilepsy, autism spectrum disorder, and attention deficit hyperactivity disorder, and novel information regarding homocarnosine-a predominant brain peptide that is a putative downstream mediator of GABA in regulating brain functions. This review will help us to understand how the gut microbiota and GABA-homocarnosine metabolism play a significant role in brain functions. Nonetheless, it could support further research on the use of GABA production-inducing microorganisms and food factors as agents to treat neurological and psychological disorders.


GABA degradation
In the presence of the GABA-transaminase (GABA-T) enzyme, GABA is catabolized to succinic semialdehyde (SSA) by transamination with the cosubstrate of α-ketoglutarate.Subsequently, SSA is oxidized by SSA dehydrogenase (SSADH) to succinate, a constituent of the tricarboxylic acid (TCA) cycle 31 .GABA-T is highly expressed in human glial cells and is responsible for clearing released GABA from the synapses to convert GABA into glutamate, which is then fed into the glutamine pool (Fig. 2) 32 .In addition to its expression in glial cells, GABA-T is also expressed in brain capillary endothelial cells, where it is believed to act as a neurotransmittermetabolizing enzyme that possibly hydrolyzes circulating GABA and protects it from entering the brain 33 .In peripheral organs, GABA-T is highly expressed in the liver and, to some extent, in the pancreas and kidneys 34 .However, why GABA-T is highly expressed in the liver remains unclear.One hypothesis is that GABA-T hydrolyzes dietary or exogenous GABA and prevents the entry of peripheral GABA into the brain.To support this hypothesis, a previous study demonstrated that 2% GABA mixed in the diet (20 g GABA/kg diet) did not increase blood GABA levels, and even at a high dose (5% GABA), it could increase blood GABA levels to only +2% of the control group 35 .Although it seems likely that peripheral GABA is highly hydrolyzed in the liver, many studies have demonstrated the beneficial effects of low doses of GABA derived from diet or gut bacteria.It would be interesting to investigate this paradox further.
Fig. 1 | Overview of the interplay of dietary factors, gut microbiota, microbial metabolites, and brain health.These dietary factors, including probiotics, prebiotics, fermented foods, and microbial enzymes, positively affect gut microbiota composition that stimulates the release of GABA and other microbial metabolites.As microbial GABA passes through the intestinal barrier, it influences brain compound levels via blood-brain barrier or vagus nerve and improves brain function.This figure was created using Biorender.com.

GABA-producing microorganisms
GABA-producing microorganisms isolated from the human gut Several gastrointestinal (GI) bacteria contain the gene encoding GAD 36,37 , which is responsible for GABA production (Table 1).Among the human microbiota, Bifidobacterium, Lactobacillus, and Bacteroides are the most well-known GABA producers 36,37 .GABA production by Bifidobacterium and Lactobacillus has been extensively studied because of their probiotic functions and the need for probiotic and fermented food development.Emerging evidence has revealed that Bacteroides may be the primary genus in gut microbiota influencing mental health through the regulation of GABA production.Compared to Bifidobacterium and Lactobacillus, Bacteroides is one of the most abundant and prevalent genera in the human gut microbiota 37,38 .Recent findings from animal and human studies have shown a strong relationship between mental health disorders and dysregulation of the gut microbiota linked to glutamate-GABA metabolism, in which changes in the composition of Bacteroides were pronounced in the mental health disorder group 37,39,40 .In this section of the review, we focus only on those that have been reported to produce GABA in humans.
A recent Integrated Microbial Genomes/Human Microbiome Project database showed that Bacteroides (31.7%) was the most abundant genus in human gut microbiota processing GAD orthologs (specifically gadB), followed by Escherichia (22.5%) and Fusobacterium (9.9%); and both Bifidobacterium and Lactobacillus processed only 2.2% 38 .A recent study identifying uncultured bacteria in the human microbiome revealed that GABA is a previously undescribed growth factor in uncultured bacteria, and the main GABA producer is Bacteroides fragilis 37 .In addition to Bacteroides, Parabacteroides, Eubacterium, and Bifidobacterium have been identified as GABA producers in human stool samples 37 .However, only Bacteroides can produce GABA under a physiological pH for the human large intestine (pH 5.7-7.4), in which generally acid-tolerant pathogens such as E. coli produce GABA at a lower pH (<5.5) 37,38 .Transcriptome analysis of stool samples from healthy individuals confirmed that Bacteroides, Parabacteroides, and E. coli are GABA producers in the human gut 38 ., comparable to GABA levels produced by high GABA-producing Lactobacillus and Bifidobacterium strains isolated from infant feces (12-300 mM) 41 .Using B. Fig. 2 | Catabolism and anabolism of GABA.The highly expressed GABA transaminase (GABA-T) from glial cells is responsible for the clearing of released GABA from the synapses to convert GABA into glutamate, which is fed into the glutamine pool.Then, glutamine is transported from the glial cells to the presynaptic element, where it is converted back to glutamate.Then, glutamate is converted to GABA by glutamic acid decarboxylase (GAD).This figure was created using Biorender.com. https://doi.org/10.1038/s41538-024-00253-2 thetaiotaomicron as a culture model, it was found that both glutamate and glutamine are substrates for GABA production, and Bacteroides can produce GABA at pH values ranging pH from 3.1 to 6.3, with the highest production ability at pH 3.1 36 .In silico analysis using a total of 961 Bacteroides genomes revealed that 96% of human-gut isolated Bacteroides genomes uniquely harbor four genes, a GAD (gadB ortholog; IPR010107), a glutaminase (glsA ortholog; IPR015868), a glutamate/GABA antiporter (gadC ortholog, IPR022520), and a K+ channel (IPR028325).These genes are involved in the GAD enzyme system 36 and may exert a protective mechanism against acid stress in Bacteroides.Bacteroides can adapt to human gut conditions during the host lifespan with the flexibility to use various energy sources from both diet-and host-derived nutrients.This makes them resilient and robust against colonization of the human gastrointestinal tract 36 .
Lactobacillus and Bifidobacterium are well known for their probiotic effects.Several studies have reported the isolation of Lactobacillus and Bifidobacterium from the human gut 37,38,[41][42][43][44] .A previous study demonstrated that GABA can be produced from human fecal fermentation, with GABA concentrations ranging from 5.4 to 56.4 µM 41 .By screening 91 strains from seven species of Lactobacillus and 13 species of Bifidobacterium isolated from infants and adults, it was found that only one Lactobacillus and three Bifidobacterium strains could produce GABA; these four GABAproducing strains were isolated from infant feces, dental carriers, and ileocecal junction areas 41 41 .A later study showed that among 135 strains from 13 species of Lactobacillus and three species of Bifidobacterium isolated from healthy adults, only two species of Lactobacillus, L. plantarum, and L. brevis, exhibited GABA production, while all three species of Bifidobacterium (B.adolescentis, B. angulatum, and B. dentium), displayed GABA production 36 .L. plantarum (30 strains) and L. brevis (three strains) produced GABA at 0.5-2.9mM and 0.5-6.5 mM, respectively, while B. adolescentis (21 strains), B. angulatum (three strains), and B. dentium (one strain) produced GABA at 4.7-58.2,25.4-33.6,and 23.9 mM, respectively 42 .The addition of exogenous PLP, a cofactor of GAD, to the culture medium was found to increase GABA production in L. plantarum but not in L. brevis, B. angulatum, or B. adolescentis 42 .Recent research suggests that the Bifidobacteriaceae family, together with Streptococcaceae, is associated with a higher abundance of fecal GABA in healthy individuals with no systemic or psychiatric illnesses 45 .This high abundance of Bifidobacterium is beneficial, especially when alpha diversity in the gut is low (associated with specific diseases) because it restores microbial diversity 46 .The key player responsible for the high abundance of GABA in feces is Bifidobacterium adolescentis, which was recently recognized as a gut microorganism involved in GABA production 44,45 .B. adolescentis 150, B. adolescentis PRL2019, and B. adolescentis HD17T2H were bifidobacterial strains that are efficient GABA producers 43,44 .A recent study suggested that some Bifidobacterium adolescentis may represent a GABA producer model due to their performance in vitro and in vivo 44 .These B. adolescentis strains were identified as B. adolescentis PRL2019 and B. adolescentis HD17T2H, which can produce 7.1 mM and 9.4 mM of GABA, respectively 44 .By de novo genome assembly, B. angulatum GT102 and B. adolescentis 150 strains contain gadB gene encoding glutamate decarboxylase, gadС gene encoding putative glutamate/gamma-aminobutyrate antiporter, and gene encoding monoamine oxidase involved in the catabolism of monoamines 47 .In addition to their GABA-producing ability, both L. plantarum 90sk and B. adolescentis 150 exhibit antibiotic resistance and antioxidant properties 43 .
In addition to Bacteroides, Lactobacillus, and Bifidobacterium, a recent study demonstrated that Lactococcus, a genus of lactic acid bacteria, can produce GABA 48 .Lactococcus garvieae MJF010 was found to be the most efficient GABA producer among 23 lactic acid bacteria strains isolated from healthy human feces 48 .The GAD enzyme of L. garvieae MJF010 showed the highest GABA-producing activity at 35 °C and pH 5, whereas exogenous PLP addition had no effect 48 .
These studies provide compelling evidence that human gut microbiota is capable of producing GABA and may play a role in mediating gut and host health.Research focusing on other gut microorganisms is of great importance to further understand their critical roles in the gastrointestinal tract.Moreover, commensal probiotic strains in the human gut can be considered delivery vehicles for GABA in specific regions of the gut 49 .

GABA-producing microorganisms isolated from foods
Extensive studies have been conducted to develop GABA-rich food supplements 50 and fermented foods 51 that leverage many health benefits of GABA 52 .Recently, GABA production has been focused on seeking highly productive GABA strains and optimizing the growth conditions of these bacteria 53 .In Japan, the food industry is mainly interested in GABA production because it is considered a bioactive compound that promotes health and can be leveraged in the development of foods for specific health use (FOSHU) 54 .
Fermenting vegetables, meat, and fruits using lactic acid bacteria (LAB) is a standard method for preserving and improving the dietary and sensory characteristics of food commodities 55 .The complex nutritional substances of food commodities are a rich source of vitamins and minerals necessary for the growth of LAB strains, which facilitate the microbial production of enzymes and other metabolites 56 .LAB efficiently and rapidly converts sugars to lactic acid as a primary metabolic product, contributing to the preservation of fermented foods.Many of these raw materials contain significant amounts of glutamate, which can be utilized by LAB to convert glutamate to GABA using the GAD enzyme to increase tolerance to acidic conditions 57 .Several GABA-producing LAB have been isolated from a wide range of fermented foods (Table 2).
Recently, microorganisms belonging to the genera Lactococcus, Lactobacillus, Leuconostoc, and Kluyveromyces were identified and isolated from Mexican milk kefir grains and showed good probiotic properties through aggregation abilities, antimicrobial activity, antibiotic susceptibility, and resistance to in vitro gastrointestinal digestion, comparable to commercial probiotics 72 .Specific isolates of Kluyveromyces (BIOTEC009 and BIOTEC010), Leuconostoc (BIOTEC011 and BIOTEC012), and Lactobacillus (BIOTEC014 and BIOTEC15) exhibited high fermentability in media supplemented with commercial prebiotics 72 .The capacity to produce GABA was classified as a medium-level GABA producer for L. lactis BIO-TEC006, BIOTEC007, BIOTEC008, K. lactis BIOTEC009, L. pseudomesenteroides BIOTEC012, and L. kefiri BIOTEC014 and was comparable to that obtained for commercial probiotics 72 .The classification system for GABA production by microorganisms was adapted from Tsukatani et al.  (2005): less than 0.5 mM was considered a low-level GABA-producer, 0.5-2.1 mM was a medium-level GABA-producer, and more than 2.1 mM was a high-level GABA-producer 73 .Moreover, Saccharomyces cerevisiae SC125 and Lactobacillus plantarum BC114, both isolated and identified from traditional Chinese paocai, yielded 23.5 mM GABA as a co-culture that promotes the production of flavor compounds and GABA in mulberry beverage brewing 74 .
Despite the great diversity of fermented food products available worldwide as products of various cultures and traditions, little is known about the microorganisms involved in the fermentation process.There may be undiscovered microorganisms in traditional fermented products that are https://doi.org/10.1038/s41538-024-00253-2more efficient producers of GABA and other compounds than those previously identified and documented.Moreover, commercially available fermented products, such as kimchi, provide more data and information on the microorganisms involved in the fermentation process.Hence, a focus on other traditional fermented products is necessary to help diversify the information on fermentation and the potential of fermenting microorganisms to produce GABA and other beneficial metabolites.

Why do microorganisms produce GABA?
It is common knowledge in this research field that bacteria, especially those with probiotic properties, can produce GABA because of their ability to express GAD genes.However, the reason by which these bacteria produce GABA remains unclear.It has been hypothesized that GABA is produced under anaerobic and acidic conditions, allowing bacteria to survive in extreme environments.As shown in Fig. 3, glycolysis takes place in the cytosol under anaerobic conditions, where two molecules each of nicotinamide adenine dinucleotide (NAD + ) and ADP are required to convert a glucose molecule into two molecules each of pyruvate nicotinamide adenine dinucleotide + hydrogen (NADH), and ATP 75 .Next, pyruvate is converted into fermentation products such as lactate, ethanol, and organic compounds, including acetate, butyrate, and propionate, in which two NAD + molecules are generated, fed back, and reutilized in the glycolysis process 75 .
The production of lactate and other acids by bacteria during fermentation lowers the pH, which leads to bacteria utilizing the GAD gene system 76 .The GAD gene system, consisting of gadB, glutaminase, gadC (glutamate/GABA antiporter), and K + channels, helps bacteria cope with changes in intracellular pH 36 .Activation of the GAD gene system owing to a decline in pH then triggers the production of GABA 76,77 .As shown in Fig. 3, free glutamate in the environment is transported into the cell by a specific transporter (glutamate/GABA antiporter), which leads to the decarboxylation of glutamate to GABA by GAD, in which intracellular H + ions are consumed 36,76,78 .GABA is then exported from the cell via the antiporter, which results in an increase in the pH of the cytoplasm due to the removal of H + ions and a slight increase in the extracellular pH due to the exchange of extracellular glutamate for more alkaline GABA 76,78 .Hence, the release of GABA helps bacteria cope with acid stress, which is crucial for the colonization of the GI tract and survival in acidic fermentation environments.

Gut-GABA-production inducing food factors
In addition to fermented food products that promote GABA production owing to the presence of GABA-producing bacteria, several researchers have explored the potential of other food factors that can induce GABA production in the gut.As mentioned above, probiotics, Bifidobacterium and Lactobacillus, and the predominant gut bacteria Bacteroides are the main GABA producers in the human gut, and food factors that can enhance the abundance of these gut bacteria are candidates for GABA productioninducing food factors.In addition to typical well-known prebiotics, such as fructooligosaccharides (FOS), emerging research suggests that Under anaerobic conditions, glycolysis takes place in the cytosol, where NAD + and ADP are required to convert glucose into pyruvate, in which NADH and ATP are produced from the process 75 .Pyruvate is then converted into lactate or other organic compounds, such as acetate, butyrate, and propionate, where NADH is utilized, and NAD + is generated in the process.Then, NAD + is fed back and reutilized in the glycolysis process 75 .The acidic fermentation by-products, lactate, and other organic compounds lower the pH, which leads bacteria to utilize the GAD gene system and triggers the production of GABA 76,77 .To produce GABA, exogenous glutamate is transported into the cell by a glutamate/GABA antiporter, then glutamate is converted into GABA by glutamic acid decarboxylase (GAD) 36,76,78 .Then, GABA is exported from the cell via the antiporter, resulting in an increase in the pH of the cytoplasm due to the removal of H + ions and a slight increase in the extracellular pH due to the exchange of extracellular glutamate for more alkaline GABA 76,78 .This figure was created using Biorender.com.microorganism-derived enzymes, such as proteases, lipases, amylases, and cellulases, have the potential to act as prebiotics to increase probiotics in the gut [79][80][81][82] .Recent studies have shown that dietary Aspergillus oryzae-derived protease markedly increases the abundance of both Bifidobacterium and Lactobacillus in the rat cecum and induces the production of various postbiotics, including GABA, which was not detected in the cecum of rats receiving no dietary protease 80,81 .Taken together with the fact that GABA is a non-proteinogenic amino acid, these findings suggest that GABA was possibly produced from elevated levels of the probiotics Bifidobacterium and Lactobacillus.More recent studies have revealed that other dietary factors, such as lipase from Penicillium camemberti, which is generally used in cheese production, also induce an increased abundance of Bifidobacterium and Lactobacillus in the rat cecum 83 .These studies imply that the digestive enzymes produced by Aspergillus and Penicillium exert prebiotic-like effects by increasing the abundance of the GABA-producing probiotics Bifidobacterium and Lactobacillus, in the gut, possibly making them efficient in GABA production.The same is true for inulin, which stimulates GABA production in the gut 84 .More work is needed to identify and investigate other food factors that have the potential to induce GABA production in the gut or brain, regardless of whether they are microorganism-derived or naturally derived.

Gaba as a mediator of the gut-brain axis
Association of gut microbiota and GABA in mental health and brain function It has been well-accepted that dysbiosis of the gut microbiota is strongly linked to human health, including mental health.Gut microbiota and probiotics impact host health through various mechanisms, including the production of metabolites, recently defined as postbiotics, such as short-chain fatty acids, peptides, and amino acids.Among these postbiotics, GABA has received much attention from researchers owing to its essential role in the nervous system and its strong correlation with the gut microbiota.Studies have suggested that peripheral or circulating GABA is mainly attributed to the gut microbiota 40,85,86 .In germ-free mice, blood (3.7 times) and fecal (1.3 times) GABA levels were lower than those in fecal-oral-inoculated germ-free mice 85 .
Another study showed that cecal GABA levels were markedly decreased in mice treated with vancomycin 86 .Oral administration of GABA-producing B. dentium ATCC 27678, but not non-GABA-producing B. breve NCIMB8807, increased cecal GABA levels and reduced colon-specific sensory neuron excitability, which are the general causes of abdominal pain 38 .Taken together, these studies indicate the potential role of GABA as a moderator in the gut-brain axis.The following section presents recent information regarding the involvement of the gut microbiota and GABA in mental health and brain diseases.A summary of this interaction is shown in Table 3.

Gut microbiota and GABA in neurological disorders
Neurological disorders such as schizophrenia (SCZ), Alzheimer's disease (AD), and Parkinson's disease (PD) have been linked to dysbiosis because of the strong connection between the gut and brain 87 .A recent study revealed that treated and non-treated SCZ patients had a decreased microbiome diversity index compared to healthy controls, where an increased abundance of Veillonellaceae, Prevotellaceae, Bacteroidaceae, Coriobacteriaceae and a decreased abundance of Lachnospiraceae, Ruminococceae, and Norank were found in SCZ patients 88 .Additionally, a lower abundance of Bacteroides and Streptococcus in the gut microbiota is a feature of SCZ, and these bacteria are associated with glutamate and GABA metabolism 89 .Furthermore, germ-free mice receiving the SCZ microbiome showed decreased glutamate but increased GABA levels in the hippocampus, displaying SCZ-relevant behaviors similar to other mouse models of SCZ involving glutamatergic hypofunction 88 .In AD, the fecal microbial composition and metabolic output were evident.Patients with AD had an increased abundance of Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Atopobiaceae, Clostridiales, Synergistaceae, Erysipelotrichaceae, and Pseudomonadaceae and a decreased abundance of Lachnospiraceae (genus Tyzzerella) and Erysipelotrichaceae (genus Erysipelatoclostridium) compared to normal controls, and these microorganisms were significantly associated with a decreased abundance of N-docosahexaenoyl GABA, 19oxoandrost-4-ene-3,17-dione, trigofoenoside F, and 22angeloylbarringtogenol C metabolites 90 .Bidirectional Mendelian randomization analysis has revealed a causal relationship between the relative abundance of Blautia, a new functional genus with potential probiotic properties 91 , and AD 92 .Elevated levels of GABA, a downstream product of Blautia-dependent arginine metabolism, in the cerebrospinal fluid (CSF) are related to a reduced risk of AD 92,93 .Patients with PD had a significant increase in Akkermansia and a decrease in Lactobacillus compared to healthy controls 94 .The differences in postural instability gait difficulty (PIGD) and tremor-dominant (TD) PD motor subtypes in basal ganglia GABA levels could be lower in TD than in PIGD, which may indicate a difference in the pathophysiological mechanisms of TD and PIGD 95 .In addition, treatment with Pediococcus pentosaceus improved the gut microbial dysbiosis and increased GABA levels in methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)-induced PD 96 .
Gut microbiota and GABA in anxiety, depression, and stress Recently, altered gut microbiota and reduced function of the GABA system in the prefrontal cortex following chronic ethanol exposure led to anxietylike behaviors 97 .Administration of Lactobacillus rhamnosus JB-1 improved stress-induced anxiety-and depression-like behaviors in mice by increasing GABA mRNA expression in the hippocampus 10 .Increased small intestine GABA level (0.03-0.04 mM) of metabolic syndrome mice model fed with diet incorporated with Lactobacillus brevis DPC6108 and DSM32386 strains improved depression-like behavior in the forced swim test and resting stress hormone corticosterone level compared to high-fat control diet 18 .Metagenomics-based analyses involving datasets collected from children with subclinical symptoms of depression and anxiety revealed high metagenomic reads of gad in groups with a high abundance of Bifidobacterium adolescentis 44 .Furthermore, the depressed phenotype had a greater prevalence of GABA-consuming microorganisms in the selected strains of Flavonifractor plautii, Pseudomonas spp., and Acinetobacter spp.then the healthy phenotype, thereby favoring GABA degradation 98 .Moreover, a decreased abundance of Bacteroides eggerthii was found to be associated with a decrease in GABA synthesis in subjects with stress and anxiety, and gut microbiota modulation through probiotic supplementation enriched GABA-synthesizing Bifidobacterium adolescentis and Bifidobacterium longum that alleviated stress-and anxiety-related symptoms 99 .

Gut microbiota and GABA in epilepsy
An imbalance in neuroactive compounds, including GABA, and intestinal dysbiosis are two important considerations in epilepsy 100,101 and are commonly observed in humans and dogs 102 .In humans, it was found that patients with four or fewer seizures per year had higher fecal Bifidobacteria and Lactobacilli than those who had more than four seizures 103 .These flora promote GABA synthesis 36,37 .In dogs, the epileptic group had a significantly reduced abundance of fecal GABA-producing (Pseudomonadales, Pseudomonadaceae, Pseudomonas, and Pseudomona_graminis) and SCFAproducing bacteria (Peptococcaceae, Ruminococcaceae and Anaerotruncus), as well as bacteria associated with a reduced risk of brain disease (Prevotellaceae) compared to the control group 102 .Despite difficulties with implementation, dietary compliance, and adverse side effects, a ketogenic diet (or low-carbohydrate, high-fat diet; KD) 104 is an effective dietary intervention to treat epilepsy.KD positively altered the gut microbiota by increasing the abundance of Akkermansia muciniphila and Parabacteriodes from 4 to 14 days of treatment, demonstrating an anti-seizure effect in a wide-range anti-epileptic drug-resistant seizure model 105 .Administration of the KD paired with Akkermansia muciniphila and Parabacteriodes significantly increased hippocampal GABA/glutamate ratios 105 .Probiotic administration (several Lactobacillus, Bifidobacterium, and Streptococcus strains) to drug-resistant epileptic (DRE) patients decreased the number of seizure occurrences and increased the serum GABA concentration after a 12-week treatment 106 .Gut microbiota and GABA in autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) Occurrence of high gut Clostridium spp. is associated with ASD in patients with gastrointestinal disease 107 .Specifically, 76-87% of beta2-toxin-producing Clostridium perfringens were significantly higher in children with ASD compared to control children, indicating that these opportunistic pathogens thrive in immature or compromised immune systems 107 .A recent study has shown that infants with increased-likelihood of ASD have a decreased abundance of Bifidobacterium but an increased abundance of Clostridium and Klebsiella compared to those with lower likelihood of ASD 108 .Moreover, fecal GABA levels of infants with increased likelihood of ASD were lower than those with lower likelihood of ASD, in which fecal GABA levels are positively correlated with Bifidobacterium, but negatively correlated with Clostridium 108 .A lower abundance of Prevotella copri, Feacalibacterium prausnitzii, and Haemophilus parainfluenzae and decreased concentrations of fecal GABA  were found in children with ADS when compared to healthy children 109 .In contrast, an increased ratio of fecal GABA/glutamate with a higher abundance in Escherichia/Shigella and a lower abundance of Bacteroides was found in mild ADS children than in healthy children 110 .Dialister, Escherichia/Shigella, and Bifidobacterium were more abundant in ASD children, while Prevotella 9, Megamonas, and Ruminococcus 2 were more abundant in healthy children, in which GABA precursors, such as N-carboxyethyl-gaminobutyric acid, glutamylproline, pyroglutamic acid, and gamma-glutamylglycine, were higher in ASD children 111 .
In ADHD, magnetic resonance spectroscopy revealed a significant reduction in brain GABA concentration in children with ADHD 112 .In contrast, increased cortical GABA concentration was observed in adults with ADHD, which suggests that GABA levels may be correlated with the age of patients with ADHD 113 .A recent study has shown that the top five depleted bacteria families in infants (6 months of age) with ADHD are Lachnospiraceae, Ruminococcus, Bacteroides, Lachnospiraceae, and Enterococcus, while the top five enriched bacteria families are Bacteroides, Dorea, Erysipelotrichaceae, Ruminococcaceae, and Dialister 114 .Interestingly, 50% of the depleted families belong to the Lactobacillales order, or lactic acid bacteria 114 .Due to the fact that lactic acid bacteria are strong GABA producers, this can suggest that the depletion of lactic acid bacteria in the gut of infants with ADHD might be related to a decrease in GABA.On the other hand, in a case study, an adult with ADHD was found to have a high abundance of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longum, Bacteroides ovatus, Bacteroides uniformis, Fusobacterium ulcerans, Enterocococcus avium, and Enterococcus gallinarumi, but fecal microbiota transplant significantly reduced the abundance of those bacteria with the relief of ADHD symptoms 115 .Since most of those bacteria (Bifidobacterium, Bacteroides, and Enterocococcus) are well-known GABA producers 37 , this may support a positive correlation of GABA with ADHD in adults as previously reported 113 .It seems likely that GABA may play a role in the pathogenesis of ADHD in children and adults, but possibly in different ways.
Brain-specific GABA-containing peptide Homocarnosine Homocarnosine (GABA-L-histidine) is a GABA-containing dipeptide that is predominantly found in the brain 116 .It is an analog of the predominant muscle dipeptide carnosine (β-alanine-L-histidine). Homocarnosine is synthesized from GABA and histidine by carnosine synthase in neurons and is degraded by carnosinase 117,118 .The occipital cortex, basal ganglia, and cervical cord have the highest human homocarnosine synthetase, currently known as carnosine synthase, activity while the cerebellar cortex has the lowest 117 .It is present in greater amounts in the human brain than in the brains of other mammals 119 .Homocarnosine concentration of the autopsied brain ranges from 0.4 mmol/kg in the corpus callosum and temporal cortex to 1.0 mmol/kg in the thalamus and basal ganglia and varies independently of GABA concentrations 120 .The homocarnosine concentration is threefold to sixfold higher in adults than in infants 121 .Recently, areas of the human central nervous system, particularly the olfactory bulb, spinal cord, medulla oblongata, thalamus, cerebellum, white matter, and frontal cortex, had a considerable amount of homocarnosine while the human CSF abundantly contains homocarnosine 122 .Although its concentration in the brain is high, the function of homocarnosine in the brain remains underexplored, which has led to a limited understanding of its high maintained concentrations in the brain.However, several biochemical properties of homocarnosine have been reported.
For instance, homocarnosine acts as a protective agent against a wide range of disease conditions, including the protection of brain endothelial cells from amyloid peptide-induced toxicity 123 and anti-inflammatory action in brain ischemic injuries 124 .Homocarnosine demonstrates similar properties to carnosine in protecting Cu and Zn superoxide dismutase from oxidative damage through a combination of copper chelation and peroxyl radical scavenging 125 .Moreover, homocarnosine, in combination with carnosine and anserine, reduces oxidative damage by decreasing lipid peroxidation and increasing antioxidant levels in the brain 126 .Many studies have explored the biological role of homocarnosine in the brain and other neurological diseases.Hence, a thorough investigation is required to better understand the role of homocarnosine.
Because homocarnosine is a GABA-containing peptide, changes in GABA levels may contribute to changes in homocarnosine homeostasis.This hypothesis is supported by the notion that homocarnosine is a possible GABA reservoir, as approximately 40% of GABA measured in human CSF is homocarnosine 127 .In addition, it has been hypothesized that the release of homocarnosine contributes to glutamate-GABA cycling and reflects an adaptive response to excess extracellular glutamate 128 , wherein a strong linear correlation between GABA and homocarnosine concentrations has been observed in healthy CSF (GABA concentration is higher than homocarnosine concentration) 129 .
Possible link of gut-brain axis and GABA-homocarnosine in brain-related diseases Homocarnosine homeostasis in the brain plays a critical role in clinical studies of neurological diseases, such as Alzheimer's disease and epilepsy 130,131 .Low homocarnosine levels may reflect decreased fractional volumes of homocarnosine-containing neurons, and homocarnosine deficits may indicate either the loss or dysfunction of GABAergic neurons 128,130 .Drugs may be administered to improve homocarnosine levels in the brain.Vigabatrin and gabapentin, known antiepileptic drugs, improve seizures by increasing levels of brain GABA and homocarnosine 131,132 .Topiramate, another anti-seizure drug, improves brain homocarnosine and GABA levels, contributing to its potent anti-epileptic action in patients with complex partial seizures 127 .Moreover, isoniazid supplementation in healthy patients elevates homocarnosine and GABA concentrations 133 .As mentioned above, homocarnosine could possibly be a good reservoir of GABA in the brain, and other neurological disorders, such as AD, ASD, and SCZ, can be associated with homocarnosine as they are characterized by low GABA levels, and GABA can induce homocarnosine production 35,88,92,134 .
It is worth mentioning that the above-mentioned neurological disorders alter gut GABA-producing microorganisms that affect GABA homeostasis in the gut and brain.An increase in the abundance of the wellknown probiotics Lactobacillus and Bifidobacterium induces gut GABA production 79,80 .However, to date, the direct interaction and correlation between homocarnosine and the gut microbiota as affected by diet remain unknown.Recently (unpublished data), our group discovered the ability of Aspergillus-derived enzymes together with FOS to exhibit a tendency to increase cecal and brain GABA levels.Moreover, the dietary intake of these prebiotic-linked enzymes and FOS increases homocarnosine levels in the brain.These findings indicate that dietary factors may act as one of the modulators of GABA and homocarnosine levels in the gut and brain.
To summarize, GABA has long been the subject of rigorous research, and its health benefits have been proven through in vitro and in vivo experiments.Although circulating GABA has long been believed to not cross the BBB, GABA's permeability through the BBB remains contested due to conflicting evidence.Recent research has demonstrated that GABA can be a potent mediator of the gut-brain axis, as it is circulating and brain levels are regulated by the microbiota, and that changes in GABA levels and microbiota composition play a role in modulating mental health.Generally, GABA is present at trace concentrations in the bloodstream.Recent studies have suggested that circulating GABA is mainly attributed to gut microbiota.Several studies have isolated GABA-producing bacteria from the human gut, such as Lactobacillus, Bifidobacterium, and Bacteroides, and from fermented foods, such as Lactobacillus, Streptococcus, Leuconostoc, and Weisella.In addition to probiotics, non-typical prebiotic food factors, such as Aspergillus-and Penicillium-derived enzymes, have been demonstrated to stimulate an increased abundance of probiotics and gut GABA production.Supplementation with probiotics and probiotic-rich products improves the cognitive function of patients with neurological disorders; eases anxiety, depression, and stress; and increases circulating and brain GABA availability.In addition to GABA, a predominant GABA-containing brain peptide, homocarnosine, has recently been demonstrated to be a possible downstream mediator of GABA in the gut-brain axis.Currently, there is limited information regarding the connections between homocarnosine, gut microbiota, and brain function.Thus, it is of great importance to further investigate this issue because this information may help clarify how the gut microbiota and GABA-homocarnosine metabolism play a role in brain function.This information will contribute to the development of functional foods and mental health interventions.

Fig. 3 |
Fig.3| Mechanism of GABA production in microorganisms.Under anaerobic and acidic conditions in the human gut and fermentation, it appears that bacteria produce GABA for their own survival purposes under these extreme environments.Under anaerobic conditions, glycolysis takes place in the cytosol, where NAD + and ADP are required to convert glucose into pyruvate, in which NADH and ATP are produced from the process75 .Pyruvate is then converted into lactate or other organic compounds, such as acetate, butyrate, and propionate, where NADH is utilized, and NAD + is generated in the process.Then, NAD + is fed back and reutilized in the glycolysis process75 .The acidic fermentation by-products, lactate, and other organic https://doi.org/10.1038/s41538-024-00253-2Review article npj Science of Food | (2024) 8:16 https://doi.org/10.1038/s41538-024-00253-2Review article npj Science of Food | (2024) 8:16

Table 1 |
GABA-producing microorganisms isolated from the human gut Microorganism Characteristic ReferencesLactobacillus brevis DPC6108 Converts 10 and 20 mg/ml of MSG to GABA at 100% conversion rate 41 Lactobacillus brevis 15 f Bifidobacterium angulatum GT102 Efficient GABA producer 42 Bifidobacterium adolescentis 150 Possess antibiotic-resistant and antioxidant activity 43 Lactobacillus plantarum 90sk GABA production is affected PLP addition; possesses antibiotic-resistant and antioxidant activity 37 Bacteroides spp.Regulation of the GABAergic system in the human gut 36 Bifidobacterium adolescentis PRL2019 Bifidobacterium adolescentis HD17T2H In vivo production of GABA with potential implication in gut-brain axis interactions 44

Table 3 |
Summary of the association between gut microbiota, GABA, and brain diseases