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1		Combining Lac^plan^bacillus plantarum and Bifidobacterium adolescen^s can
2		improve GABA produc^on in faecal fermenta^ons.
3		Rihua Xu1,2, Melinda J. Mayer2*, Mark Philo3, Gwénaëlle Le Gall4, Guesh Mulaw5, Alise
4		Ponsero2, Arjan Narbad2
5
6		1School of Life Science, Inner Mongolia University, Hohhot 010070, China
7		2Food, Microbiome and Health Ins^tute Strategic Programme, Quadram Ins^tute Bioscience,
8		Norwich NR4 7UQ, UK
9		3Core Science Resources, Quadram Ins^tute Bioscience, Norwich NR4 7UQ, UK
10		4Norwich Medical School, University of East Anglia, Norwich NR4 7UQ, UK
11		5College of Natural and Computa^onal Sciences, Aksum University, Aksum 1010, Ethiopia
12
13		*corresponding author Dr Melinda Mayer, Quadram Ins^tute Bioscience, Rosamund Franklin
14		Road, Norwich Research Park, Colney, Norwich NR4 7UQ, UK melinda.mayer@quadram.ac.uk.
15
16		Running ^tle GABA produc^on by beneficial bacteria

© The Author(s) 2026. Published by Oxford University Press on behalf of Applied Microbiology International. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

18 Abstract
19 Aims This project aimed to inves^gate produc^on of the inhibitory neurotransmi^er γ-
20	aminobutyric acid (GABA) from poten^al probio^c strains. We studied produc^on in co-
21	cultures and faecal fermenta^ons and examined the effect of selected strains on the faecal
22 microbiome composi^on and metabolome in vitro.
23 Methods and results Strains of intes^nally-derived Bifidobacterium adolescen^s and
24 Lac^plan^bacillus plantarum from fermented cereals were grown singly, in co-culture and in
25 faecal fermenta^ons designed to simulate colonic condi^ons.
26		Isolates synthesised varing amounts of GABA in vitro; GABA produc^on could be increased by
27		co-culture, lac^c acid or reduced pH but was decreased in the presence of high buffering. In
28		faecal fermenta^ons, selected strains inoculated singly or in combina^on persisted over 24
29		hours and increased the GABA concentra^on without causing major disrup^ons in the
30		microbiome or metabolome. B. adolescen^s supplementa^on increased short chain fa^y
31		acids acetate and propionate and L. plantarum was associated with increased succinate levels,
32 while all treatments exhibited a reduc^on in Escherichia compared to the controls.
33 Conclusions GABA produc^on from these lac^c acid bacteria is strain-specific and the
34 combina^on of these two species shows poten^al for future next-genera^on probio^c
35 development.
Impact Statement
37 Bacteria derived from the human gut or fermented foods can produce GABA in vitro in the
38 context of a gut microbiome without causing significant disrup^on to its composi^on and

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39 func^on.

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41 Keywords: Gamma-aminobutyric acid, Bifidobacterium, Lac^plan^bacillus, psychobio^c,
42 faecal fermenta^on, metagenome, metabolome.
43
44 Introduc^on
45		Microbes hold a vast poten^al to improve human health, producing beneficial chemicals and
46		nutrients in fermented foods, being harnessed for produc^on of pharmaceu^cals, or being
47		u^lised in vivo as probio^cs. There is increasing interest in mining the gut microbiome and
48		fermented foods for beneficial bacteria which can persist and func^on in the human gut to
49		improve health. The benefits of fermented foods can also be increased by using bacteria which
50		produce valuable micronutrients or bioac^ves, such as GABA (Ko et al. 2013, Yu et al. 2020,
51		Kaur et al. 2023, Tames et al. 2023). Microbes which produce neurotransmi^ers may present
52		an opportunity to combat brain disease or psychological condi^ons, used either as probio^cs
53		(Sarkar et al. 2016, Gholian et al. 2024), or in the produc^on of func^onal fermented foods
54 (Diez-Gu^errez et al. 2019).
55		The composi^on and func^on of the gut microbiome has far-reaching effects on human
56		health, influencing condi^ons including Parkinson’s Disease, ageing, and depression (Valles-
57		Colomer et al. 2019, Romano et al. 2021, Parker et al. 2022). Many members of the gut
58		microbiome can both produce and consume neurotransmi^ers, poten^ally affec^ng host
59		physiology and behaviour and forming a key part of the gut-brain axis (Sarkar et al. 2016,
60		Strandwitz 2018, Chen et al. 2021). Faecal microbiota transplanta^on has demonstrated that
61		the microbiome can have a profound effect on both the gut and the central nervous system

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62 (Parker et al. 2022), and affect plasma GABA levels (Koo^e et al. 2017). GABA is the main
63 inhibitory neurotransmi^er in the brain, regula^ng many physiological and psychological
64 processes, including sleep-wake cycle regula^on, motor ac^vity, memory func^on, cogni^on
65 and inflamma^on (Bhat et al. 2010, Strandwitz 2018, Cui et al. 2020). GABA and its receptors
66 play an essen^al role in the pathogenesis of human neurological disorders (Sarkar et al. 2016,
67 Chen et al. 2021). Many gut microbes have the capacity to produce GABA (Yunes et al. 2016,
68 Pokusaeva et al. 2017, Strandwitz et al. 2019, Otaru et al. 2021). In many bacteria, the use of
69 protons to generate GABA from glutamate is a key mediator of acid tolerance, and the
70 glutamate decarboxylase (GAD) system has also been linked to responses to other stresses
71 (Feehily and Karatzas 2013, Otaru et al. 2021).
72 Several reports indicate that neurochemical modula^on by bacteria within the gut can
73 influence behaviour in both humans and animal model systems (Strandwitz 2018, Strandwitz
74 et al. 2019, Valles-Colomer et al. 2019, Averina et al. 2020). Although GABA is reported to be 75 unable to cross the blood-brain barrier (Chen et al. 2021), it can s^ll have effects on the
76 microbiome (Strandwitz et al. 2019, Yu et al. 2020). In addi^on, a link between probio^c 77 Lac^caseibacillus rhamnosus applica^on and GABA receptor expression in the brain has been
78 demonstrated in mice (Bravo et al. 2011).
79 Psychobio^cs include probio^cs and prebio^cs that have posi^ve psychiatric effects (Sarkar 80 et al. 2016). Many researchers have focused on beneficial bacteria such as lac^c acid bacteria 81 (LAB) for psychobio^c discovery, and in vivo tests have demonstrated their ability to modulate 82 behaviour (Messaoudi et al. 2011, Sarkar et al. 2016, Luang-In et al. 2020). GABA produc^on 83 has been reported from intes^nal strains of Lactobacilli and Bifidobacteria and species from 84 the genera Lactococcus, Streptococcus, Enterococcus and Leuconostoc (Yunes et al. 2016, Cui

85 et al. 2020, Laroute et al. 2022) Fermented foods can also be a good source of producers
86 (Raethong et al. 2022). GABA is synthesized by LAB with the enzyme GAD (EC 4.1.1.15) via
87 decarboxyla^on of L-glutamate; bacterial glutamate import and GABA export generally occurs
88 simultaneously via a specific glutamate/GABA an^porter (Yunes et al. 2016, Diez-Gu^errez et
89 al. 2019).
90 GABA-producing probio^cs have been shown to have behavioural and physiological effects 91 in animal models (Pokusaeva et al. 2017, Pa^erson et al. 2019, Laroute et al. 2022). Similarly, 92 inges^on of foods enriched with GABA via microbial fermenta^on has demonstrated 93 beneficial effects on states such as depressive behaviour, insomnia or hypertension (Hayakawa 94 et al. 2004, Ko et al. 2013, Diez-Gu^errez et al. 2019, Yu et al. 2020). These results provide 95 evidence that microbial endocrinology plays a role in microbiota-gut-brain interac^ons and 96 changes in behaviour (Ko et al. 2013). In addi^on to the posi^ve effects on the host, GABA- 97 producing probio^cs can also modulate the gut microbiome (Yu et al. 2020, De Oliveira et al. 98 2023, Kaur et al. 2023, Casertano et al. 2024, Tames et al. 2024). It is important to understand 99 not only what levels are produced by probio^cs in situ, but what effect they have on the na^ve 100 composi^on of the microbiome.
101 In previous analyses (unpublished work), GAD genes were iden^fied in a large number of
102 strains in the Quadram Ins^tute Bioscience culture collec^on. The aim of our work was to
103 inves^gate GABA produc^on from selected strains, isolated from the human gut and
104 fermented foods, to determine their produc^on in condi^ons simula^ng the colon and to

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105 study their effect on the faecal microbiome composi^on and metabolome in vitro.

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108	Materials and Methods
109	Strains and growth condi^ons
110	Strains which carried glutamate decarboxylase genes (EC 4.1.1.15) were selected from the
111	Quadram Ins^tute Bioscience culture collec^on: 7 Bifidobacterium adolescen^s strains
112	isolated from healthy human faeces and 7 strains of Lac^plan^bacillus plantarum isolated
113	from African fermented foods (Table 1). Lac^plan^bacillus pentosus DSM20314 was from the
114	Deutsche Sammlung von Mikroorganismen und Zellculturen (DSMZ), Braunschweig. B.
115	adolescen^s was grown in BHI+C (Brain heart infusion (Oxoid) with complements (0.5% v/v
116	vitamin K solu^on in ethanol, resazurin (1 mg l-1), hemin (5 mg l-1) and L-cysteine hydrochloride
117	(0.5 g l-1) in pre-reduced media in an anaerobic cabinet (Don Whitley, UK) at 37oC with an
118	atmosphere of 5% CO2, 10% H2 in N2; Lactobacilli were grown aerobically in MRS (de Man,
119 Rogosa, Sharp, Oxoid) at 37oC.
120 Screening in nutrient and colon model media
121 All strains were sub-cultured twice in their preferred media and condi^ons, then inoculated 122 at 2% (v/v) into fresh media with 2% monosodium glutamate (MSG, Merck). Growth in
123 nutrient media (BHI+C or MRS broth) was tested a^er 24, 48, and 72 h using separate bo^les 124	for each ^me point. Growth in colon model medium (CMM, peptone water 2 g l-1, yeast extract
125	2g l-1, NaHCO3 2g l-1, NaCl 0.1g l-1, K2HPO4 0.04 g l-1, KH2PO4 0.04 g l-1, MgSO4.7H2O 0.01 g l-1,
126	CaCl2.6H2O 0.01 g l-1, Tween 80 2 ml, hemin 0.02 g l-1, D-glucose 2%, vitamin K1 10 μl, cysteine
127	HCl 0.5 g l-1 and bile salts 0.5 g l-1, pH 6.0 (Mandalari et al. 2007) or high buffer colon model
128 medium (HBCM adapted from CMM to increase buffering capacity (Peck 2024), peptone water

129

-1, Na2HPO4 0.04 g l-1 l-1, MgSO4.7H2O 0.01 g l-1, CaCl2.6H2O 0.01 g l-1, Tween 80 2 ml, hemin 0.02 g l-1, vitamin K1 10 μl, cysteine HCl 0.5 g l-1 and bile salts 0.5 g l-1, pH 6.8, supplemented with 1% D-glucose) was performed anaerobically for 24 h with 2% MSG. Chemicals were obtained from Merck unless stated otherwise. The cell free supernatants (CFS) were collected by centrifuga^on at 1699 x
g for 10 min at 4oC followed by filter steriliza^on with 0.22 µm filters (Millipore) into sterile
glass bo^les.
GABA produc^on in co-culture and effect of pH
Strains were first grown anaerobically in single culture in their preferred medium using a 2% inoculum, measuring both op^cal density (OD600) with an Infinite F50 microplate reader (Tecan), and viable counts, by pla^ng in triplicate 10 μl spots of 10-fold dilu^ons in PBS on the appropriate solid media. Replicate experiments (n=2) were used to es^mate the ^me cultures
took to reach mid exponen^al phase and the average cell count at this ^me. Strains at mid exponen^al phase (B. adolescen^s OD600 0.35-0.5, L. plantarum OD600 0.5-0.6) were adjusted to c. 1x107 cfu ml-1, inoculated at 2% each and co-cultured anaerobically in a 1:1 v/v mix of BHI+C:MRS for 72 h. Viable counts were taken on aerobic MRS plates (L. plantarum) or anaerobic BHI (B. adolescen^s) and GABA produc^on measured by LC-MS. To assess the effect of pH, B. adolescen^s QI0300 was grown in 1:1 BHI+C:MRS mixed media with the ini^al pH adjusted to 6.0, 5.5, 5.0, 4.5, 4.0 or 3.5 using 3 M HCl, or in co-culture with LAB, or supplemented with either 175 mM D/L lac^c acid or an equal volume of CFS from overnight

149

cultures

grown without MSG. GABA yield, viable counts and final pH were measured

150

as below a^er 24 h.

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153 Serum bo^le faecal fermenta^ons
154	Faecal samples used in the colon model experiments were provided by a single donor from a
155	study approved by the QIB Human Research Governance commi^ee (IFR01/2015) registered
156 at h^p://www.clinicaltrials.gov (QIB Colon Model study, NCT02653001). The par^cipant
157 provided their wri^en informed consent to par^cipate in this study and the research was
158	conducted in accordance with the Declara^on of Helsinki. The donor was healthy, without
159	known metabolic or gastrointes^nal condi^ons, and had not received any an^bio^c, probio^c,
160 or prebio^c interven^on for at least 3 months prior to the experiment. The sample was
161 processed within 4 h of stool collec^on.
162	20 g of faeces were placed in a Circulator 400 stomacher bag (Seward, UK) with 100 ml
163 phosphate buffered saline (PBS), and homogenised using a Stomacher 400 at 230 rpm for 30
164	s. A^er filtering and homogenizing, the faecal slurry was centrifuged (4000 × g, 25 min) and
165	resuspended in PBS to prepare a 20% faecal slurry. Sterile glycerol was added to aliquots to a
166 final concentra^on of 25% and aliquots were stored at -80°C un^l required. Faecal slurries
167 were defrosted at 37°C before use.
168 Serum bottle fermentations were conducted anaerobically at 37 °C using 30 ml reduced
169	HBCM with final concentrations of faecal slurry and MSG of 1% and 2% respectively.
170	Experiments consisted of 4 groups: control (HBCM with faecal slurry), Bif group (HBCM with
171 faecal slurry and B. adolescentis QI0300, approximately 1×108 cells ml-1), Lac group (HBCM
172 with faecal slurry and L. plantarum FI11205, approximately 1×108 cells ml-1), and Bif+Lac mix
173 group (HBCM with faecal slurry and B. adolescentis QI0300 and L. plantarum FI11205 at the
174 ratio of 1:1, approximately 3×108 cells ml-1 each species). Each group was set up in triplicate.

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175 Aliquots (1 ml) were withdrawn from each bo^le at 0 h and 24 h (T0, T24) and frozen at -80°C
176 for further analysis.
177 GABA measurement by LC-MS/MS
178 Samples were analysed using a Acquity I-Class UPLC coupled to a Xevo TQ Absolute mass 179 spectrometer (Waters, UK). A Raptor Polar 100 x 2.1 mm x 2.7 µm column (Restek, UK) was 180 used in HILIC mode at 35°C for chromatographic separa^on. Mobile phase consisted of solvent 181 A, water with 20mM ammonium formate (> 99% LC-MS grade, Merck) and 0.5% v/v formic 182 acid (LC-MS grade, Greyhound Chromatography) and solvent B, 90% v/v acetonitrile: water 183 (both LC-MS grade, Fisher Scien^fic) with 20 mM ammonium formate and 0.5% v/v formic 184 acid pumped at 400 µl per minute. Sample injec^ons were 0.2 µl with a gradient of 96% B at 185 injec^on for 2 min, 30% B at 10 min, 5% B at 10.1 min for 1 min and return to ini^al condi^ons 186 for 3 min. The mass spectrometer source was set with a cone temperature of 120°C and cone 187 gas flow of 150 l h-1. The desolva^on temperature was 500°C with gas flow of 1000 l h-1. The 188 nebuliser gas was 90 l h-1 and capillary set at 2.5 kV. The quadrupole was run in mul^ple 189 reac^on posi^ve mode with target mass transi^on for GABA of 104/69 amu and secondary 190 ion of 104/86 amu. Cone voltage was 15 V for both and collision energies of 14 and 14 eV, 191 respec^vely. Calibra^on standard GABA (> 99%, Merck) was prepared in solvent B at 10 to 192 10000 ng ml-1 and all dilu^ons of samples were made in solvent B. Under the condi^ons 193 applied, GABA eluted at between 2.5-3 min (Supplementary Fig. S1).
194 Evalua^on of microbiota by metagenomic analysis
195 DNA was extracted from faecal fermenta^ons using the Maxwell® RSC PureFood GMO and 196 Authen^ca^on Kit and Maxwell® RSC 48 instrument (Promega) according to the 197 manufacturer’s instruc^ons, with some modifica^ons. 1 ml samples from faecal fermenta^ons

198

were thawed on ice then

at

x g at 4°C for 15 min.

Supernatants were removed and the pellets resuspended in 800 μl CTAB Buffer and vortexed for 30 s. Samples were heated at 95°C for 5 min, allowed to cool for 2 min then vortexed thoroughly for 1 min. The suspension was transferred into a Lysing Matrix E tube (MP Biomedicals) and homogenised using a Fastprep FR120 cell disrupter (MP Biomedicals) for 60 s at 6.5 m s-1 for 3 ^mes, le^ng the samples cool down on ice for 5 min between runs. Samples were then treated as in the Maxwell® protocol. DNA was quan^fied using a Qubit Broad Range DNA Quan^fica^on Kit (Thermo Fisher Scien^fic) and Qubit 3.0 fluorometer (Life Technologies).
Metagenomic shotgun sequencing was performed at QIB using an Illumina Nextseq500 as described previously (Parker et al. 2022). Sequencing was performed on one of each triplicate samples for each of two biological replicates at T0 h and T24 h. Sequences were quality controlled using the QIB Clean-up pipeline v1.4 (available at h^ps://github.com/tela^n/cleanup), and included a step of adapter trimming and read quality filtering using fastp v.0.22.4 (Chen 2023) and mul^QC v.1.25.0 (Ewels et al. 2016). Poten^al human DNA read contamina^on was further removed using Hos^le v.1.1.0 (Constan^nides et al. 2023) against the human-t2t-hla reference database. A^er QC and human read exclusions, the samples had on median 30 million read pairs, with a minimum of 15 million and a maximum of 51 million read pairs. Metaphlan v.4.0.1 (Blanco-Miguez et al. 2023) against the Jun23_CHOCOPhlAnSGB_202307 database was run on the sequences a^er quality control in order to profile the samples.

219

Community composi^on analysis was performed using the Microbiome R package v.1.24.0,
220 and visualisa^ons were generated using ggplot2 v.3.5. Ordina^on of the samples was

221 performed using Principal Coordinates Analysis (PCoA) with species level Bray-Cur^s distances
222 between samples, using the Microbiome R package.
Metabolomic analysis by 1H-NMR
224 Metabolites from faecal fermenta^ons were analysed by proton Nuclear Magne^c Resonance
225	(1H NMR) spectroscopy. Supernatants (400 μl) were mixed with 200 μl NMR buffer (0.26 g
226	NaH2PO4 and 1.41 g K2HPO4 made up in 100 ml D2O mixed with 1 mM sodium 3
227 (Trimethylsilyl)-propionate-d4, (TSP) (17 mg) as a chemical shi^ reference). Five hundred μl
228 was transferred into a 5 mm NMR tube for spectral acquisi^on. The 1H NMR spectra were
229 recorded at 600 MHz on a Bruker Advance spectrometer (Bruker BioSpin GmbH, Germany)
230	running Topspin 2.0 so^ware and fi^ed with a cryoprobe and a 24-slot autosampler. Each 1H
231	NMR spectrum was acquired with 32 scans, a spectral width of 12,500 Hz, and an acquisi^on
232 ^me of 2.6 s. The noesypr1d pre-satura^on sequence was used to suppress the residual water
233	signal with a low-power selec^ve irradia^on at the water frequency during the recycle delay
234	(D1 = 4 s) and mixing ^me (D8 = 0.01 s). Spectra were transformed with a 0.1 Hz line
235 broadening, and were manually phased, baseline corrected and referenced by se^ng the TSP
236 methyl signal to 0 ppm. The metabolites were quan^fied using the so^ware Chenomx® NMR 237 Suite 10.0™.
Results
Produc^on of GABA in nutrient media varies between strains
241 The GABA-producing ability of strains was assessed at 72 h in their preferred nutrient media.

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242 Ini^al screening showed that GABA levels from some cultures were not notably greater than

243	background levels in media (BHI+C 0.013 mM; MRS 0.094 mM). However, 7 strains of B.
244	adolescen^s and 6 strains of L. plantarum produced detectable levels of GABA, with L.
245 plantarum strains showing the highest yields (Fig. 1A).
246 To assess GABA produc^on over ^me, 5 strains of each species showing the highest GABA
247 produc^on were grown in their preferred nutrient media for 24, 48 or 72 h. B. adolescen^s
248	QI0300, L. plantarum FI11223 and L. plantarum FI11205 produced the highest amounts of
249	GABA (1.65 ± 0.09 mM, 16.16 ± 3.21 mM, and 2.95 ± 0.05 mM respec^vely) at 72 h (Fig. 1B)
250	while L. plantarum FI11208 produced its highest levels at 24 and 48 h. In most strains, GABA
251 produc^on increased over ^me, but it was notable that yields were lower than in previous
252 experiments in the same condi^ons.
253 The final concentra^on of GABA a^er 72 h ranged between 0.08 ± 0.01 mM to 6.08 ± 0.23
254 mM for B. adolescen^s and between 0.24 ± 0.02 mM to 26.05 ± 0.37 mM for L. plantarum
255	(Fig. 1). The diversity of produc^on between the different Bifidobacterium and
256	Lac^plan^bacillus strains demonstrates that GABA produc^on in vitro is highly strain
257 dependent. Repeated analyses also showed that although some strains showed consistent
258 levels, produc^on o^en varied between different biological replicates. When 2 strains were
259 grown in triplicate in three separate experiments, GABA levels were found to vary by up to 3
260 fold (Fig. 1C).
261 Co-culture improves GABA produc^on
262 Co-culture of B. adolescen^s and L. plantarum strains could give notable increases in GABA 263	produc^on, with growth and produc^on varying with different strain combina^ons (Fig. 2).
264	GABA yield was highest when B. adolescen^s QI0300 was paired with L. plantarum strains

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265	FI11223, FI11206 or FI11205, giving yields at 24 h which were higher than those seen in single

cultures of either

± 5.36 mM, 62.55 ± 2.06 mM and 53.97 ± 0.63 mM
respec^vely). Although at the inoculum stage the ra^o of the Bifidobacteria and Lactobacilli in co-culture was 1:1, a^er 24 h incuba^on the Lactobacilli tended to dominate in most combina^ons. However, L. plantarum FI11205 maintained a balanced ra^o of cell number with B. adolescen^s strains QI0300, QI0281 and QI0450 (Fig. 2).
Effect of acid stress on GABA produc^on
B. adolescen^s QI0300 exhibited lower growth and total GABA yield as the culture pH dropped from 6.0 to 3.5 (Fig. 3A). However, when GABA yield was corrected for cell numbers, produc^on was shown to increase steeply below pH 4.5 (Fig. 3A). To assess the effect of coculture or lac^c acid on GABA produc^on, B. adolescen^s QI0300 was co-cultured in 1:1 BHI+C:MRS with either acid- and GABA-producing strain L. plantarum FI11205, acid-producing , but non-GABA-producing strain L. pentosus DSM20314, the cell-free supernatants (CFS) of these bacteria, or D/L lac^c acid alone, with the final GABA produc^on corrected to the viable cell count. At 24 h, coculture with both Lac^plan^bacillus species gave a notable drop in pH and an increase in GABA produc^on (Fig. 3B, C). Addi^on of lac^c acid failed to reduce the final pH compared to B. adolescen^s alone, but did lead to higher GABA levels. Co-culture with CFS from L. plantarum FI11205 grown without MSG led to a low ini^al and final pH and the highest GABA level (Fig. 3D). Increased produc^on was also seen when the CFS was from nonGABA producing L. pentosus DSM20314, but although this treatment gave similar star^ng and final pH to the L. plantarum CFS, GABA produc^on was higher with the la^er. These results indicate that lac^c acid produc^on from the Lactobacilli may be a factor in GABA yield in co-
cultures, but the pH of the culture is not the sole determinant.
290 Strains produce GABA in colon model media
291 The GABA-producing ability of the top producers from the screening stage were assessed for
292	24 h in a complex medium designed to simulate colon condi^ons (colon model media). The
293 results demonstrated that some GABA-producing strains can perform well in colon model
294	condi^ons, although produc^on was lower than in nutrient media (Fig. 4). L. plantarum strains
295	commonly had be^er produc^on at 24 h. B. adolescen^s QI0300 and L. plantarum FI11205
296 resulted in the highest produc^on of GABA, 0.587 ± 0.138 mM and 1.142 ± 0.276 mM, 297 respec^vely.
298 Strain supplementa^on can increase GABA in faecal fermenta^ons
299	To inves^gate whether these strains might produce GABA in gastrointes^nal condi^ons, B. 300	adolescen^s QI0300 and L. plantarum FI11205 were included in simple faecal fermenta^ons, 301	both singly (Bif and Lac respec^vely) and in combina^on (Bif+Lac), using a colon model 302	medium adapted for increased buffering capacity (HBCM). The ini^al pH of cultures was 6.8. 303	GABA produc^on levels in this medium supplemented with 2% MSG were lower than in 304	nutrient media or colon model medium with ordinary buffering capacity; in one experiment 305	a^er 24 h B. adolescen^s QI0300 produced 0.107 ± 0.001 mM and L. plantarum FI11205 only 306	0.041 ± 0.002 mM (n=2) compared to 0.030 mM in uninoculated broth, while in a second 307	experiment yields were 0.077 ± 0.001 mM and 0.011 ± 0.002 mM respec^vely at 24 h and 308	0.074 ± 0.004 mM and 0.011 ± 0.005 mM at 48 h (n=2). In two separate faecal fermenta^ons, 309	using the same cryopreserved faecal sample with one month storage between experiments, 310	GABA levels at 24 h were 0.51 ± 0.08 mM and 0.76 ± 0.10 mM (n=3) in controls. In the first

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311 experiment, addi^on of L. plantarum FI11205 or B. adolescen^s QI0300 gave only modest

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312 increases compared to the second fermenta^on (Fig. 5). However, the presence of both 313 bacteria with a 3-fold higher inoculum gave notably higher levels, giving mean fold increases 314 in GABA levels of 2.6 and 9.0 in the two experiments. Given the increased inoculum used in 315 the Bif+Lac sample, the first experiment did not demonstrate synergis^c produc^on when 316 both strains were present. However, in the second experiment the mean GABA yield in the dual inoculum samples (26.88 mM) was more than 3 ^mes the sum of the yields in the single samples (14.17 mM). Despite extra buffering, the presence of L. plantarum led to a reduced pH at 24 h, but there was not a direct rela^onship between pH and GABA yield.
Strain supplementa^on did not disrupt faecal microbiome diversity
321 The microbial composi^on in single samples from both colon model experiments was 322 characterised according to species-level alpha-diversity (Shannon index) and global 323 composi^on. For all experimental condi^ons, a clear diminu^on of the alpha-diversity was 324 observed between the 0 h and 24 h sampling points (Fig. 6A). This decrease was caused by 325 reduc^ons in both species richness (Fig. 6B) and evenness (Fig. 6C). A clear shi^ in the beta 326	diversity between the two ^me points was observed for all experimental condi^ons (Fig. 6D). 327	All samples displayed a similar change in their global community composi^on between 0 and 328	24 h, with all samples taken a^er 24h of incuba^on separated to the baseline samples when 329 visualized using an ordina^on plot (Fig. 6D).

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At baseline, the samples differed principally in terms of the two taxa of interest added to the samples (Bifidobacterium and Lac^plan^bacillus) (Fig. 6E, Supplementary Fig. S2A), and displayed high rela^ve abundance of Blau^a, Ruminococcus, Roseburia and Faecalibacterium. A^er 24 h of incuba^on, all samples and the controls showed an increased rela^ve abundance 334 of Bifidobacterium and Escherichia, while the rela^ve abundance of other families such as
335	Oscillospiraceae, Lachnospiraceae and Bacteroidaceae were reduced. Although 336	Lac^plan^bacillus species are common gut commensals, they were only prevalent in samples 337	where L. plantarum FI11205 was added. Apart from Bifidobacterium and Lactobacillus, the 338	species profile in the control samples was similar to that in the treated samples, with the main 339	change being in Escherichia coli which, although present in all samples, was more abundant 340 at 24 h in un-supplemented control samples (Fig. 6E). 341 In order to inves^gate the change in individual species rela^ve abundances between ^me 342	points, we computed the Log2 fold change between the mean rela^ve abundance of the 343	species at 0 and 24 h ^me points. This allowed us to iden^fy 25 species that had a log2 fold 344	change above 2 in one or more of the experimental condi^ons (Supplementary Fig. S2B). As 345	expected, we observed a large number of bacterial species that show a decreased log2 fold 346	change between 0 and 24 h ^me points, in par^cular taxa from the Lachnospiraceae, 347	Oscillospiraceae, Bacteriaceae and Clostridiaceae, confirming the previous observa^ons. On 348	the other hand, we observe the increase of several species of Bifidobacteriaceae between the 349	two ^me points as well as an increase in rela^ve abundance of E. coli, once again in 350 accordance with the pa^erns observed at the genus and family level (Supplementary Fig. S2B). 351 Most samples showed broadly similar pa^erns, indica^ng that incuba^on ^me was a larger 352 factor than treatment and that the added bacteria could increase GABA concentra^ons 353 without causing a significant disrup^on in the microbiome. Notably, L. plantarum was found 354 to be stable between the ^me points in both the Lac (Log2FC=0.13) and Bif+Lac (Log2FC=-
355	0.12) condi^ons.

Strain supplementa^on increases some beneficial metabolites
1H-NMR detected 50 measurable compounds (Supplementary Table 1). The most abundant metabolites were acetate, formate, glucose, lactate and glutamate. Addi^on of B. adolescen^s QI0300, alone or with L. plantarum FI11205, gave greater increases in acetate and formate as well as minor compounds propionate and 3-Methyl-2-oxovalerate over 24 h, and lower levels of lactate than in control or Lac samples (Fig. 7, Supplementary Table 1). Several other metabolites were also reduced in the presence of B. adolescen^s QI0300 compared to other samples at 24 h, including 1, 3-dihydroxyacetone and phenylacetate. L. plantarum FI11205 was associated with slightly larger increases in succinate, but despite the stability of this strain in the metagenome, lactate levels were not consistently higher in Lac-supplemented samples than in the control, possibly indica^ng its metabolism by the microbiota.
Discussion

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Analysis of selected strains containing GAD genes showed that GABA produc^on from intes^nal B. adolescen^s and fermented food L. plantarum isolates was strain-specific and variable between replicate experiments. Average yields from B. adolescen^s and L. plantarum cultures were similar to those from strains reported recently when incubated in nutrient media with MSG (Zhuang et al. 2018, Monteagudo-Mera et al. 2023, Tames et al. 2023). Kaur and colleagues (2023) reported that L. fermentum L18 produced the highest yield of GABA at 24 h, and the level decreased at 72 h, sugges^ng consump^on or degrada^on in some strains (Kaur et al. 2023). In our experiments, one L. plantarum strain also showed a maximum yield 378 at 48 h and two B. adolescen^s strains showed lowered levels with longer incuba^ons, but in 379 general maximum yields were found at 72 h.
380 GABA produc^on is known to be affected by culture condi^ons and medium composi^on, 381 with pH being a key factor (Diez-Gu^errez et al. 2019, Cui et al. 2020). Selected producer 382 strains performed well in a medium designed to simulate colon condi^ons compared to 383 nutrient media at the same ^me point, but when increased buffering was included yields were 384 decreased, par^cularly from L. plantarum. The rela^onship between pH and GABA produc^on 385 has been well described and the GAD system has been demonstrated to protect Bacteroides 386 against acid stress (Feehily and Karatzas 2013, Otaru et al. 2021). Many GAD enzymes have 387 their highest ac^vity at pH 4-5, with ac^vity frequently reducing as the pH rises to neutral (Cui 388 et al. 2020). We also found higher produc^on of GABA per cell at lower pH, however in terms 389 of total yield GABA was decreased due to increasingly poorer growth as the pH was reduced 390 from 5. 391 Co-culture can have synergis^c effects on bacterial growth and metabolism (Oliveira et al. 392	2012); probio^c formula^ons including mixes of Lactobacilli and Bifidobacteria have shown 393 promising results (Messaoudi et al. 2011, Luang-In et al. 2020, De Oliveira et al. 2023) and 394 several reports have demonstrated improved GABA produc^on with combina^ons of species 395 (Cui et al. 2020). This study also found that combining B. adolescen^s with L. plantarum strains 396 gave notable increases in GABA levels, and results indicated that lac^c acid may play a role in 397 improving produc^on. 398 In faecal fermenta^ons, GABA was detectable in control samples – the GAD system has 399 been described in many families, including the gastrointes^nally-prevalent Bacteroides (Otaru 400	et al. 2021), and GABA synthe^c pathways were found to be widespread in gut microbes 401	(Strandwitz et al. 2019, Valles-Colomer et al. 2019). Increases in GABA levels a^er addi^on of 402 B. adolescen^s and/or L. plantarum varied between repeated experiments using the same
403 preserved faecal sample from a single donor, but in general B. adolescen^s addi^on was more 404 effec^ve than L. plantarum, while a higher inoculum of both strains gave the best yield, with 405 some indica^on of synergy in the second experiment. Time had a greater effect on alpha and 406 beta diversity than treatment. Metagenomic sequencing showed both strains persisted over 407 24 h, but did not cause large effects on the most prevalent genera, apart from a reduc^on in 408 Escherichia. It is important to remember that these metagenomic and metabolomic results 409 are derived from analysis of single samples from one donor and further analysis is required 410 before firm conclusions can be drawn. Microbiome composi^on and func^on can be very 411 donor-specific; survival and ac^vity of these strains may vary in different backgrounds. In a 412 pH-controlled batch culture colon model, addi^on of L. fermentum also did not cause large 413 disrup^on of the microbiome diversity or composi^on, although increases in Lactobacillaceae 414 and Bifidobacteriaceae were noted (Kaur et al. 2023). However, increased GABA levels, both 415 from bacterial treatments or fermented milk, did affect gut bacterial composi^on both in the 416 SHIME® in vitro intes^nal model system (De Oliveira et al. 2023, Casertano et al. 2024) and in 417 mice (Yu et al. 2020, Tames et al. 2024). Several of these studies also noted changes in SCFA: 418 Lactobacillus helve^cus-Bifidobacterium longum treatment gave increased total SCFA in vitro, 419 due primarily to ace^c acid increase (De Oliveira et al. 2023); mouse treatment with B. 420 adolescen^s gave decreases in total SCFA (Tames et al. 2024), while GABA-rich milk gave 421 increased total SCFA, par^cularly butyric acid (Yu et al. 2020). Both GABA and SCFA are 422 important signalling molecules in the gut-brain axis (Sarkar et al. 2016, Belelli et al. 2025). 423 Interac^ons between GABA intake and gastrointes^nal SCFA levels have been reported in mice 424 (Xie et al. 2017) and SCFA have also been implicated in mood adjustment (reviewed in (Belelli 425 et al. 2025)). Our faecal fermenta^ons showed increases in acetate, par^cularly in the higher 426 level two-strain treatment, and an increase in the less abundant propionate when B.
427 adolescen^s was added. However, butyrate was not consistently affected. As microbial SCFA 428 produc^on is very species-specific these effects may well vary in different faecal backgrounds. 429 We observed increases in succinate a^er supplementa^on with L. plantarum, but not with 430 B. adolescen^s alone; many bacteria u^lise the GABA shunt pathway to produce succinate 431 from GABA (Feehily and Karatzas 2013) and control of this conversion might affect final GABA concentra^ons in the gut. An inves^ga^on of the transcriptome of L. plantarum CGMCC1.2437 found that genes associated with pyruvate metabolism and glycolysis/gluconeogenesis pathways were induced by MSG, and the authors hypothesised that this could lead to synthesis of succinate from glucose (Zhuang et al. 2018). 436 For produc^on of GABA as a pharmaceu^cal or a food addi^ve, high produc^on is of key 437	concern. However, for produc^on in the human gut, it might be more beneficial to deliver 438	consistent and controlled levels. Both L. plantarum and B. adolescen^s are good candidates 439 for GABA-producing probio^cs or for produc^on of GABA-enriched foods (Echegaray et al. 440	2023, Tames et al. 2023, Gholian et al. 2024, Tames et al. 2024). However, it is important to 441	take into considera^on the effect of the environment, eg pH and precursors, on the regula^on 442	of produc^on, and the effect of GABA produc^on on the metabolism and persistence of the 443	producer strain. Transcriptomic analysis showed that MSG-induced high GABA produc^on 444	affected several other biological processes in L. plantarum (Zhuang et al. 2018). Probio^cs 445	themselves can have significant impacts on the gut microbiome and metabolome, immune 446	responses and the gut-brain axis (Plaza-Diaz et al. 2019, Gholian et al. 2024), and changes in 447	the microbiome may have unan^cipated effects on endogenous GABA levels. The effect of 448	GABA-producing probio^cs on the composi^on and func^on of the gut microbiome must be
449 taken into account in any future therapeu^c studies, to ensure that final levels remain 450 consistent and within a beneficial range. 451 This study is limited by the use of simple faecal fermenta^ons, which only allow short term 452 assessment of microbial community structure. The metagenomic and metabolomic results are 453	derived from a single donor, and although they provide valuable preliminary insights, follow- 454	up studies with larger cohorts are needed to confirm these findings. Future work will benefit 455 from expanding into more detailed, longer term in vivo colon fermenta^on models, including 456 upper diges^ve tract models to assess the survival and ac^vity of the bacteria through the 457 whole gastrointes^nal tract. 458 Data availability statement 459 Data from metagenomic sequencing of faecal fermenta^ons is available in the European 460 Nucleo^de Archive (ENA) database under project PRJEB85806 461 h^ps://www.ebi.ac.uk/ena/browser/view/PRJEB85806. Genome accessions are available 462	from the Na^onal Center for Biotechnology Informa^on (NCBI) under Bioprojects 463	PRJNA1377923 (Bifidobacterium sp. (ID 1377923) - BioProject - NCBI), PRJNA1377924 464	(Lac^plan^bacillus plantarum (ID 1377924) - BioProject - NCBI) and PRJNA1377925 465 (Lac^plan^bacillus plantarum (ID 1377925) - BioProject - NCBI). 466 Ethical approvement statement 467 Faecal samples were provided by a single donor from the QIB Colon Model study approved by 468 the QIB Human Research Governance commi^ee (IFR01/2015) registered at 469 h^p://www.clinicaltrials.gov (NCT02653001). The par^cipant provided their wri^en informed
470 consent to par^cipate in this study and the research was conducted in accordance with the 471 Declara^on of Helsinki. 472 Acknowledgements
473 We are very grateful to Nwanneka Akinyemi (University of Lagos, Nigeria), Valeria Rizello 474 (University of Messina, Italy) Maria Diaz and Carmen Nueno-Palop (Quadram Ins^tute 475 Bioscience (QIB)) for strain isola^on at QIB, to fermented food collaborator Professor Folarin 476 Oguntoyinbo (University of Lagos, Nigeria and Boone Appalachian State University, North 477 Carolina, USA) and to QIB colleagues David Baker and Rhiannon Evans, for whole genome 478 and metagenomic sequencing, and James Choi and Dr Sumeet Tiwari for prior genome 479 sequencing and analysis. We thank Dr Emad Shehata (QIB) for facilita^ng faecal sample 480 collec^on and Stephanie Ong for technical assistance. 481 Conflict of interest 482 The authors declare that they have no conflicts of interest. 483 Funding 484 This work was supported by the Biotechnology and Biological Sciences Research Council 485 (BBSRC) Ins^tute Strategic Programme grant Food, Microbiome and Health BB/X011054/1 486 theme BBS/E/QU/230001D and by the China Scholarship Council. 487 References

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601 Xie M, Chen HH, Nie SP. et al. Gamma-Aminobutyric Acid Increases the Produc^on of Short- 602 Chain Fa^y Acids and Decreases pH Values in Mouse Colon. Molecules 2017;22:653. 603	h^ps://doi:10.3390/molecules22040653

604 Yu L, Han X, Cen S. et al. Beneficial effect of GABA-rich fermented milk on insomnia involving 605 regula^on of gut microbiota. Microbiol Res 2020;233:126409. 606	h^ps://doi:10.1016/j.micres.2020.126409
607 Yunes RA, Poluektova EU, Dyachkova MS. et al. GABA produc^on and structure of gadB/gadC 608 genes in Lactobacillus and Bifidobacterium strains from human microbiota. Anaerobe 609	2016;42:197-204. h^ps://doi:10.1016/j.anaerobe.2016.10.011
610 Zhuang K, Jiang Y, Feng X. et al. Transcriptomic response to GABA-producing Lactobacillus 611 plantarum CGMCC 1.2437T induced by L-MSG. PLoS One 2018;13:e0199021. 612	h^ps://doi:10.1371/journal.pone.0199021
613 Author contribu^ons
614	Rihua Xu (Conceptualisa^on, Formal analysis, Inves^ga^on, Methodology, Wri^ng – original 615	dra^, Wri^ng – review and edi^ng), Melinda J. Mayer (Conceptualisa^on, Methodology, 616	Supervision, Wri^ng – original dra^, Wri^ng – review and edi^ng), Mark Philo (Formal 617	analysis, Inves^ga^on, Methodology, Wri^ng – review and edi^ng), Gwénaëlle Le Gall (Formal 618	analysis, Inves^ga^on, Methodology, Wri^ng – review and edi^ng), Guesh Mulaw 619	(Methodology, Wri^ng – review and edi^ng), Alise Ponsero (Data cura^on, Formal analysis,
620	Inves^ga^on, Methodology, Wri^ng – review and edi^ng), Arjan Narbad (Conceptualisa^on, 621 Funding acquisi^on. Supervision, Wri^ng – review and edi^ng).

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622
623
624 Table 1 GABA-producing bacterial strains.
Species	Strain	Isola^on source	Genbank accession number	16S rRNA gene	
Bifidobacterium adolescen^s	QI0148	Healthy human faeces	JBSXXZ000000000	PX683695	
Bifidobacterium adolescen^s	QI0195	Healthy human faeces	JBSXXY000000000	PX683696	
Bifidobacterium adolescen^s	QI0281	Healthy human faeces	JBSXXX000000000	PX683697	
Bifidobacterium adolescen^s	QI0300	Healthy human faeces	JBSXXW000000000	PX683698	
Bifidobacterium adolescen^s	QI0448	Healthy human faeces	JBSXXV000000000	PX683699	
Bifidobacterium adolescen^s	QI0449	Healthy human faeces	JBSXXU000000000	PX683700	
Bifidobacterium adolescen^s	QI0450	Healthy human faeces	JBSXXT000000000	PX683701	
Lac^plan^bacillus plantarum	FI11123	Nigerian fermented maize ogi	JBSXYB000000000	PX683717	
Lac^plan^bacillus plantarum	FI11125	Nigerian fermented millet kunu zaki	JBSXYA000000000	PX683718	
Lac^plan^bacillus plantarum	FI11205	Ethiopian teff dough	JBSXZE000000000	PX684482	
Lac^plan^bacillus plantarum	FI11206	Ethiopian teff dough	JBSXZD000000000	PX684483	
Lac^plan^bacillus plantarum	FI11208	Ethiopian ergo	JBSXZC000000000	PX684484	
Lac^plan^bacillus plantarum	FI11212	Ethiopian teff dough	JBSXZB000000000	PX684485	
Lac^plan^bacillus plantarum	FI11223	Ethiopian kocho	JBSXZA000000000	PX684486	
625
626
627
628
629
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Figure Legends

Figure 1 GABA produc^on in nutrient media with MSG. A, producer strains of B. adolescen^s

and L. plantarum at 72 h, results are the mean of triplicate samples +/- sd; B, GABA produc^on n selected strains of B. adolescen^s or L. plantarum respec^vely a^er 24, 48 or 72 h ncuba^on, results are the mean of duplicate cultures +/- sd; C, GABA produc^on from two
637 strains over three separate experiments, results are the mean of triplicate samples +/- sd. 638 Background media levels were subtracted from all measurements.

Figure 2 GABA produc^on in co-culture. A, total produc^on from 72 h co-cultures of

640
641
642

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639 Bifidobacterium and Lac^plan^bacillus in mixed nutrient media; B, counts of Bifidobacterium 643 and Lac^plan^bacillus strains from selec^ve pla^ng showed that Lac^plan^bacillus strains
644 commonly dominated the co-cultures a^er 72 h. Results are the mean of duplicate samples
645	+/- sd.
646

647

648	Figure 3 Effect of pH and co-culture on GABA produc^on of B. adolescen^s QI0300. A, a^er 24 649	h growth in pH-adjusted media showing viable cell counts, GABA produc^on (mM) and GABA 650 produc^on corrected to cell count (pg cell-1); B-C, a^er 24 h co-cultures with LAB, LAB

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651 supernatant or lac^c acid, showing GABA produc^on (B) and ini^al and final pH (C). Results 652 are the mean of triplicate samples +/- sd.

654
655
Figure 4 GABA produc^on of B. adolescen^s and L. plantarum in colon
model media with MSG
656
at 24 h
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660
661
662
faecal slurry and 2% MSG. Small circles, experiment 1, large circles, experiment 2. Each point
is from a single vessel.
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Figure 6 Alpha-diversity and global sample composi^on. A, Alpha-diversity (Shannon index),
B, Richness (Observed richness) and C, Evenness (Pielou evenness index) at the species level
666 across the experimental condi^ons and ^me points. D, PCoA using a Bray-Cur^s distance on
667 species-level counts. Samples are colored according to their experimental condi^ons and
668 shapes represent the two sampling ^me points. E, Genus-level taxonomic composi^on of the
669 samples. The taxonomic counts were aggregated at the genus level before TSS normaliza^on.

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671

were grouped as ’Others’.

672

670 Families that accounted for less than 0.5% abundance and 25% prevalence in the samples
673
674	Figure 7 1H-NMR analysis of faecal fermenta^ons. A, heatmap showing changes of fermen^ng
products and SCFA in both faecal fermenta^on experiments between 0 h and 24 h (colours
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676 indicate concentra^on (mM)); B, concentra^ons of fermenta^on products and SCFA between

677
treatment groups at 0 and 24 h.
678
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