Seaweed Fermentation Modulates Gut Microbiome Composition Without Altering Metabolite Output In Vitro

Seaweed Fermentation Modulates Gut Microbiome Composition Without Altering Metabolite Output In Vitro | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Seaweed Fermentation Modulates Gut Microbiome Composition Without Altering Metabolite Output In Vitro Amée Johanna Haeften, Irene Saez Devis, Matthias Kasimir, Evangelia Zioga, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7794319/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Seaweed consumption gains popularity due to its high micronutrient, protein and fiber content. While seaweed extracts show promising effects on the gut, it remains unclear if processing of raw seaweed changes the nutrient accessibility for the gut microbiota. This study aimed to investigate how different processing methods affect the prebiotic potential of a common brown seaweed, Saccharina latissima . Methods The seaweed was processed (blanched, oven-dried or fermented by Lactiplantibacillus plantarum ) and underwent simulated upper-gastrointestinal digestion with INFOGEST 2.0 prior to in vitro fermentation, using four adult human fecal donors. Samples were analyzed for their microbiome composition and produced metabolites. Results Seaweed-supplemented media changed the fecal microbial composition compared to low-fiber and high-fiber controls. Specifically, seaweed increased the relative abundances of Bacteroidales, Escherichia/Shigella and unclassified Lachnospiraceae, while Bifidobacteria decreased. Regardless of processing, seaweed-supplemented cultures showed similar alpha and beta diversity indicating that processing did not modulate seaweed’s impact on the microbial composition. The short-chain fatty acid and amino acid concentrations were similar in the seaweed-supplemented media and the low-fiber medium after fermentation, while formic acid and lactic acid were significantly higher in the high-fiber medium, compared to other media, and negatively correlated with the final pH after fermentation. Conclusion Seaweed altered the microbiome composition, however with minor effects of seaweed processing. These compositional shifts did not translate into metabolic changes, suggesting that seaweed components were largely inaccessible to microbes under the tested conditions. Further studies are recommended to investigate alternative processing and co-formulations to enhance seaweed fermentability. seaweed gut microbiome SCFA aromatic amino acids Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction While the demand for sustainable and healthy food increases, seaweed shows potential as a sustainable alternative protein source, due to its high protein content and its status as a rich source of essential amino acids and healthy omega-3 fatty acids [ 1 ]. Moreover, seaweed has a 100–200× lower CO 2 equivalent emission (~ 0.08–0.17 kg CO 2eq /kg fresh) [ 2 ] than beef (~ 16.6 kg CO 2eq /kg live weight) [ 3 ], highlighting its low environmental impact. Commercial seaweed aquaculture in Denmark focuses mostly on Saccharina latissima [ 4 ], which can be found in coastal areas of Northern Europe, as well as northern of Spain and Portugal, and on the coastlines of Canada and the United States [ 5 ]. This brown macroalga is cultivated for multiple applications, such as food products [ 6 ], its bioactive compounds, like phenolics, flavonoids and pigments [ 7 ], and as a biofuel [ 8 ]. It has a 10–16% dry matter content (DM) and is rich in carbohydrates (42–46% DM), ash (26–45% DM) and proteins (8–11% DM), depending on seasonal changes, age and locations [ 9 , 10 ]. The four main carbohydrates in S. latissima are alginate, laminarin, cellulose and fucoidan [ 11 ], which are fibers that cannot be digested in upper-gastrointestinal tract. Together with the other dietary fibers, they account for 27.8–40.9% of the dry weight of S. latissima [ 12 , 13 ]. Dietary fibers can stimulate the growth and activities of beneficial gut bacteria [ 14 ], which metabolize fibers into short-chain fatty acids (SCFAs). These SCFAs promote gut barrier integrity, reduce inflammation, improve glucose metabolism and insulin sensitivity, and lower the risk of colon cancer [ 15 , 16 ]. Additionally, fiber intake alters the amino acid metabolism by gut bacteria, stimulating the production of health-promoting tryptophan metabolites and indirectly suppressing indole production [ 17 ]. Beyond these metabolic effects, fibers also improve bowel movements, alleviate constipation, enhance digestion, and reduce abdominal discomfort, due to their high water-holding capacity and lubricating properties [ 18 , 19 ]. Previous studies found that in vitro fermentation of seaweed polysaccharide extracts increased SCFAs production [ 20 – 23 ] and promoted relative abundances of beneficial bacteria like Bifidobacterium, Bacteroides and Lactobacillus [ 20 , 21 , 24 ]. Specifically, Bacteroides spp. metabolized alginate, resulting in an increased production of SCFAs in vitro [ 21 ]. Furthermore, fecal fermentation of laminarin increased propionic and butyric acid production [ 25 ], and fucoidan increased the abundances of SCFA-producing bacteria from the genera Blautia , Bacteroides and Alloprevotella [ 26 ]. While these studies highlight the effect of extracted polysaccharides on the gut microbiota, they do not reflect real-world scenarios where whole seaweed, rather than isolated extracts, is consumed. Processing of seaweed is essential to reduce its naturally high iodine content before consumption, which in S. latissima can reach concentrations of up to 6500 mg·kg⁻¹ dw -1 [ 9 ]. Exceeding the upper intake level of 600 µg/day can cause adverse health effects, eventually leading to for example thyroid dysfunction or cancer [ 27 ]. Currently, seaweed is mostly processed by blanching, grinding or drying. Blanching is an effective way to increase the release of iodine and other matrix-entrapped compounds [ 10 ], and, for instance, increases the carbohydrate content (% DM) in S. latissima without affecting protein or fat content (% DM) [ 10 ]. Furthermore, seaweed’s particle size reduction, by e.g. grinding or bead-beating, increases lipid digestibility by 32% and protein digestibility by 5–16% [ 28 , 29 ], due to the release of compounds bound to polysaccharides in the cell walls [ 30 ]. In this study, we investigate the effects of processing of S. latissima on the gut microbiome in vitro . We hypothesize that processing (freeze-drying, blanching, fermentation with lactic acid bacteria and oven-drying) increases the nutrient accessibility to gut bacteria, thereby improving the fermentability of seaweed in the colon. The fermentability was assessed as the impact on the gut microbiome composition and metabolic activity was assessed by the production of SCFAs and amino acid metabolites during fermentation. Materials and Methods Seaweed biomass The seaweed, S. latissima , was harvested off the coast of Stavanger, Norway, at the end of June 2024 by Arctic Seaweed A/S and kept at 4°C until further use. Processing of the seaweed The “oven” seaweed samples were oven-dried for 4 h at 60°C in the SalvisLAB TC-40S Thermocenter Oven (Rotkreuz, Switzerland). The “fermentation” seaweed samples were fermented for 2 days at 30°C by Lactiplantibacillus plantarum. The L. plantarum NFICC983 glycerol stock was revived in Man Rogosa Sharpe (MRS) broth and fresh pre-cultures were used as inoculum for the seaweed fermentations as described previously [ 31 ]. The “blanch” seaweed samples were blanched in a water bath with seawater (salinity: 32‰) for 1 minute at 45°C and immediately put on ice. After processing, samples were stored at -20°C until freeze-drying and homogenization (Fig. 1 ). Freeze-drying and homogenization The four seaweed samples were freeze-dried for 52 h in the Christ LMC-1 freeze-dryer (Martin Christ Gefriertorcknungsanlagen GmbH, Osterode am Harz, Germany). The freeze-dried samples were then homogenized using a KN295 Kniftec™ rotor mill (FOSS Analytical, Hillerød, Denmark) and stored at -20°C until further use. Upper-gastrointestinal digestion using INFOGEST 2.0 INFOGEST 2.0 was used to mimic digestion of seaweed in the upper-gastrointestinal tract, following a standardized protocol [ 32 ]. In short, in the oral phase, 0.25 g of seaweed was mixed 0.75 g of water, followed by mixing with simulated salivary fluid (ratio 1:1 vol/vol), without enzymes, and incubated for 2 minutes at 37°C and pH 7. Then, the oral bolus was mixed with simulated gastric fluid (ratio 1:1 vol/vol) and gastric lipase and incubated for 2 h at 37°C and pH 3. In the intestinal phase, the gastric chyme was mixed with simulated intestinal fluid (ratio 1:1 vol/vol), pancreatin and bile salts, and incubated for 2 h at 37°C and pH 7. The samples were centrifuged for 45 minutes at 5,500 g at 37°C and the pellets were collected and stored at -20°C. After INFOGEST 2.0, the pellets were freeze-dried and homogenized as described above, to standardize the input material added to the fermentation media. Anaerobic batch fermentation Six different media (four seaweed, one high-fiber and one low-fiber control) were prepared according to a described protocol [ 33 ]. The high-fiber control contained a mix of glucose (0.4 g/L), xylan (0.8 g/L), apple pectin (0.8 g/L), arabinogalactan (0.8 g/L), and starch (5 g/L), while no polysaccharides were added to the seaweed-enriched media and the low-fiber medium (negative control). After autoclaving, the four different seaweed samples (7.8 g/L) were added to the seaweed media. One day before the fecal fermentation, 12 mL of each medium (in quadruplicates) was transferred into 15 mL Falcon tubes and kept in the anaerobic workstation at 37°C overnight to make the medium anoxic. On day 1, 250 µL of fecal slurry was inoculated into the media in the anaerobic workstation. Four adult human fecal samples, selected for their high abundance of Bifidobacterium , a genus well known as efficient fiber-fermenters (Table 1 ), were obtained from the PRIMA human baseline study [ 34 ]. Samples were collected at 0 h, 24 h and 48 h and centrifuged for 10 minutes at 10,000 g at 4°C. The pH of the supernatant was measured on the sampling day with the SevenCompact™ pH/ion meter S220 (Mettler Toledo™, Ohio, USA). The remaining supernatant and the pellets were stored at -20°C until analysis. Table 1 Fecal samples, selected for their dominant relative abundance of Bifidobacterium . Donor Bifidobacterium (%) Dominant Bifidobacterium spp. 1 58.6 B. adolescentis 2 62.2 B. adolescentis + B. longum 3 68.9 B. catenulatum 4 56.4 B. catenulatum Bacterial 16S rRNA Sequencing DNA extraction, PCR and library preparation DNA was extracted from fecal pellets using the DNeasy® PowerLyzer® PowerSoil® Kit (Qiagen, Hilden, Germany), following the Quick-Start protocol [ 35 ]. DNA concentration was measured with the Qubit™ dsDNA high sensitivity (HS) fluorescence assay (Thermo Fisher Scientific, Waltham, USA) in a 96-well plate, according to the manufacturer’s protocol. Fluorescence was measured at an excitation/emission wavelength of 495/525 nm using Kaleido 3.0 software (Kaleido Technologies, New Delhi, India) and the Perkin Elmer Enspire 2300 Multi-mode Microplate Reader (New Life Scientific Inc., Cridersville, USA). PCR amplification for library preparation and subsequent sequencing on the Ion Torrent was done, as previously described [ 36 ], using1 µM forward primer (PBU) and 1 µM reverse primer (PBR; 5′-trP1-adaptor-ATTACCGCGGCTGCTGG-3′) and 2 µL extracted DNA (5 ng/µL) in a total reaction volume of 20 µL. The unique sequence of each primer is provided in Supplementary Table S1 . DNA was amplified by PCR with the Veriti® 96-Well Thermal Cycler Model 9902 (Life Technologies, Carlsbad, USA). Then, the DNA was purified using the HighPrep™ PCR Clean Up (MagBio Genomics, Gaithersburg, USA), following the manufacturer’s protocol, after which the DNA concentration was measured again. The DNA samples were mixed to an equimolar mixture and sent to Ion Torrent sequencing on a 318-chip using the Ion OneTouch™ 200bp Template Kit v2 DL (Life Technologies, Carlsbad, USA). 16S rRNA gene amplicon sequencing bioinformatic analyses The 16S rRNA gene amplicon data were processed with our in-house-pipeline [ 37 ]. In short, raw amplicon sequences were demultiplexed using cutadapt (v. 4.1) [ 38 ], denoised using DADA2 (v. 1.22) [ 39 ]. Then, ASVs amplicon sequence variants were classified against RDP training set_18 [ 40 ]. The downstream processing was done with Phyloseq (v. 1.42.0) in R (v. 4.4.3) [ 41 ] for further data processing. ASVs were decontaminated with the decontam package [ 42 ]. All ASVs on genus level can be found in Supplementary Table S2 . Metabolites quantification The SCFAs in the supernatants were analyzed by MS-Omics Aps (Vedbæk, Denmark). In short, the supernatants were transferred to Spin-X® centrifuge tubes (pore size of 0.22 µm) and centrifuged at 15,000 g for 5 minutes at 4°C. Sample analysis was carried out by MS-Omics as follows. Samples were acidified using hydrochloride acid, and deuterium labelled internal standards were added. All samples were analyzed in a randomized order. Analysis was performed using a high polarity column (Zebron™ ZB-FFAP, GC Cap. Column 30 m × 0.25 mm × 0.25 µm) installed in a GC (7890B, Agilent) coupled with a time-of-flight MS (Pegasus® BT, LECO)]. The system was controlled by [ChromaTOF® (LECO)]. Raw data was converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2021b (Mathworks, Inc.) using the PARADISe software as previously described [ 43 ]. Amino acid metabolites were analyzed using liquid chromatography-mass spectrometry (LC-MS) with L-tryptophan (indole-d5, 98%) as internal standard, as previously described [ 44 ]. In short, 2 µL of sample was analyzed with an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry system consisting of a Dionex Ultimate 3000 RS liquid chromatograph (Thermo Scientific) coupled to a Bruker maXis time-of-flight mass spectrometer equipped with an electrospray interphase (Bruker Daltonics) operating in positive mode to analyze tyramine and tryptamine and in the negative mode to analyze all other amino acid metabolites. The analytes were separated on a Poroshell 120 SB-C18 column with a dimension of 2.1×100 mm and 2.7 µm particle size (Agilent Technologies) as previously described [ 45 ]. Quality control samples and standard mix solutions were analyzed before and after the sample set, with two standards measured after every ten samples. Data was processed using QuantAnalysis v.2.2 (Bruker Daltonics), applying calibration curves (with quadratic regression) generated for each metabolite from all standards. Calibration curves were made by plotting analyte-to-internal standard peak area ratios against calibration standard concentrations, as previously described [ 17 ]. Lactic acid content was measured with High-Pressure Liquid Chromatography (HPLC) with a refractive index detector. Supernatant was mixed with 5 mM sulfuric acid solution (1:4 ratio), vortexed at 8,000 rpm for 5 minutes and filtered through a 0.2 µm syringe filters before transferring 500 µL sample into HPLC vials. The HPLC was equipped with an Aminex HPX-87H column (Bio-Rad, USA) in an Ultimate HPLC system (Dionex, Thermo Fisher Scientific, USA) and coupled to a Shodex RI-101 refractive index detector (Showa Denko K.K., Isesaki, Japan) for detection. The mobile phase consisted of 5 mM sulfuric acid. The column was set at 60°C, with a flow rate of 0.5 mL/min and an injection volume of 20 µL. Standards were prepared at concentrations of 50, 100, 500, and 1,000 µg/mL (ppm) in 5 mM sulfuric acid and filtered through a 0.22 µm syringe filter. Chromeleon 2.0 software (Thermo Fisher Scientific, Waltham, USA) was used for data analysis. Data analysis All data was analyzed, and figures were made using R (v. 4.4.3) [ 41 ] and figures were combined in Inkscape (v. 0.92.1). Normal distributions were checked using the Shapiro-Wilk test. The significant differences between short-chain fatty acid and amino acid concentrations in the different media at a certain time point were determined with groupwise ANOVA and Tukey's honestly significant difference (HSD). Significance is defined as p ≤ 0.05 and a trend is defined as 0.05 < p ≤ 0.10, as used in the results and discussion. The media pH values were compared using emmeans corrected for false discovery rate (FDR) within each time point and within each fermentation medium. Subsequently, Pearson’s correlation test was done to test Pearson’s correlation coefficient between pH and the produced SCFAs and lactic acid, combining all measurements from each medium and each replicate. To determine the beta dispersion in the beta diversity analysis, one-way ANOVA was used to identify possible differences between the groups, followed by pairwise PERMANOVA with FDR correction, using the ecole package [ 46 ]. The full statistical analyses can be found on https://github.com/ameevh/Sea4Gut . Results Processed seaweed (blanched, oven-dried, or fermented with Lactiplantibacillus plantarum ) was digested (INFOGEST 2.0), and the undigested material was added to fecal fermentation media. Fecal samples of four donors were inoculated, and samples were collected at 0, 24, and 48 h for pH, 16S rRNA sequencing, and metabolite profiling (Fig. 1 ). Seaweed supplementation affects fecal microbiota composition in vitro At baseline (0 h), alpha diversity did not differ between fermentation media (Fig. 2 A). In the low-fiber medium, however, both Shannon index and observed richness increased significantly after 24 h (p = 0.007, p = 0.033) and further after 48 h (p = 0.003, p = 0.003) compared to baseline. The seaweed-supplemented media had similar alpha diversities over time, except for the oven-dried medium where the Shannon index increased significantly from 24 to 48 h (p = 0.044). After 24 h of fermentation, the Shannon index in the low-fiber medium was significantly higher compared to the high-fiber medium (p = 0.048), the fermented seaweed medium (p = 0.013) and the oven-dried medium (p = 0.048), and observed richness in the low-fiber medium was significantly higher compared to all other media (p ≤ 0.05). After 48 h, the Shannon index of the low-fiber medium was significantly higher than the high-fiber medium (p = 0.002) and the fermented seaweed medium (p = 0.025), and the observed richness was significantly higher in the low-fiber medium compared to all other media with the strongest difference with the high-fiber medium (p = 4.7*10 − 5 ). Despite visual differences in the PCoA plots (Fig. 2 B), there were no significant differences in the Aitchison distance between the different media at all time points (determined with PERMANOVA, Supplementary Table S3). Significant differences in Aitchison distance between all donors were observed at all time points (p < 0.01; Supplementary Table S4), indicating a strong donor effect on the beta diversity. On the order level, Bifidobacteriales and Clostridiales had the highest relative abundances at baseline (Fig. 2 C). After 24 h, the relative abundance of Bifidobacteriales decreased in the low-fiber medium and the four seaweed-supplemented media, while the abundances of Clostridiales and Enterobacterales increased mostly in the seaweed-supplemented media. In donors 2, 3 and 4, more distinct differences were also observed between the seaweed-supplemented media and the low-fiber medium. Similar to beta diversity, microbiome compositional changes appeared mostly donor-driven and largely independent of seaweed processing. Bifidobacterium was the most abundant genus at baseline (Fig. 2 D). Changes in the microbial composition were strongly donor-dependent, but the seaweed-supplemented media also showed a different microbial composition compared to the high-fiber and low-fiber media. The high-fiber media inoculated with donors 3 and 4 had a large relative abundance of Bifidobacterium during fermentation, while the relative abundance of Bifidobacterium decreased in most samples. In the high-fiber medium inoculated with donor 1, a high relative abundance of unclassified Clostridiaceae was observed after 24 and 48 h, while the high-fiber medium inoculated with donor 2 showed a high relative abundance of Clostridium sensu stricto during fermentation. The relative abundance of Escherichia/Shigella increased in the seaweed-supplemented media inoculated with donor 2, 3 and 4 and in the seaweed-supplemented media inoculated with donor 1, the relative abundance of unclassified Enterobacteriaceae increased after 24 and 48 h. Furthermore, an increase in the relative abundance of unclassified Lachnospiraceae was observed in donor 2 increased after 24 and 48 h. Seaweed-supplemented media inoculated with donors 1 and 3 showed an increase in relative abundance of Butyrivibrio after 24 h, except for the LAB fermented seaweed in donor 1. After 48 h, an increase in relative abundance of Bacteroides was observed in all seaweed-supplemented media for all donors, and mostly in donors 1, 3 and 4. Bacteroides species differed in the samples grown in seaweed-supplemented media, compared to samples grown in the low-fiber medium (Table 2 and Supplementary Table S5). Although not-significantly different (p < 0.05), the highest abundance of Bacteroides spp. was Bacteroides kribbi/koreensis/ovatus in all seaweed media and the high-fiber medium, compared to Bacteroides uniformis in the low-fiber medium. Interestingly, Bacteroides ovatus has been shown to degrade alginate, a complex carbohydrate found in seaweed [ 47 ]. Table 2 Three most abundant Bacteroides spp. in the high-fiber, low-fiber and the four seaweed-supplemented media, combining the 4 donors at 24 and 48 h (mean ± standard deviation, n = 8). Medium ASV Species Relative abundance (%) High-fiber ASV_0015 kribbi/koreensis/ovatus 0.0064 ± 0.0138 ASV_0011 uniformis 0.0014 ± 0.0044 ASV_0017 xylanisolvens 0.0006 ± 0.0013 Low-fiber ASV_0011 uniformis 0.9772 ± 3.2355 ASV_0062 thetaiotaomicron 0.3801 ± 0.2893 ASV_0090 intestinalis/cellulosyliticus 0.3424 ± 0.4324 Untreated ASV_0015 kribbi/koreensis/ovatus 4.7383 ± 10.0577 ASV_0011 xylanisolvens 1.6931 ± 5.9346 ASV_0017 uniformis 0.7392 ± 2.2170 Blanch ASV_0015 kribbi/koreensis/ovatus 2.1961 ± 3.9356 ASV_0017 xylanisolvens 1.2308 ± 4.4284 ASV_0011 uniformis 0.5951 ± 1.9409 Ferment ASV_0015 kribbi/koreensis/ovatus 7.8594 ± 14.9231 ASV_0017 xylanisolvens 1.8855 ± 6.5986 ASV_0011 uniformis 0.6341 ± 1.6449 Oven ASV_0015 kribbi/koreensis/ovatus 3.2904 ± 5.6847 ASV_0011 uniformis 0.6337 ± 1.9311 ASV_0017 xylanisolvens 0.4071 ± 1.6038 Seaweed supplementation does not affect short-chain and branched short-chain fatty acid production in adult human fecal samples After 24 h of fermentation, formic acid (3.46 ± 2.94 mM) and lactic acid (24.01 ± 17.02 mM) were detected in significantly higher concentrations in the high-fiber medium than all other media (p < 0.05, Fig. 3 A). Lactic acid was only detected in the high-fiber medium after 24 and 48 h, and in one sample from the fermented seaweed medium after 24 h (3.27 mM, Supplementary Table S6). Total SCFAs increased in most media from 24 to 48 h and, except the high-fiber medium where the concentration of total SCFAs did not increase significantly (p > 0.05) from 24 to 48 h (Fig. 3 A). Acetic acid was significantly higher in the seaweed media compared to the high-fiber medium, with highest concentrations found in the blanched seaweed medium (51.09 ± 5.11 mM). No differences in butyric acid were observed between the different media at both 24 and 48 h. Propanoic acid increased significantly from 24 to 48 h in the untreated seaweed medium and the fermented seaweed medium. After 48 h, the propanoic acid remained low in the high-fiber medium (0.58 ± 0.41 mM), which was significantly lower than the propanoic acid concentration in all other media (p < 0.01). The most abundant branched short-chain fatty acid (BCFA) was isobutyric acid, which was significantly lower in the high-fiber medium compared to the low-fiber medium (p = 0.006), the untreated seaweed medium (p = 0.022), the blanched seaweed medium (p = 0.009) and the oven-dried seaweed medium (p = 0.031, Supplementary Fig. 1, Supplementary Table S7). Following a similar pattern, the concentrations of isovaleric acid and 2-methyl butanoic acid remained low after 48 h in the high-fiber medium and were significantly lower than in the low-fiber and seaweed media (p < 0.05). The BCFA 3-methyl pentanoic acid was also found in low concentrations in all media and was significantly lower after 48 h in the high-fiber medium than the low-fiber medium (p = 0.035) and the blanched seaweed medium (p = 0.036). Despite the higher concentrations of total SCFAs, acetic acid and butyric acid in the low-fiber medium and the seaweed media, the pH only decreased significantly in the high-fiber medium from baseline (pH = 6.6) to 24 (pH = 4.6) and 48 h (pH = 4.6, Supplementary Table S8). Using Pearson’s correlation, a strong negative correlation was observed between pH and the formic acid concentration (r s =-0.61, p < 0.01) and a very strong negative correlation was observed between pH and the lactic acid concentration (r s =-0.86, p < 0.01, Fig. 3 B). Seaweed supplementation does not affect the production of phenylalanine, tryptophan and tyrosine To get a broader idea of the effects of seaweed on host-microbiome interactions, we also investigated the microbial metabolism of aromatic amino acids and their derivatives. After 24 h, phenylalanine tended to be higher in the high-fiber medium, compared to the low-fiber medium and seaweed-supplemented media (both p = 0.10), after which the phenylalanine concentration decreased after 48 h in all media (Fig. 4 ). Similarly, its derivative phenyllactic acid was found in significantly higher concentrations in the high-fiber medium after 48 h (p < 0.05, Supplementary Fig. 2). Tryptophan was highest in the oven-dried seaweed medium and significantly lower in the low-fiber medium (p = 0.050) after 24 h. After 48 h, a decrease in tryptophan concentration was observed in all samples except the high-fiber medium. No significant differences were observed in the concentrations of tryptophan derivatives between the different media. The highest tyrosine concentration was found in the untreated seaweed medium after 24 h, which was significantly higher than in the high-fiber medium (p = 0.044) and the low-fiber medium (p = 0.041). A decrease in tyrosine concentration was observed in all samples after 48 h with non-significant differences between all media, while the tyrosine derivatives 4-hydroxyphenyllactic acid and 4-hydroxyphenylpropionic acid were produced in significantly higher concentrations in the fiber-rich medium (Supplementary Fig. 2). Discussion We found that alpha diversity was highest after 24 and 48 h in the low-fiber medium for both the Shannon index and the observed richness. High-protein/low-fiber diets have previously shown to increase alpha diversity in vitro [ 48 ]. Furthermore, the reduction in observed richness after 24 and 48 h in the high-fiber medium can be a result of the pH reduction in this medium, since not all bacteria can survive under acidic conditions [ 49 ] which therefore reduces richness [ 50 , 51 ]. Despite a visual difference between the high-fiber medium and the other media in the PCoA plot, no significant differences were found between the Aitchison distance of the different media, possibly due to the low sample size (n = 4) or the strong donor-dependent effect. The different microbial compositions of the seaweed-supplemented media on order and genus levels after 24 and 48 h, compared to the high-fiber and low-fiber media, indicate seaweed fermentation by the fecal bacteria. Contrary to findings of increased relative abundance of Bifidobacterium after in vitro fermentation of seaweed extracts [ 20 , 22 , 24 ], our results show a decrease in relative abundance of Bifidobacterium across the seaweed-supplemented media and in the low-fiber medium. This decrease may be explained by the high relative baseline abundance of Bifidobacterium , leading to a decrease in relative abundance without necessarily affecting the absolute concentrations. Furthermore, we observed increased the relative abundances of unclassified Lachnospiraceae in the seaweed media of donor 2. Similarly, increased relative abundances of Lachnospiraceae were reported after fermentation of another brown alga Laminiaria digitata [ 52 ]. Lachnospiraceae are common fiber degraders that have beneficial effects on the gut microbiome and host health by fermenting non-digestible plant polysaccharides, such as cellulose, into acetic acid, butyric acid and propionic acid [ 53 , 54 ]. Escherichia/Shigella abundances increased after seaweed supplementation in all donors. Although high abundances of Escherichia/Shigella are sometimes linked to low nutrient availability, increased Escherichia coli abundances were previously reported after in vitro fermentation of the brown seaweed Ecklonia radiata [ 20 ]. The higher relative abundance of Bacteroides spp. in the seaweed-supplemented media may be attributed to the laminarin and alginate in seaweed, which promotes the growth of species such as B. ovatus and B. dorei and is linked to the production of acetic and propionic acids. [ 24 , 47 ]. Our study found that the low-fiber medium and the seaweed-supplemented media had significantly higher SCFA concentrations compared to the high-fiber medium after 48 h, except for formic and lactic acid which were significantly higher in the high-fiber medium compared to all other media. The significantly higher concentration of acetic acid in our processed seaweed media could also be caused by fermentation of seaweed fibers, since the high-fiber and low-fiber medium controls had similar acetic acid concentrations. However, the acetic acid concentration in the low-fiber medium suggests the presence of bacteria that are capable of fermenting amino acids into acetic acid, most likely through proteolytic fermentation or the Stickland fermentation of glycine into acetic acid by Cluster XI Clostridia [ 55 ]. The butyric acid concentration in the seaweed-supplemented media was not significantly higher than in the high-fiber medium or low-fiber medium, possibly due to the large outlier of donor 2 in the high-fiber medium. The higher, though not significant, concentrations of butyric acid in the seaweed media may be linked to Butyrivibrio and other members of the Lachnospiraceae family, known butyric acid producers [ 56 ] that were relatively abundant in donors 2 and 3 after 24 and 48 h. Proteolytic fermentation could explain why SCFA concentrations in the low-fiber medium did not differ significantly from those in the seaweed media. The higher concentrations of propanoic acid, isobutyric acid and isovaleric acid in the low-fiber medium and seaweed-supplemented media compared to the high-fiber medium can also be a result of proteolytic fermentation [ 57 ], and Bacteroidetes can, for example, produce propanoic acid from peptides [ 58 ]. This could explain the higher relative abundances of Bacteroides in the media with less nutrient accessibility, like the low-fiber and the seaweed-supplemented media. Phenyllactic acid (PLA), the aromatic lactic acid of phenylalanine, was significantly higher in the high-fiber medium than all other media at 48 h. Similarly, 4-hydroxyphenyllactic acid, the aromatic lactic acid of tyrosine, was significantly higher in the high-fiber medium than in all other media at both 24 and 48 h. No differences were observed in the concentration of indole lactic acid; the aromatic lactic acid produced from tryptophan. High concentrations of aromatic lactic acids are associated with fiber degradation, such as fructo-oligosaccharides and galacto-oligosaccharides, which favor increased abundance bacteria such as Clostridium sensu stricto , Lachnospiraceae [ 59 ] and Bifidobacterium spp.[ 44 , 60 ]. In our study, high relative abundances of Clostridum sensu stricto , unclassified genera of Clostridiaceae and Bifidobacterium were observed in the high-fiber medium, compared to the other media, for all donors after both 24 and 48 h, which may have contributed to the production of phenyllactic acid in this medium. The aromatic propionic acids of phenylalanine and tryptophan, phenylpropionic acid and indole-3-propionic acid, were detected in low amounts in the high-fiber medium. In the other samples, the concentrations were slightly higher but due to the high standard deviations between the samples, no significant differences were observed between the media. The aromatic propionic acid of tyrosine, 4-hydroxyphenylpropionic acid, was detected in significantly higher concentrations in the high-fiber medium compared to the other media after 48 h. This aromatic amino acid is known to be produced during fiber degradation [ 61 ], for example by Lachnospiraceae [ 62 ]. In our study, however, the fiber-fermenting unclassified Lachnospiraceae had the highest relative abundance in the seaweed-supplemented media in donor 2 and was hardly present in the samples from the high-fiber medium (Fig. 2 D). The decarboxylase products of tryptophan and tyrosine, namely tryptamine and tyramine, were not detected in significantly different concentrations between the different media or time points. Tryptamine was produced in higher concentrations in the high-fiber medium and remained relatively low in the seaweed-supplemented media, without significant differences between the media due to the large standard deviations. Similarly, the other indole-derived metabolites were detected without significant differences between the media. Overall, we conclude that seaweed supplementation modifies the composition of the microbiome, regardless of the pre-digestive processing methods, but does not significantly affect metabolite production. A follow-up study testing alternative processing or a combined intake of Saccharina latissima with enzymes or probiotics are recommended to study how to enhance the fermentability of seaweed polysaccharides in the gut. Declarations Competing interests The authors declare that they have no competing interests. Funding The authors would like to thank Katja Ann Kristensen, Bodil Madsen and Steffen Valencia Bach for their technical support in the experiments and Mikael Pedersen for his valuable contribution performing the LC-MS. We thank Matthew Robert Carey for his help with R and Josep Rubert for his external supervision during the first part of the project. We thank Nicola Procházková and Henrik Roager for providing the fecal samples that were collected in the PRIMA human baseline study (NCT04804319). Finally, we thank M. D. Dalgaard at the DTU in-house facility (DTU Multi-Assay Core, DMAC) for performing the 16S rRNA gene sequencing. This study was part of the project Seaweed for gut health, funded by Ekhagastifelsen (Seaweed for gut health, grant number 69, year-2022). The fecal samples used in the study were collected in the PRIMA human baseline study (NCT04804319). Author Contribution AJVH, AKS, MFL, SLH and ISD designed the experiments. ISD and EZ processed the seaweed. AJVH, MK and ISD performed the INFOGEST 2.0. AJVH performed and analyzed the in vitro fermentation experiments and prepared the samples for LC-MS, GC-MS and HPLC. EZ performed the HPLC experiment and initial analyses. AJVH prepared the samples for sequencing, and AJVH and MSM analyzed the data. AJVH, AKS and MFL drafted the manuscript. Acknowledgement This study was part of the project Seaweed for gut health, funded by Ekhagastifelsen (Seaweed for gut health, grant number 69, year-2022). The authors would like to thank Katja Ann Kristensen, Bodil Madsen and Steffen Valencia Bach for their technical support in the experiments and Mikael Pedersen for his valuable contribution performing the LC-MS. We thank Matthew Robert Carey for his help with R and Josep Rubert for his external supervision during the first part of the project. We thank Nicola Procházková and Henrik Roager for providing the fecal samples that were collected in the PRIMA human baseline study (NCT04804319). Finally, we thank M. D. Dalgaard at the DTU in-house facility (DTU Multi-Assay Core, DMAC) for performing the 16S rRNA gene sequencing. Data Availability The sequencing data have been deposited into the Sequence Read Archive under BioProject PRJNA1337439. 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10:02:06","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69635,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/33b035611a9a6b2dd2bfa30c.png"},{"id":96405332,"identity":"9cb0dfd8-ed69-48e4-9cde-5bfcae2e9887","added_by":"auto","created_at":"2025-11-20 16:58:02","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":62876,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/8d0af3c28b05fc0760f9354e.png"},{"id":96453677,"identity":"43872df2-594d-45b9-a8f0-9c9cde34a470","added_by":"auto","created_at":"2025-11-21 10:01:16","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26881,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/6660fc8f999f2b191747e7d7.png"},{"id":96405330,"identity":"4c47add8-fbb5-4d26-b5d3-906c4b8e6a00","added_by":"auto","created_at":"2025-11-20 16:58:02","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143191,"visible":true,"origin":"","legend":"","description":"","filename":"e7dd739741cb42fd87d364b62ebe06c31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/96ca9d4460053401a4c068dc.xml"},{"id":96454593,"identity":"859bf589-700b-4a3b-b697-3a336481a088","added_by":"auto","created_at":"2025-11-21 10:02:57","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":156407,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/1b7098dd927d6a990f2373a2.html"},{"id":96405317,"identity":"b97e1871-3fa2-4c06-a856-8d3bd8a1753e","added_by":"auto","created_at":"2025-11-20 16:58:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214092,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic of the experimental plan for \u003cem\u003ein vitro \u003c/em\u003egut fermentation\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/9099d0cb144c3f7d4a3b1ef1.png"},{"id":96453741,"identity":"3af7257c-12cf-49ea-b2d5-7f0bab036585","added_by":"auto","created_at":"2025-11-21 10:01:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":216753,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobiome composition analyses, including alpha diversity with Shannon index and observed richness (A), beta diversity shown in PCoA plots with Aitchison distance (B), relative abundance (%) on order level for 4 fecal donor samples at 0, 24 and 48 h in six different fermentation media (C) and relative abundance (%) on genus level for 4 fecal donor samples at 0, 24 and 48 h in six different fermentation media (D). Significant differences are indicated with * (p≤0.05), ** (p≤0.01) and *** (p≤0.001).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/21f4cf3adbc88e2bbb49b39e.png"},{"id":96405319,"identity":"f0fc1c58-59d9-43e1-83cb-0a2bfea3786b","added_by":"auto","created_at":"2025-11-20 16:58:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":191315,"visible":true,"origin":"","legend":"\u003cp\u003eShort-chain fatty acid (SCFA) produced in the different seaweed media after 24 and 48 h. SCFAs with p≤0.10 between the different media and butyric acid are plotted, where * indicates p≤0.05, ** p≤0.01 and *** p≤0.001 (n=4) (A). Correlation plot of SCFA and lactic acid concentrations (mM) to pH, determined with Pearson’s correlation coefficient (B).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/508ed5aa66783f435e33df13.png"},{"id":96454077,"identity":"938149b0-08fd-4703-9f3c-521ba60c4e3f","added_by":"auto","created_at":"2025-11-21 10:02:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81124,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations (µM) of the three aromatic amino acids phenylalanine, tryptophan and tyrosine at 24 and 48 h, where * indicates p\u003cstrong\u003e≤\u003c/strong\u003e0.05, ** p\u003cstrong\u003e≤\u003c/strong\u003e0.01 and *** p\u003cstrong\u003e≤\u003c/strong\u003e0.001 (n=4).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/775fd7b5953201fcd6383b31.png"},{"id":96880267,"identity":"560feae4-3a9b-436a-8d8d-39d34cc8e85a","added_by":"auto","created_at":"2025-11-27 06:53:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1555144,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/bc9f9e07-f6e9-47e2-ac77-bfed2088578f.pdf"},{"id":96405325,"identity":"9ec4937d-f3bf-4fd7-8382-22005f6d4b4a","added_by":"auto","created_at":"2025-11-20 16:58:02","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":330398,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/9db453de52d4edd33f02c496.xlsx"},{"id":96405318,"identity":"c65f3c6f-6351-4890-aa6b-d42f3d3aea31","added_by":"auto","created_at":"2025-11-20 16:58:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":150861,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7794319/v1/77b970e4fa5db4c35dfa4462.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seaweed Fermentation Modulates Gut Microbiome Composition Without Altering Metabolite Output In Vitro","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWhile the demand for sustainable and healthy food increases, seaweed shows potential as a sustainable alternative protein source, due to its high protein content and its status as a rich source of essential amino acids and healthy omega-3 fatty acids [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Moreover, seaweed has a 100\u0026ndash;200\u0026times; lower CO\u003csub\u003e2\u003c/sub\u003e equivalent emission (~\u0026thinsp;0.08\u0026ndash;0.17 kg CO\u003csub\u003e2eq\u003c/sub\u003e/kg fresh) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] than beef (~\u0026thinsp;16.6 kg CO\u003csub\u003e2eq\u003c/sub\u003e/kg live weight) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], highlighting its low environmental impact.\u003c/p\u003e\u003cp\u003eCommercial seaweed aquaculture in Denmark focuses mostly on \u003cem\u003eSaccharina latissima\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which can be found in coastal areas of Northern Europe, as well as northern of Spain and Portugal, and on the coastlines of Canada and the United States [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This brown macroalga is cultivated for multiple applications, such as food products [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], its bioactive compounds, like phenolics, flavonoids and pigments [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and as a biofuel [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It has a 10\u0026ndash;16% dry matter content (DM) and is rich in carbohydrates (42\u0026ndash;46% DM), ash (26\u0026ndash;45% DM) and proteins (8\u0026ndash;11% DM), depending on seasonal changes, age and locations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The four main carbohydrates in \u003cem\u003eS. latissima\u003c/em\u003e are alginate, laminarin, cellulose and fucoidan [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], which are fibers that cannot be digested in upper-gastrointestinal tract. Together with the other dietary fibers, they account for 27.8\u0026ndash;40.9% of the dry weight of \u003cem\u003eS. latissima\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDietary fibers can stimulate the growth and activities of beneficial gut bacteria [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which metabolize fibers into short-chain fatty acids (SCFAs). These SCFAs promote gut barrier integrity, reduce inflammation, improve glucose metabolism and insulin sensitivity, and lower the risk of colon cancer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, fiber intake alters the amino acid metabolism by gut bacteria, stimulating the production of health-promoting tryptophan metabolites and indirectly suppressing indole production [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Beyond these metabolic effects, fibers also improve bowel movements, alleviate constipation, enhance digestion, and reduce abdominal discomfort, due to their high water-holding capacity and lubricating properties [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious studies found that \u003cem\u003ein vitro\u003c/em\u003e fermentation of seaweed polysaccharide extracts increased SCFAs production [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and promoted relative abundances of beneficial bacteria like \u003cem\u003eBifidobacterium, Bacteroides\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Specifically, \u003cem\u003eBacteroides\u003c/em\u003e spp. metabolized alginate, resulting in an increased production of SCFAs \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, fecal fermentation of laminarin increased propionic and butyric acid production [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and fucoidan increased the abundances of SCFA-producing bacteria from the genera \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eAlloprevotella\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While these studies highlight the effect of extracted polysaccharides on the gut microbiota, they do not reflect real-world scenarios where whole seaweed, rather than isolated extracts, is consumed.\u003c/p\u003e\u003cp\u003eProcessing of seaweed is essential to reduce its naturally high iodine content before consumption, which in \u003cem\u003eS. latissima\u003c/em\u003e can reach concentrations of up to 6500 mg\u0026middot;kg⁻\u0026sup1; dw\u003csup\u003e-1\u003c/sup\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Exceeding the upper intake level of 600 \u0026micro;g/day can cause adverse health effects, eventually leading to for example thyroid dysfunction or cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Currently, seaweed is mostly processed by blanching, grinding or drying. Blanching is an effective way to increase the release of iodine and other matrix-entrapped compounds [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and, for instance, increases the carbohydrate content (% DM) in \u003cem\u003eS. latissima\u003c/em\u003e without affecting protein or fat content (% DM) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, seaweed\u0026rsquo;s particle size reduction, by e.g. grinding or bead-beating, increases lipid digestibility by 32% and protein digestibility by 5\u0026ndash;16% [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], due to the release of compounds bound to polysaccharides in the cell walls [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we investigate the effects of processing of \u003cem\u003eS. latissima\u003c/em\u003e on the gut microbiome \u003cem\u003ein vitro\u003c/em\u003e. We hypothesize that processing (freeze-drying, blanching, fermentation with lactic acid bacteria and oven-drying) increases the nutrient accessibility to gut bacteria, thereby improving the fermentability of seaweed in the colon. The fermentability was assessed as the impact on the gut microbiome composition and metabolic activity was assessed by the production of SCFAs and amino acid metabolites during fermentation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSeaweed biomass\u003c/h2\u003e\u003cp\u003eThe seaweed, \u003cem\u003eS. latissima\u003c/em\u003e, was harvested off the coast of Stavanger, Norway, at the end of June 2024 by Arctic Seaweed A/S and kept at 4\u0026deg;C until further use.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProcessing of the seaweed\u003c/h3\u003e\n\u003cp\u003eThe \u0026ldquo;oven\u0026rdquo; seaweed samples were oven-dried for 4 h at 60\u0026deg;C in the SalvisLAB TC-40S Thermocenter Oven (Rotkreuz, Switzerland). The \u0026ldquo;fermentation\u0026rdquo; seaweed samples were fermented for 2 days at 30\u0026deg;C by \u003cem\u003eLactiplantibacillus plantarum.\u003c/em\u003e The \u003cem\u003eL. plantarum NFICC983\u003c/em\u003e glycerol stock was revived in Man Rogosa Sharpe (MRS) broth and fresh pre-cultures were used as inoculum for the seaweed fermentations as described previously [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The \u0026ldquo;blanch\u0026rdquo; seaweed samples were blanched in a water bath with seawater (salinity: 32\u0026permil;) for 1 minute at 45\u0026deg;C and immediately put on ice. After processing, samples were stored at -20\u0026deg;C until freeze-drying and homogenization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eFreeze-drying and homogenization\u003c/h3\u003e\n\u003cp\u003eThe four seaweed samples were freeze-dried for 52 h in the Christ LMC-1 freeze-dryer (Martin Christ Gefriertorcknungsanlagen GmbH, Osterode am Harz, Germany). The freeze-dried samples were then homogenized using a KN295 Kniftec\u0026trade; rotor mill (FOSS Analytical, Hiller\u0026oslash;d, Denmark) and stored at -20\u0026deg;C until further use.\u003c/p\u003e\n\u003ch3\u003eUpper-gastrointestinal digestion using INFOGEST 2.0\u003c/h3\u003e\n\u003cp\u003eINFOGEST 2.0 was used to mimic digestion of seaweed in the upper-gastrointestinal tract, following a standardized protocol [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In short, in the oral phase, 0.25 g of seaweed was mixed 0.75 g of water, followed by mixing with simulated salivary fluid (ratio 1:1 vol/vol), without enzymes, and incubated for 2 minutes at 37\u0026deg;C and pH 7. Then, the oral bolus was mixed with simulated gastric fluid (ratio 1:1 vol/vol) and gastric lipase and incubated for 2 h at 37\u0026deg;C and pH 3. In the intestinal phase, the gastric chyme was mixed with simulated intestinal fluid (ratio 1:1 vol/vol), pancreatin and bile salts, and incubated for 2 h at 37\u0026deg;C and pH 7. The samples were centrifuged for 45 minutes at 5,500 \u003cem\u003eg\u003c/em\u003e at 37\u0026deg;C and the pellets were collected and stored at -20\u0026deg;C. After INFOGEST 2.0, the pellets were freeze-dried and homogenized as described above, to standardize the input material added to the fermentation media.\u003c/p\u003e\n\u003ch3\u003eAnaerobic batch fermentation\u003c/h3\u003e\n\u003cp\u003eSix different media (four seaweed, one high-fiber and one low-fiber control) were prepared according to a described protocol [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The high-fiber control contained a mix of glucose (0.4 g/L), xylan (0.8 g/L), apple pectin (0.8 g/L), arabinogalactan (0.8 g/L), and starch (5 g/L), while no polysaccharides were added to the seaweed-enriched media and the low-fiber medium (negative control). After autoclaving, the four different seaweed samples (7.8 g/L) were added to the seaweed media.\u003c/p\u003e\u003cp\u003eOne day before the fecal fermentation, 12 mL of each medium (in quadruplicates) was transferred into 15 mL Falcon tubes and kept in the anaerobic workstation at 37\u0026deg;C overnight to make the medium anoxic. On day 1, 250 \u0026micro;L of fecal slurry was inoculated into the media in the anaerobic workstation. Four adult human fecal samples, selected for their high abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e, a genus well known as efficient fiber-fermenters (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), were obtained from the PRIMA human baseline study [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Samples were collected at 0 h, 24 h and 48 h and centrifuged for 10 minutes at 10,000 \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C. The pH of the supernatant was measured on the sampling day with the SevenCompact\u0026trade; pH/ion meter S220 (Mettler Toledo\u0026trade;, Ohio, USA). The remaining supernatant and the pellets were stored at -20\u0026deg;C until analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFecal samples, selected for their dominant relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDonor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eBifidobacterium\u003c/em\u003e (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDominant \u003cem\u003eBifidobacterium\u003c/em\u003e spp.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e58.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eB. adolescentis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e62.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eB. adolescentis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. longum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e68.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eB. catenulatum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e56.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eB. catenulatum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBacterial 16S rRNA Sequencing\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eDNA extraction, PCR and library preparation\u003c/h2\u003e\u003cp\u003eDNA was extracted from fecal pellets using the DNeasy\u0026reg; PowerLyzer\u0026reg; PowerSoil\u0026reg; Kit (Qiagen, Hilden, Germany), following the Quick-Start protocol [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. DNA concentration was measured with the Qubit\u0026trade; dsDNA high sensitivity (HS) fluorescence assay (Thermo Fisher Scientific, Waltham, USA) in a 96-well plate, according to the manufacturer\u0026rsquo;s protocol. Fluorescence was measured at an excitation/emission wavelength of 495/525 nm using Kaleido 3.0 software (Kaleido Technologies, New Delhi, India) and the Perkin Elmer Enspire 2300 Multi-mode Microplate Reader (New Life Scientific Inc., Cridersville, USA).\u003c/p\u003e\u003cp\u003ePCR amplification for library preparation and subsequent sequencing on the Ion Torrent was done, as previously described [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], using1 \u0026micro;M forward primer (PBU) and 1 \u0026micro;M reverse primer (PBR; 5\u0026prime;-trP1-adaptor-ATTACCGCGGCTGCTGG-3\u0026prime;) and 2 \u0026micro;L extracted DNA (5 ng/\u0026micro;L) in a total reaction volume of 20 \u0026micro;L. The unique sequence of each primer is provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. DNA was amplified by PCR with the Veriti\u0026reg; 96-Well Thermal Cycler Model 9902 (Life Technologies, Carlsbad, USA). Then, the DNA was purified using the HighPrep\u0026trade; PCR Clean Up (MagBio Genomics, Gaithersburg, USA), following the manufacturer\u0026rsquo;s protocol, after which the DNA concentration was measured again. The DNA samples were mixed to an equimolar mixture and sent to Ion Torrent sequencing on a 318-chip using the Ion OneTouch\u0026trade; 200bp Template Kit v2 DL (Life Technologies, Carlsbad, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S rRNA gene amplicon sequencing bioinformatic analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 16S rRNA gene amplicon data were processed with our in-house-pipeline [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In short, raw amplicon sequences were demultiplexed using cutadapt (v. 4.1) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], denoised using DADA2 (v. 1.22) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Then, ASVs amplicon sequence variants were classified against RDP training set_18 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The downstream processing was done with Phyloseq (v. 1.42.0) in R (v. 4.4.3) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] for further data processing. ASVs were decontaminated with the decontam package [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. All ASVs on genus level can be found in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eMetabolites quantification\u003c/h3\u003e\n\u003cp\u003eThe SCFAs in the supernatants were analyzed by MS-Omics Aps (Vedb\u0026aelig;k, Denmark). In short, the supernatants were transferred to Spin-X\u0026reg; centrifuge tubes (pore size of 0.22 \u0026micro;m) and centrifuged at 15,000 \u003cem\u003eg\u003c/em\u003e for 5 minutes at 4\u0026deg;C. Sample analysis was carried out by MS-Omics as follows. Samples were acidified using hydrochloride acid, and deuterium labelled internal standards were added. All samples were analyzed in a randomized order. Analysis was performed using a high polarity column (Zebron\u0026trade; ZB-FFAP, GC Cap. Column 30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m) installed in a GC (7890B, Agilent) coupled with a time-of-flight MS (Pegasus\u0026reg; BT, LECO)]. The system was controlled by [ChromaTOF\u0026reg; (LECO)]. Raw data was converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2021b (Mathworks, Inc.) using the PARADISe software as previously described [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmino acid metabolites were analyzed using liquid chromatography-mass spectrometry (LC-MS) with L-tryptophan (indole-d5, 98%) as internal standard, as previously described [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In short, 2 \u0026micro;L of sample was analyzed with an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry system consisting of a Dionex Ultimate 3000 RS liquid chromatograph (Thermo Scientific) coupled to a Bruker maXis time-of-flight mass spectrometer equipped with an electrospray interphase (Bruker Daltonics) operating in positive mode to analyze tyramine and tryptamine and in the negative mode to analyze all other amino acid metabolites. The analytes were separated on a Poroshell 120 SB-C18 column with a dimension of 2.1\u0026times;100 mm and 2.7 \u0026micro;m particle size (Agilent Technologies) as previously described [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Quality control samples and standard mix solutions were analyzed before and after the sample set, with two standards measured after every ten samples. Data was processed using QuantAnalysis v.2.2 (Bruker Daltonics), applying calibration curves (with quadratic regression) generated for each metabolite from all standards. Calibration curves were made by plotting analyte-to-internal standard peak area ratios against calibration standard concentrations, as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLactic acid content was measured with High-Pressure Liquid Chromatography (HPLC) with a refractive index detector. Supernatant was mixed with 5 mM sulfuric acid solution (1:4 ratio), vortexed at 8,000 rpm for 5 minutes and filtered through a 0.2 \u0026micro;m syringe filters before transferring 500 \u0026micro;L sample into HPLC vials. The HPLC was equipped with an Aminex HPX-87H column (Bio-Rad, USA) in an Ultimate HPLC system (Dionex, Thermo Fisher Scientific, USA) and coupled to a Shodex RI-101 refractive index detector (Showa Denko K.K., Isesaki, Japan) for detection. The mobile phase consisted of 5 mM sulfuric acid. The column was set at 60\u0026deg;C, with a flow rate of 0.5 mL/min and an injection volume of 20 \u0026micro;L. Standards were prepared at concentrations of 50, 100, 500, and 1,000 \u0026micro;g/mL (ppm) in 5 mM sulfuric acid and filtered through a 0.22 \u0026micro;m syringe filter. Chromeleon 2.0 software (Thermo Fisher Scientific, Waltham, USA) was used for data analysis.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eAll data was analyzed, and figures were made using R (v. 4.4.3) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and figures were combined in Inkscape (v. 0.92.1). Normal distributions were checked using the Shapiro-Wilk test. The significant differences between short-chain fatty acid and amino acid concentrations in the different media at a certain time point were determined with groupwise ANOVA and Tukey's honestly significant difference (HSD). Significance is defined as p\u0026thinsp;\u003cb\u003e\u0026le;\u003c/b\u003e\u0026thinsp;0.05 and a trend is defined as 0.05\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u003cb\u003e\u0026le;\u003c/b\u003e\u0026thinsp;0.10, as used in the results and discussion. The media pH values were compared using emmeans corrected for false discovery rate (FDR) within each time point and within each fermentation medium. Subsequently, Pearson\u0026rsquo;s correlation test was done to test Pearson\u0026rsquo;s correlation coefficient between pH and the produced SCFAs and lactic acid, combining all measurements from each medium and each replicate.\u003c/p\u003e\u003cp\u003eTo determine the beta dispersion in the beta diversity analysis, one-way ANOVA was used to identify possible differences between the groups, followed by pairwise PERMANOVA with FDR correction, using the \u003cem\u003eecole\u003c/em\u003e package [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The full statistical analyses can be found on \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ameevh/Sea4Gut\u003c/span\u003e\u003cspan address=\"https://github.com/ameevh/Sea4Gut\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eProcessed seaweed (blanched, oven-dried, or fermented with \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e) was digested (INFOGEST 2.0), and the undigested material was added to fecal fermentation media. Fecal samples of four donors were inoculated, and samples were collected at 0, 24, and 48 h for pH, 16S rRNA sequencing, and metabolite profiling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSeaweed supplementation affects fecal microbiota composition\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt baseline (0 h), alpha diversity did not differ between fermentation media (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the low-fiber medium, however, both Shannon index and observed richness increased significantly after 24 h (p\u0026thinsp;=\u0026thinsp;0.007, p\u0026thinsp;=\u0026thinsp;0.033) and further after 48 h (p\u0026thinsp;=\u0026thinsp;0.003, p\u0026thinsp;=\u0026thinsp;0.003) compared to baseline. The seaweed-supplemented media had similar alpha diversities over time, except for the oven-dried medium where the Shannon index increased significantly from 24 to 48 h (p\u0026thinsp;=\u0026thinsp;0.044). After 24 h of fermentation, the Shannon index in the low-fiber medium was significantly higher compared to the high-fiber medium (p\u0026thinsp;=\u0026thinsp;0.048), the fermented seaweed medium (p\u0026thinsp;=\u0026thinsp;0.013) and the oven-dried medium (p\u0026thinsp;=\u0026thinsp;0.048), and observed richness in the low-fiber medium was significantly higher compared to all other media (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). After 48 h, the Shannon index of the low-fiber medium was significantly higher than the high-fiber medium (p\u0026thinsp;=\u0026thinsp;0.002) and the fermented seaweed medium (p\u0026thinsp;=\u0026thinsp;0.025), and the observed richness was significantly higher in the low-fiber medium compared to all other media with the strongest difference with the high-fiber medium (p\u0026thinsp;=\u0026thinsp;4.7*10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eDespite visual differences in the PCoA plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), there were no significant differences in the Aitchison distance between the different media at all time points (determined with PERMANOVA, Supplementary Table S3). Significant differences in Aitchison distance between all donors were observed at all time points (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Supplementary Table S4), indicating a strong donor effect on the beta diversity.\u003c/p\u003e\u003cp\u003eOn the order level, Bifidobacteriales and Clostridiales had the highest relative abundances at baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). After 24 h, the relative abundance of Bifidobacteriales decreased in the low-fiber medium and the four seaweed-supplemented media, while the abundances of Clostridiales and Enterobacterales increased mostly in the seaweed-supplemented media. In donors 2, 3 and 4, more distinct differences were also observed between the seaweed-supplemented media and the low-fiber medium.\u003c/p\u003e\u003cp\u003eSimilar to beta diversity, microbiome compositional changes appeared mostly donor-driven and largely independent of seaweed processing. \u003cem\u003eBifidobacterium\u003c/em\u003e was the most abundant genus at baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Changes in the microbial composition were strongly donor-dependent, but the seaweed-supplemented media also showed a different microbial composition compared to the high-fiber and low-fiber media. The high-fiber media inoculated with donors 3 and 4 had a large relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e during fermentation, while the relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e decreased in most samples. In the high-fiber medium inoculated with donor 1, a high relative abundance of unclassified Clostridiaceae was observed after 24 and 48 h, while the high-fiber medium inoculated with donor 2 showed a high relative abundance of \u003cem\u003eClostridium sensu stricto\u003c/em\u003e during fermentation. The relative abundance of \u003cem\u003eEscherichia/Shigella\u003c/em\u003e increased in the seaweed-supplemented media inoculated with donor 2, 3 and 4 and in the seaweed-supplemented media inoculated with donor 1, the relative abundance of unclassified Enterobacteriaceae increased after 24 and 48 h. Furthermore, an increase in the relative abundance of unclassified Lachnospiraceae was observed in donor 2 increased after 24 and 48 h. Seaweed-supplemented media inoculated with donors 1 and 3 showed an increase in relative abundance of \u003cem\u003eButyrivibrio\u003c/em\u003e after 24 h, except for the LAB fermented seaweed in donor 1.\u003c/p\u003e\u003cp\u003eAfter 48 h, an increase in relative abundance of \u003cem\u003eBacteroides\u003c/em\u003e was observed in all seaweed-supplemented media for all donors, and mostly in donors 1, 3 and 4. \u003cem\u003eBacteroides\u003c/em\u003e species differed in the samples grown in seaweed-supplemented media, compared to samples grown in the low-fiber medium (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Table S5). Although not-significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the highest abundance of \u003cem\u003eBacteroides\u003c/em\u003e spp. was \u003cem\u003eBacteroides kribbi/koreensis/ovatus\u003c/em\u003e in all seaweed media and the high-fiber medium, compared to \u003cem\u003eBacteroides uniformis\u003c/em\u003e in the low-fiber medium. Interestingly, \u003cem\u003eBacteroides ovatus\u003c/em\u003e has been shown to degrade alginate, a complex carbohydrate found in seaweed [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThree most abundant \u003cem\u003eBacteroides\u003c/em\u003e spp. in the high-fiber, low-fiber and the four seaweed-supplemented media, combining the 4 donors at 24 and 48 h (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;8).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRelative abundance (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eHigh-fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ekribbi/koreensis/ovatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.0064\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0138\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003euniformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.0014\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0044\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003exylanisolvens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.0006\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0013\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eLow-fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003euniformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.9772\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2355\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0062\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ethetaiotaomicron\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.3801\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2893\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0090\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eintestinalis/cellulosyliticus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.3424\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4324\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eUntreated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ekribbi/koreensis/ovatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.7383\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0577\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003exylanisolvens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.6931\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9346\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003euniformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.7392\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2170\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eBlanch\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ekribbi/koreensis/ovatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.1961\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9356\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003exylanisolvens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.2308\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4284\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003euniformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.5951\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9409\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eFerment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ekribbi/koreensis/ovatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.8594\u0026thinsp;\u0026plusmn;\u0026thinsp;14.9231\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003exylanisolvens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.8855\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5986\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003euniformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.6341\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6449\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eOven\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ekribbi/koreensis/ovatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.2904\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6847\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003euniformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.6337\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9311\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eASV_0017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003exylanisolvens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.4071\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6038\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSeaweed supplementation does not affect short-chain and branched short-chain fatty acid production in adult human fecal samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter 24 h of fermentation, formic acid (3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94 mM) and lactic acid (24.01\u0026thinsp;\u0026plusmn;\u0026thinsp;17.02 mM) were detected in significantly higher concentrations in the high-fiber medium than all other media (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Lactic acid was only detected in the high-fiber medium after 24 and 48 h, and in one sample from the fermented seaweed medium after 24 h (3.27 mM, Supplementary Table S6). Total SCFAs increased in most media from 24 to 48 h and, except the high-fiber medium where the concentration of total SCFAs did not increase significantly (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) from 24 to 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Acetic acid was significantly higher in the seaweed media compared to the high-fiber medium, with highest concentrations found in the blanched seaweed medium (51.09\u0026thinsp;\u0026plusmn;\u0026thinsp;5.11 mM). No differences in butyric acid were observed between the different media at both 24 and 48 h. Propanoic acid increased significantly from 24 to 48 h in the untreated seaweed medium and the fermented seaweed medium. After 48 h, the propanoic acid remained low in the high-fiber medium (0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 mM), which was significantly lower than the propanoic acid concentration in all other media (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The most abundant branched short-chain fatty acid (BCFA) was isobutyric acid, which was significantly lower in the high-fiber medium compared to the low-fiber medium (p\u0026thinsp;=\u0026thinsp;0.006), the untreated seaweed medium (p\u0026thinsp;=\u0026thinsp;0.022), the blanched seaweed medium (p\u0026thinsp;=\u0026thinsp;0.009) and the oven-dried seaweed medium (p\u0026thinsp;=\u0026thinsp;0.031, Supplementary Fig.\u0026nbsp;1, Supplementary Table S7). Following a similar pattern, the concentrations of isovaleric acid and 2-methyl butanoic acid remained low after 48 h in the high-fiber medium and were significantly lower than in the low-fiber and seaweed media (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The BCFA 3-methyl pentanoic acid was also found in low concentrations in all media and was significantly lower after 48 h in the high-fiber medium than the low-fiber medium (p\u0026thinsp;=\u0026thinsp;0.035) and the blanched seaweed medium (p\u0026thinsp;=\u0026thinsp;0.036).\u003c/p\u003e\u003cp\u003eDespite the higher concentrations of total SCFAs, acetic acid and butyric acid in the low-fiber medium and the seaweed media, the pH only decreased significantly in the high-fiber medium from baseline (pH\u0026thinsp;=\u0026thinsp;6.6) to 24 (pH\u0026thinsp;=\u0026thinsp;4.6) and 48 h (pH\u0026thinsp;=\u0026thinsp;4.6, Supplementary Table S8). Using Pearson\u0026rsquo;s correlation, a strong negative correlation was observed between pH and the formic acid concentration (r\u003csub\u003es\u003c/sub\u003e=-0.61, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a very strong negative correlation was observed between pH and the lactic acid concentration (r\u003csub\u003es\u003c/sub\u003e=-0.86, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSeaweed supplementation does not affect the production of phenylalanine, tryptophan and tyrosine\u003c/h2\u003e\u003cp\u003eTo get a broader idea of the effects of seaweed on host-microbiome interactions, we also investigated the microbial metabolism of aromatic amino acids and their derivatives.\u003c/p\u003e\u003cp\u003eAfter 24 h, phenylalanine tended to be higher in the high-fiber medium, compared to the low-fiber medium and seaweed-supplemented media (both p\u0026thinsp;=\u0026thinsp;0.10), after which the phenylalanine concentration decreased after 48 h in all media (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, its derivative phenyllactic acid was found in significantly higher concentrations in the high-fiber medium after 48 h (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Supplementary Fig.\u0026nbsp;2). Tryptophan was highest in the oven-dried seaweed medium and significantly lower in the low-fiber medium (p\u0026thinsp;=\u0026thinsp;0.050) after 24 h. After 48 h, a decrease in tryptophan concentration was observed in all samples except the high-fiber medium. No significant differences were observed in the concentrations of tryptophan derivatives between the different media. The highest tyrosine concentration was found in the untreated seaweed medium after 24 h, which was significantly higher than in the high-fiber medium (p\u0026thinsp;=\u0026thinsp;0.044) and the low-fiber medium (p\u0026thinsp;=\u0026thinsp;0.041). A decrease in tyrosine concentration was observed in all samples after 48 h with non-significant differences between all media, while the tyrosine derivatives 4-hydroxyphenyllactic acid and 4-hydroxyphenylpropionic acid were produced in significantly higher concentrations in the fiber-rich medium (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe found that alpha diversity was highest after 24 and 48 h in the low-fiber medium for both the Shannon index and the observed richness. High-protein/low-fiber diets have previously shown to increase alpha diversity \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, the reduction in observed richness after 24 and 48 h in the high-fiber medium can be a result of the pH reduction in this medium, since not all bacteria can survive under acidic conditions [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] which therefore reduces richness [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Despite a visual difference between the high-fiber medium and the other media in the PCoA plot, no significant differences were found between the Aitchison distance of the different media, possibly due to the low sample size (n\u0026thinsp;=\u0026thinsp;4) or the strong donor-dependent effect.\u003c/p\u003e\u003cp\u003eThe different microbial compositions of the seaweed-supplemented media on order and genus levels after 24 and 48 h, compared to the high-fiber and low-fiber media, indicate seaweed fermentation by the fecal bacteria. Contrary to findings of increased relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e after \u003cem\u003ein vitro\u003c/em\u003e fermentation of seaweed extracts [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], our results show a decrease in relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e across the seaweed-supplemented media and in the low-fiber medium. This decrease may be explained by the high relative baseline abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e, leading to a decrease in relative abundance without necessarily affecting the absolute concentrations. Furthermore, we observed increased the relative abundances of unclassified Lachnospiraceae in the seaweed media of donor 2. Similarly, increased relative abundances of Lachnospiraceae were reported after fermentation of another brown alga \u003cem\u003eLaminiaria digitata\u003c/em\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Lachnospiraceae are common fiber degraders that have beneficial effects on the gut microbiome and host health by fermenting non-digestible plant polysaccharides, such as cellulose, into acetic acid, butyric acid and propionic acid [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. \u003cem\u003eEscherichia/Shigella\u003c/em\u003e abundances increased after seaweed supplementation in all donors. Although high abundances of \u003cem\u003eEscherichia/Shigella\u003c/em\u003e are sometimes linked to low nutrient availability, increased \u003cem\u003eEscherichia coli\u003c/em\u003e abundances were previously reported after \u003cem\u003ein vitro\u003c/em\u003e fermentation of the brown seaweed \u003cem\u003eEcklonia radiata\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The higher relative abundance of \u003cem\u003eBacteroides\u003c/em\u003e spp. in the seaweed-supplemented media may be attributed to the laminarin and alginate in seaweed, which promotes the growth of species such as \u003cem\u003eB. ovatus\u003c/em\u003e and \u003cem\u003eB. dorei\u003c/em\u003e and is linked to the production of acetic and propionic acids. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur study found that the low-fiber medium and the seaweed-supplemented media had significantly higher SCFA concentrations compared to the high-fiber medium after 48 h, except for formic and lactic acid which were significantly higher in the high-fiber medium compared to all other media. The significantly higher concentration of acetic acid in our processed seaweed media could also be caused by fermentation of seaweed fibers, since the high-fiber and low-fiber medium controls had similar acetic acid concentrations. However, the acetic acid concentration in the low-fiber medium suggests the presence of bacteria that are capable of fermenting amino acids into acetic acid, most likely through proteolytic fermentation or the Stickland fermentation of glycine into acetic acid by Cluster XI \u003cem\u003eClostridia\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The butyric acid concentration in the seaweed-supplemented media was not significantly higher than in the high-fiber medium or low-fiber medium, possibly due to the large outlier of donor 2 in the high-fiber medium. The higher, though not significant, concentrations of butyric acid in the seaweed media may be linked to \u003cem\u003eButyrivibrio\u003c/em\u003e and other members of the Lachnospiraceae family, known butyric acid producers [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] that were relatively abundant in donors 2 and 3 after 24 and 48 h. Proteolytic fermentation could explain why SCFA concentrations in the low-fiber medium did not differ significantly from those in the seaweed media.\u003c/p\u003e\u003cp\u003eThe higher concentrations of propanoic acid, isobutyric acid and isovaleric acid in the low-fiber medium and seaweed-supplemented media compared to the high-fiber medium can also be a result of proteolytic fermentation [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and Bacteroidetes can, for example, produce propanoic acid from peptides [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. This could explain the higher relative abundances of \u003cem\u003eBacteroides\u003c/em\u003e in the media with less nutrient accessibility, like the low-fiber and the seaweed-supplemented media.\u003c/p\u003e\u003cp\u003ePhenyllactic acid (PLA), the aromatic lactic acid of phenylalanine, was significantly higher in the high-fiber medium than all other media at 48 h. Similarly, 4-hydroxyphenyllactic acid, the aromatic lactic acid of tyrosine, was significantly higher in the high-fiber medium than in all other media at both 24 and 48 h. No differences were observed in the concentration of indole lactic acid; the aromatic lactic acid produced from tryptophan. High concentrations of aromatic lactic acids are associated with fiber degradation, such as fructo-oligosaccharides and galacto-oligosaccharides, which favor increased abundance bacteria such as \u003cem\u003eClostridium sensu stricto\u003c/em\u003e, Lachnospiraceae [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and \u003cem\u003eBifidobacterium\u003c/em\u003e spp.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In our study, high relative abundances of \u003cem\u003eClostridum sensu stricto\u003c/em\u003e, unclassified genera of Clostridiaceae and \u003cem\u003eBifidobacterium\u003c/em\u003e were observed in the high-fiber medium, compared to the other media, for all donors after both 24 and 48 h, which may have contributed to the production of phenyllactic acid in this medium. The aromatic propionic acids of phenylalanine and tryptophan, phenylpropionic acid and indole-3-propionic acid, were detected in low amounts in the high-fiber medium. In the other samples, the concentrations were slightly higher but due to the high standard deviations between the samples, no significant differences were observed between the media. The aromatic propionic acid of tyrosine, 4-hydroxyphenylpropionic acid, was detected in significantly higher concentrations in the high-fiber medium compared to the other media after 48 h. This aromatic amino acid is known to be produced during fiber degradation [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], for example by Lachnospiraceae [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In our study, however, the fiber-fermenting unclassified Lachnospiraceae had the highest relative abundance in the seaweed-supplemented media in donor 2 and was hardly present in the samples from the high-fiber medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The decarboxylase products of tryptophan and tyrosine, namely tryptamine and tyramine, were not detected in significantly different concentrations between the different media or time points. Tryptamine was produced in higher concentrations in the high-fiber medium and remained relatively low in the seaweed-supplemented media, without significant differences between the media due to the large standard deviations. Similarly, the other indole-derived metabolites were detected without significant differences between the media.\u003c/p\u003e\u003cp\u003eOverall, we conclude that seaweed supplementation modifies the composition of the microbiome, regardless of the pre-digestive processing methods, but does not significantly affect metabolite production. A follow-up study testing alternative processing or a combined intake of \u003cem\u003eSaccharina latissima\u003c/em\u003e with enzymes or probiotics are recommended to study how to enhance the fermentability of seaweed polysaccharides in the gut.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Katja Ann Kristensen, Bodil Madsen and Steffen Valencia Bach for their technical support in the experiments and Mikael Pedersen for his valuable contribution performing the LC-MS. We thank Matthew Robert Carey for his help with R and Josep Rubert for his external supervision during the first part of the project. We thank Nicola Proch\u0026aacute;zkov\u0026aacute; and Henrik Roager for providing the fecal samples that were collected in the PRIMA human baseline study (NCT04804319). Finally, we thank M. D. Dalgaard at the DTU in-house facility (DTU Multi-Assay Core, DMAC) for performing the 16S rRNA gene sequencing.\u003c/p\u003e\u003cp\u003eThis study was part of the project Seaweed for gut health, funded by Ekhagastifelsen (Seaweed for gut health, grant number 69, year-2022). The fecal samples used in the study were collected in the PRIMA human baseline study (NCT04804319).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAJVH, AKS, MFL, SLH and ISD designed the experiments. ISD and EZ processed the seaweed. AJVH, MK and ISD performed the INFOGEST 2.0. AJVH performed and analyzed the in vitro fermentation experiments and prepared the samples for LC-MS, GC-MS and HPLC. EZ performed the HPLC experiment and initial analyses. AJVH prepared the samples for sequencing, and AJVH and MSM analyzed the data. AJVH, AKS and MFL drafted the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was part of the project Seaweed for gut health, funded by Ekhagastifelsen (Seaweed for gut health, grant number 69, year-2022). The authors would like to thank Katja Ann Kristensen, Bodil Madsen and Steffen Valencia Bach for their technical support in the experiments and Mikael Pedersen for his valuable contribution performing the LC-MS. We thank Matthew Robert Carey for his help with R and Josep Rubert for his external supervision during the first part of the project. We thank Nicola Proch\u0026aacute;zkov\u0026aacute; and Henrik Roager for providing the fecal samples that were collected in the PRIMA human baseline study (NCT04804319). Finally, we thank M. D. Dalgaard at the DTU in-house facility (DTU Multi-Assay Core, DMAC) for performing the 16S rRNA gene sequencing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe sequencing data have been deposited into the Sequence Read Archive under BioProject PRJNA1337439. The full statistical analyses for this project has been uploaded to github and are available at the following URL: https://github.com/ameevh/Sea4Gut.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGull\u0026oacute;n P, Astray G, Gull\u0026oacute;n B, et al. Inclusion of seaweeds as healthy approach to formulate new low-salt meat products. 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Environ Microbiol. 2016;18:2214\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1462-2920.13158\u003c/span\u003e\u003cspan address=\"10.1111/1462-2920.13158\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"seaweed, gut microbiome, SCFA, aromatic amino acids","lastPublishedDoi":"10.21203/rs.3.rs-7794319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7794319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eSeaweed consumption gains popularity due to its high micronutrient, protein and fiber content. While seaweed extracts show promising effects on the gut, it remains unclear if processing of raw seaweed changes the nutrient accessibility for the gut microbiota. This study aimed to investigate how different processing methods affect the prebiotic potential of a common brown seaweed, \u003cem\u003eSaccharina latissima\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe seaweed was processed (blanched, oven-dried or fermented by \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e) and underwent simulated upper-gastrointestinal digestion with INFOGEST 2.0 prior to \u003cem\u003ein vitro\u003c/em\u003e fermentation, using four adult human fecal donors. Samples were analyzed for their microbiome composition and produced metabolites.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSeaweed-supplemented media changed the fecal microbial composition compared to low-fiber and high-fiber controls. Specifically, seaweed increased the relative abundances of Bacteroidales, \u003cem\u003eEscherichia/Shigella\u003c/em\u003e and unclassified Lachnospiraceae, while \u003cem\u003eBifidobacteria\u003c/em\u003e decreased. Regardless of processing, seaweed-supplemented cultures showed similar alpha and beta diversity indicating that processing did not modulate seaweed\u0026rsquo;s impact on the microbial composition. The short-chain fatty acid and amino acid concentrations were similar in the seaweed-supplemented media and the low-fiber medium after fermentation, while formic acid and lactic acid were significantly higher in the high-fiber medium, compared to other media, and negatively correlated with the final pH after fermentation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eSeaweed altered the microbiome composition, however with minor effects of seaweed processing. These compositional shifts did not translate into metabolic changes, suggesting that seaweed components were largely inaccessible to microbes under the tested conditions. Further studies are recommended to investigate alternative processing and co-formulations to enhance seaweed fermentability.\u003c/p\u003e","manuscriptTitle":"Seaweed Fermentation Modulates Gut Microbiome Composition Without Altering Metabolite Output In Vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-20 16:57:57","doi":"10.21203/rs.3.rs-7794319/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"469c0e79-2e82-4c1b-87e6-bb3e04215a60","owner":[],"postedDate":"November 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-27T06:53:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-20 16:57:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7794319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7794319","identity":"rs-7794319","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}