HPLC-MS-MS quantification of short-chain fatty acids secreted by probiotic strains

HPLC-MS-MS quantification of short-chain fatty acids secreted by probiotic strains | 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 HPLC-MS-MS quantification of short-chain fatty acids secreted by probiotic strains Marco Calvigioni, Andrea Bertolini, Simone Codini, Diletta Mazzantini, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2128764/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 Short-chain fatty acids (SCFAs) are the main by-products of microbial fermentations occurring in the human intestine and are directly involved in the host’s physiological balance. As impaired gut concentrations of acetic, propionic, and butyric acids are often associated with systemic disorders, the administration of SCFA-producing microorganisms has been suggested as attractive approach to solve symptoms related to SCFAs deficiencies. In this research, nine probiotic strains ( Bacillus clausii NR, OC, SIN, and T , Bacillus coagulans ATCC 7050 , Bifidobacterium breve DSM 16604 , Limosilactobacillus reuteri DSM 17938, Lacticaseibacillus rhamnosus ATCC 53103, and Saccharomyces boulardii CNCM I-745) commonly included in commercial formulations were tested for their ability to secrete SCFAs by using an improved and sensitive protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS). All tested microorganisms were shown to secrete acetic acid, with only B. clausii and S. boulardii additionally able to produce propionic and butyric acids. Quantitative differences in the secretion of SCFAs were also evidenced. The application of HPLC-MS-MS may help in the analysis of SCFA production by probiotics, especially for their administration as targeted bacteriotherapy to improve SCFAs deficiencies. short-chain fatty acids probiotics HPLC-MS-MS acetic acid propionic acid butyric acid secretion Figures Figure 1 Figure 2 Introduction The human gut microbiota actively cooperates in maintaining physiological balance, in nutrient catabolism and absorption, as well as in the production of short-chain fatty acids (SCFAs) [ 1 ]. SCFAs are small carboxylic organic acids with a backbone of at most six carbon atoms. They represent the largest group of metabolic products obtained by microbial fermentation of dietary complex carbohydrates, otherwise undegradable by the human digestive system [ 2 ]. Faecalibacterium prausnitzii , Eubacterium rectale , Akkermansia muciniphila , Clostridium spp., Bifidobacterium spp., Lactobacillus spp., and members belonging to families Ruminococcaceae , Lachnospiraceae , and Bacteroidaceae are the main actors able to produce SCFAs in the human gut as result of bacterial metabolism [ 3 ]. The highest intestinal SCFA concentrations are reached by acetic, propionic, and butyric acids, which are known to exert countless beneficial effects and orchestrate several physiological functions [ 4 , 5 ]. Acetic acid is the structurally simplest fatty acid and can, therefore, be used as metabolic substrate in fatty acid biosynthesis and Krebs’ cycle [ 6 ]. Besides being a major energy source for skeletal muscle [ 6 ], acetic acid is also involved in lipid metabolism of the liver and adipose tissue [ 7 ]. The administration of acetate was reported to decrease food intake, body weight gain, blood cholesterol, and triglyceride levels [ 7 ]. Propionic acid improves the barrier function and epithelial integrity in the gut, and impacts on glucose and lipid liver homeostasis [ 8 ]. Previous studies reported the ability of propionic acid to indirectly modulate gene expression and cell metabolism through immunomodulatory mechanisms [ 8 , 9 ]. Butyric acid is probably the most multifunctional SCFA, acting as energy source for colonic epithelial cells and promoting beta oxidation rather than glycolysis [ 10 ]. Enhancing the expression of tight-junction proteins, butyric acid facilitates the maintenance of the intestinal barrier integrity, thus hampering epithelial invasion by pathogenic microorganisms [ 10 ]. It has also been revealed to inhibit tumoral cell expansion and release of pro-inflammatory cytokines [ 11 , 12 ], thus demonstrating anti-carcinogenic and anti-inflammatory properties. All the main SCFAs were found able to differently modulate appetite and energy intake by increasing colonic glucagon-like peptide-1 secretion [ 13 ], thus reducing food craving [ 14 ]. Moreover, they are also directly involved in the microbiota-gut-brain axis neuroendocrine signaling, brain physiology, and neuronal damage prevention [ 15 ]. A correlation between lower intestinal SCFA concentrations and the exacerbation of different pathological conditions, such as inflammatory bowel diseases and neurological and neuropsychiatric disorders ( e.g ., autism, depression, Alzheimer and Parkinson’s diseases, multiple sclerosis), has been confirmed [ 15 – 19 ]. The administration of properly selected probiotic microorganisms able to counteract SCFA deficiencies appeared as a promising novel approach to co-adjuvate the management of these conditions in humans and improve pathology-associated symptoms. For instance, psychobiotics producing SCFAs, neurotransmitters, and neuroendocrine hormones were revealed to provide wide health benefits to patients suffering from mental and neurological disorders [ 20 ]. Several studies demonstrated the ability of some probiotics to shift the gut microbiota composition towards higher abundances of SCFA-producing species [ 3 ]. Acetic, propionic, and butyric acids were also scarcely demonstrated to be secreted by probiotic microorganisms themselves in different amounts and in a species- or even strain-specific modality [ 1 , 20 – 22 ], thus pointing out SCFA production by probiotics as beneficial additional feature for certain clinical conditions. In this study, the amount of acetic acid, propionic acid, and butyric acid actively secreted by nine probiotic strains was quantified by using an optimized and sensitive protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS). Materials And Methods Materials and chemicals Acetic acid (purity ≥ 99.8%), propionic acid (≥ 99.5%), propionic acid-2,3- 13 C 2 (99 atom % 13 C) and butyric acid (≥ 99.5%) analytical standards, water LC-MS grade, acetonitrile LC-MS grade (ACN), formic acid (FA, ≧ 98%), hydrochloric acid (ACS reagent, 37% w/w), NaOH solution 1 M, diethyl-ether (≥ 99%), 3-nitrophenylhydrazine (3NPH) hydrochloride, N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide (EDC) hydrochloride, Bifidus Selective agar (BSM), BSM supplement, and Sabouraud-2% dextrose agar (SDA) were bought from Merck KGaA (Darmstadt, Germany). Acetic acid 13 C 2 (99 atom % 13 C) analytic standard was purchased from Cambridge Isotope Laboratories (Tewksbury, USA). Brain Heart Infusion agar (BHI) was obtained from Biolife (Monza, Italy), while Trypticase Soy agar (TSA) supplemented with 5% horse blood (TSH) was from bioMérieux (Paris, France). De Man, Rogosa, and Sharpe (MRS) agar was acquired from Thermo Fisher Scientific (Waltham, USA). Microbial strains and growth conditions Bacillus clausii NR, OC, SIN, and T (isolated from Enterogermina, Sanofi, Paris, France, and declared on the product label), Bacillus coagulans ATCC 7050 (isolated from Lactò Più, Recordati, Milan, Italy, and declared on the product label), Bifidobacterium breve DSM 16604 (isolated from Neovaxitiol, IBSA Farmaceutici, Lodi, Italy, and declared on the product label), Limosilactobacillus reuteri DSM 17938 (isolated from Reuflor, Italchimici, Brescia, Italy, and declared on the product label with the old nomenclature Lactobacillus reuteri DSM 17938), Lacticaseibacillus rhamnosus ATCC 53103 (isolated from Dicoflor, Dicofarm, Roma, Italy, and declared on the product label with the old nomenclature Lactobacillus rhamnosus ATCC 53103), and Saccharomyces boulardii CNCM I-745 (isolated from Codex, Zambon, Bresso, Italy, and declared on the product label) were tested in the present investigation. B. clausii NR, OC, SIN, and T were isolated from Enterogermina as described in a previous work [ 23 ]. Stock solutions of NR, OC, SIN, and T kept at -80°C were thawed and streaked on BHI agar plates containing 1% (w/v) of glucose (BHIG). Plates were incubated at 37°C for 48h. B. coagulans was seeded on TSH and plates were incubated at 37°C for 48-72h. B. breve was propagated on BSM containing 0.116 g/L of BSM supplement and grown at 37°C for 48-72h in anaerobic atmosphere, generated by using Thermo Scientific™ AnaeroGen™ Compact (Thermo Fisher Scientific). L. reuteri and L. rhamnosus were streaked on MRS agar and plates were incubated at 37°C for 48h in 5% CO 2 -enriched atmosphere, generated by using Thermo Scientific™ CO 2 Gen™ Compact (Thermo Fisher Scientific). S. boulardii was seeded on Sabouraud-2% dextrose agar and plates were incubated at 30°C for 48-72h. Preparation of culture supernatants Different liquid culture media ( i.e. , Luria Bertani broth, BHI and BHIG broths, RPMI 1640 medium) were tested for microbial propagation. BHIG broth was selected as the most suitable culture medium, as it allowed an optimal uniform replication of all microorganisms and contained amino acids and glucose, which are important substrates for SCFA biosynthesis [ 24 ]. Thus, for each strain, a well-isolated colony was inoculated in 5 mL of BHIG broth. Suspensions were incubated overnight at 37°C in constant shaking. Subsequently, 100 µL of microbial cultures were inoculated in 25 mL of fresh BHIG medium. Cultures were incubated at 37°C up to an optical density at 600 nm (OD 600 ) of 1.8 and then centrifuged at 3,870 rcf for 20 min at 4°C. Supernatants were collected and filtered using 0.22 µm filters to completely remove microbial cells. Supernatants were produced three times in separate days and stored at -80°C until use. Sample preparation Microbial supernatants underwent a liquid-liquid extraction procedure before HPLC-MS-MS analysis, as previously described by De Baere and colleagues [ 25 ] with protocol modifications. Briefly, 200 µL of each sample were placed in a 2 mL tube and added with 10 µL of a 10 µg/mL internal standard mixture made of 13 C 2 -acetic acid and 13 C 2 -propionic acid. The former was used as the internal standard (IS) for the quantification of acetic acid, while the latter for both propionic and butyric acids. Samples were mixed and equilibrated at room temperature for 5 min. Thereafter, 20 µL of HCl 37% w/w were added, samples were mixed for 15 sec, and extracted for 20 min by gently shaking in an orbital shaker, using 1 mL of diethyl-ether. After a centrifugation step of 5 min at 1,230 rcf, the organic phase was transferred to new tubes and 100 µL of NaOH 1 M were added. Samples were shaked again for 20 min and then centrifuged. The organic phase was removed and the aqueous phase containing SCFAs was added with 10 µL of HCl 37% in order to obtain a pH value in the range 4–7. Actually, pH is one of the limiting conditions of the derivatization process that was carried out to change the analytes’ structure, thus improving chromatographic separation and enhancing instrumental sensitivity. It can be achieved as follows: 50 µL of each sample was added with 50 µL of HPLC-MS water and then derivatization was performed adding 50 µL of 3-nitrophenylhydrazine (3NPH) hydrochloride 200 mM and 50 µL of N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride 120 mM. 3NPH was employed to convert SCFAs to their 3-nitrophenylhydrazone form, which had showed an excellent in-solution chemical stability [ 26 ]. Solutions were incubated at 40°C for 30 min in constant shaking. Afterwards, the derivatization reaction was quenched adding 200 µL of 0.1% formic acid, and derivatized samples were ready to be injected into the HPLC-MS-MS system for analysis. Quantification made use of calibration curves, prepared by serial dilution with water of stock standard solutions at the concentration of 1 µg/mL (for propionic and butyric acids) and 5 µg/mL (for acetic acid), to obtain concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.90 and 1.95 ng/mL for acetic acid, and 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 ng/mL for propionic and butyric acids. Each calibration point (50 µL) was diluted with 50 µL of sterile BHIG, previously extracted according to the procedure used for samples, and added with a proportional amount of the IS mixture in order to achieve, in all calibrators, the same initial concentration of IS in the samples. The use of the sample matrix to build the calibration curves makes calibrators similar to the samples, providing more reliable and reproducible results. Derivatization and injection of calibration points were then performed as described above. HPLC-MS-MS analysis The instrumental layout consisted in a 1290 UHPLC Infinity II system (Agilent, Santa Clara, USA), including a binary pump, a column oven set at 40°C, and a thermostated autosampler, coupled to a QTRAP 6500 + LC-MS-MS (Sciex, Ontario, Canada), working as a triple quadrupole and equipped with an IonDrive™ Turbo V source (Sciex). Chromatographic separation was achieved by using a 110 Å, 2×50 mm, 3µm particle size, Gemini C18 HPLC column (Phenomenex, Torrance, USA) protected by a C18 SecurityGuard™ cartridge (Phenomenex) and using acetonitrile containing 0.1% formic acid (solvent A, A) and water with 0.1% formic acid (solvent B, B) as mobile phases. Gradient elution, with a 500 µL/min flow rate, was performed as follows: 0.0-0.3 min (A) 10%, 2.5–3.5 min (A) 20%, 3.6–4.5 min (A) 90%, 4.6–5.5 (A) 10%. Injection volume was set at 5 µL. System control, data acquisition, and data processing were performed using Sciex Analyst® software (version 1.7.2). A mass spectrometry selected reaction monitoring method was operated in negative ion mode. For each compound, after the optimization of declustering potential (DP, -80 V), collision energy, and collision exit potential ( Table S1 , Supplementary Information), three transitions were considered in the analysis. Based on the highest signal/noise ratios, one of them was used as quantifier (Q) and the other two as qualifiers (q) ( Table S1 ). Further operative parameters were set as follows: gas source 1, 45 arbitrary units; gas source 2, 30 arbitrary units; ion spray voltage, -4.5 kV; source temperature, 500°C; curtain gas, 35 arbitrary units; collision gas, N 2 ; operative pressure with collision gas on, 3 mPa; entrance potential, -10V. Method validation To evaluate the method ability to differentiate the molecules of interest from other possible components and interferents present in samples, specificity was checked by repeated injections of analytes into the system and their retention times were monitored. Linearity was evaluated within the calibration curve range built as aforementioned, while instrumental sensitivity was assessed by evaluating limits of analyte detection (LOD) and quantification (LOQ). Using the S-to-N Script tool of Sciex Analyst® software, concentrations providing a S/N ratio close to 3 and 10 were assumed as LOD and LOQ, respectively. Recovery and matrix effect were calculated as reported by Matuszewski and colleagues [ 27 ]. Recovery was evaluated by comparing the peak areas of analytes added to blank BHIG broth before and after the extraction procedure, while the estimation of matrix effect was performed comparing the peak areas of the analytes added to water (A) and blank BHIG broth (B) previously subjected to the extraction process [(B/A) x 100]. Precision (%), expressed as relative standard deviation (RSD%), intra-day and inter-day accuracies, calculated with the formula [(measured concentration/nominal concentration spiked) x 100], were measured in blank samples spiked with three different concentration levels of analytes (5 ng/mL, 50 ng/mL and 250 ng/mL for acetic acid, 1 ng/mL, 10 ng/mL, and 50 ng/mL for propionic and butyric acids). Stability of analytes as a result of a freeze-thaw cycle was evaluated, as well. Aliquots of freshly prepared samples, spiked at low, medium, and high concentrations (already used for the calculation of accuracies) were immediately injected and results were compared to those from a second aliquot of the same concentration frozen at -20°C and thawed at room temperature before the assay. Statistical analysis Data are expressed as the mean ± standard deviation. Three biological replicates with three technical replicates each were performed. All statistical analyses were performed with GraphPad Prism 8 (GraphPad Software Inc., USA). Statistical significance was set at a P-value of < 0.05. A one-way ANOVA followed by Tukey’s test for multiple comparisons was applied to infer statistically significant differences in the production of acetic, propionic, and butyric acids, separately. Results Method validation Traces and specific retention times of acetic, propionic, and butyric acids from standard solutions and samples were achieved as shown in Figure 1 . Each compound exhibited three traces, each corresponding to a specific MRM transition: the one possessing the higher signal to noise ratio was used for quantification (quantifier, Q) of the analyte, while the others, which are representative of the specific analyte structure, confirmed its identity (qualifier, q). Retention time further confirmed the peak correspondence. These features demonstrated an excellent specificity and sensitivity (LOD and LOQ values) of the HPLC-MS-MS method for the quantification of SCFAs. Their values are reported in Table 1 together with the recovery of the extraction process and contribution of matrix effect. Linearity resulted ≥ 0.9997 for each analyte. Methodological inter-day and intra-day accuracy was in the optimal range of 85-115%, which is in compliance with the European Medicines Agency guidelines [28]. Precision, representing the closeness among repeated individual measurements of the analytes, was always < 7%. Re-analysis of samples after storage showed no degradation of analytes or their relative internal standards, thus confirming the stability of our protocol. Table 1 Analyte Retention time (min) LOD/LOQ (pg/mL) Recovery (%) Matrix Effect (%) Acetic acid 1.82 ± 0.01 4.90/9.80 91.93 ± 9.02 91.17 ± 7.85 Propionic acid 2.62 ± 0.02 1.95/3.90 117.75 ± 13.20 95.96 ± 14.49 Butyric acid 3.76 ± 0.01 1.95/3.90 104.40 ± 4.78 117.25 ± 3.89 Quantification of the SCFA amount in culture supernatants B. clausii , B. coagulans , B. breve , L. reuteri , L. rhamnosus , and S. boulardii supernatants collected from actively replicating cells were subjected to a HPLC-MS-MS analysis to determine the amount of acetic, propionic, and butyric acids secreted in the culture medium. Regarding acetic acid ( Figure 2A ), B. clausii T and L. reuteri resulted the highest producers among the tested strains, secreting 602.00 ± 54.15 ng/mL and 644.33 ± 7.15 ng/mL, respectively. No differences were highlighted among the four B. clausii strains. Secretion of acetic acid from B. coagulans , L. rhamnosus , and S. boulardii was significantly lower compared to B. clausii NR (BC: P = 0.0023; LRh and SB: P = 0.0003), OC (BC: P = 0.0215; LRh: P = 0.0031; SB: P = 0.0029), SIN (BC: P = 0.0132; LRh: P = 0.0019; SB: P = 0.0018), T (BC: P = 0.0002; LRh and SB: P < 0.0001), B. breve (BC: P = 0.0042; LRh and SB: P = 0.0006), and L. reuteri (P < 0.0001). Propionic acid concentrations were found to be three orders of magnitude lower than acetic acid in the culture supernatants ( Figure 2B ). B. coagulans , B. breve , L. reuteri , and L. rhamnosus did not secrete propionic acid at all in our conditions. B. clausii T was able to produce the highest levels of propionic acid (1.21 ± 0.38 ng/mL), resulting significantly different from NR (P = 0.0374), SIN (P = 0.0112), and S. boulardii (P = 0.0007). The levels of butyric acid were found to be slightly higher than those of propionic acid ( Figure 2C ). B. coagulans , B. breve , and L. rhamnosus were unable to secrete butyric acid, while all B. clausii strains showed a comparable secretion (NR: 2.72 ± 0.47 ng/mL; OC: 2.70 ± 0.31 ng/mL; SIN: 2.70 ± 0.06 ng/mL; T: 3.04 ± 0.25 ng/mL). A higher concentration of butyric acid was found in B. clausii NR, OC, SIN, and T culture supernatants compared to L. reuteri (NR: P = 0.0380; OC and T: P = 0.0002; SIN: P = 0.0012) and S. boulardii (NR: P = 0.0029; OC: P = 0.0001; SIN and T: P < 0.0001). Discussion Different HPLC technologies have been used over years to quantify SCFAs [ 25 , 29 , 30 ], especially in studies where the intestinal microbiota was altered in association with various pathological conditions [ 31 – 35 ]. In our study, coupling tandem mass spectrometry to HPLC (HPLC-MS-MS) to quantify acetic, propionic, and butyric acids in microbial culture supernatants led to a very specific and sensitive detection, which is properly required when low analyte concentrations are present, as in the case of SCFAs [ 36 , 37 ]. The evaluation of SCFA secretion by probiotic strains can provide dissimilar results, even when the same microbial strain is tested, probably due to the culture conditions and media used for microbial growth [ 1 , 21 ]. In fact, experimental protocols and environmental conditions influence the outcomes of analyses in terms of both quality and quantity of secreted SCFAs. For this reason, to guarantee a uniform microbial growth and obtain comparable results from the different tested strains, in this study a unique culture medium ( i.e ., BHIG) containing substantial concentrations of glucose and amino acids, which are the main essential substrates for SCFA synthesis, was selected and the same culture conditions were applied. Among members of the Bacillus genus, B. clausii and B. coagulans have been used as probiotics for years considering the beneficial effects exerted in several gastrointestinal disorders [ 38 – 40 ]. Since no evidence is present in the literature regarding their direct production of SCFAs, the present study highlights new metabolic features of these species. The ability of B. clausii NR, OC, SIN, and T to secrete acetic, propionic, and butyric acids in our in vitro model suggests their potential to produce SCFA also in vivo . These compounds could contribute to the properties these strains have demonstrated as adjuvant treatment in several gastrointestinal dysfunctions [ 41 – 46 ]. B. coagulans strains are worldwide recognized as effective probiotics, and the role of B. coagulans SANK 70258 in ameliorating ulcerative colitis and leading the gut microbiota composition towards the enrichment in butyrate-producing bacteria has recently been shown [ 47 ]. Herein, B. coagulans ATCC 7050 was proven to be able to secrete acetic acid, while propionic and butyric acids were not detected in its culture supernatant. Bifidobacterium spp. have been demonstrated to confer many benefits to the human health, mainly when administered for pediatric pathologies, such as allergies, obesity, diarrhea, colic, and celiac disease [ 48 ]. Different strains of B. breve are widely effective in preventing or ameliorating symptoms of several diseases, including Alzheimer’s disease ( i.e ., B. breve A1) and obesity-associated insulin sensitivity ( i.e ., B. breve BR03 and B632) by directly or indirectly modulating the local concentration of SCFAs [ 49 , 50 ]. In the present investigation, high concentrations of acetic acid were found in the culture supernatant of B. breve DSM 16604. As expected, propionic and butyric acids were not detected, since the biosynthetic pathways for propionate and butyrate are not present in Bifidobacterium species [ 51 ]. Numerous lactobacilli are commonly administered as probiotics due to their beneficial properties [ 52 ]. Among lactobacilli, L. reuteri DSM 17938 is a well-characterized and largely commercialized probiotic microorganism, found to be suitable for the prevention and co-treatment of chronic constipation, colic, diarrhea, and gastroenteritis, especially in children [ 53 – 56 ]. The ability of L. reuteri to produce SCFAs is a strain-dependent feature, as previously evidenced for L. reuteri NCIMB 11951, 701359, 701089, 702655, and 702656 [ 22 ]. Herein, we demonstrated the ability of L. reuteri DSM 17938 to secrete large amounts of acetic acid and butyric acid to a lesser extent, thus suggesting a possible mechanism of action for reaching the health benefits associated to its administration. L. rhamnosus , whose persistence on the international market has lasted for more than 30 years due to its efficacy in managing several clinical conditions, is another bacterial species considered to have excellent probiotic properties [ 57 ]. L. rhamnosus strains were often demonstrated to be able to promote butyrogenesis and shape the gut microbiota with increased abundances of butyrate-producing bacteria [ 58 ]. L. rhamnosus GG turned out to secrete acetic, propionic, and butyric acids in skim milk supplemented with prebiotics, as reported by Asarat and colleagues [ 21 ], while another study showed the release of only propionic acid by this strain in MRS [ 1 ]. In this study, L. rhamnosus ATCC 53103 was able to secrete acetic acid in BHIG, but propionic and butyric acids were not detected in its culture supernatant. Our findings on B. breve, L. reuteri , and L. rhamnosus are in line with previous observations reporting Bifidobacterium and Lactobacillus species as mainly acetate producers [ 20 ]. Several species of Saccharomyces are intrinsically able to determine an enrichment of SCFA-producing bacteria in the gut microbiota [ 59 , 60 ], but only a few acidify the intestinal environment through the secretion of high levels of acetic acid themselves [ 60 ]. Up to date, no information about secretion of propionic and butyric acid by S. boulardii is available in the literature. Although many efforts have been made on S. cerevisiae strains for enhancing the production of SCFAs by genetic engineering [ 61 , 62 ], a clear characterization of S. boulardii ability to secrete SCFAs is still lacking. S. boulardii CNCM I-745 was shown to release both acetic, propionic, and butyric acids in its culture supernatant, confirming its potency as regards SCFA metabolism. In conclusion, the application of a novel sensitive HPLC-MS-MS protocol for the detection and quantification of SCFAs allowed us to establish that all the tested probiotic strains are able to actively secrete acetic acid in vitro and a part of them all the three main short-chain fatty acids. Although our study cannot exclude a different microbial behavior in vivo , we believe that the in vitro production of SCFAs should be taken into consideration as key feature when next generation probiotics and psychobiotics are evaluated for their potential clinical effectiveness. An in-depth characterization of strains contained in probiotic formulations as regards SCFA secretion could be a novel aspect to consider in the probiotic research and contribute to the spread of more targeted and personalized bacteriotherapy strategies to promote human health and manage diseases. Declarations Acknowledgments The Centre for Instrumentation Sharing of University of Pisa (CISUP) is kindly acknowledged for providing the Sciex QTrap 6500+ mass spectrometer used for the spectrometric assays. Funding and conflict of interest This work was sponsored and funded by Sanofi S.p.A. MC, AB, SC, DM, AP, FC, RZ, and AS have no conflict of interest to declare. EG has been a lecturer for Sanofi S.p.A. Data availability statement Datasets generated during the current study will be made available from the corresponding author on reasonable request. 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Leber C, Da Silva NA (2014) Engineering of Saccharomyces cerevisiae for the synthesis of short chain fatty acids. Biotechnol Bioeng 111(2):347–58. doi: 10.1002/bit.25021 . Yu AQ, Pratomo Juwono NK, Foo JL, Leong SSJ, Chang MW (2016) Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids. Metab Eng 34:36–43. doi: 10.1016/j.ymben.2015.12.005 . Additional Declarations Competing interest reported. This work was sponsored and funded by Sanofi S.p.A. M.C., A.B., S.C., D.M., A.P., F.C., R.Z., and A.S. have no conflict of interest to declare. E.G. has been a lecturer for Sanofi S.p.A. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2128764","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":141671510,"identity":"775288ae-2739-4a52-b338-e2d455963a57","order_by":0,"name":"Marco Calvigioni","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Calvigioni","suffix":""},{"id":141671511,"identity":"9d3fa0e7-94ab-43a8-92a6-d9e32515dc2c","order_by":1,"name":"Andrea Bertolini","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Bertolini","suffix":""},{"id":141671512,"identity":"ec58c05f-3ac7-4a9f-af54-c05f3aa83132","order_by":2,"name":"Simone Codini","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Simone","middleName":"","lastName":"Codini","suffix":""},{"id":141671513,"identity":"d37d72dc-01a7-47d3-810f-b15cf9762253","order_by":3,"name":"Diletta Mazzantini","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Diletta","middleName":"","lastName":"Mazzantini","suffix":""},{"id":141671515,"identity":"68b8d1c6-9296-4aba-a5ea-773048e4d679","order_by":4,"name":"Adelaide Panattoni","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Adelaide","middleName":"","lastName":"Panattoni","suffix":""},{"id":141671517,"identity":"1411921d-f399-4dcd-bb9f-1fcfb76a2d54","order_by":5,"name":"Francesco Celandroni","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Celandroni","suffix":""},{"id":141671520,"identity":"096a3106-ead6-46de-b472-4decac19a0c6","order_by":6,"name":"Riccardo Zucchi","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Zucchi","suffix":""},{"id":141671521,"identity":"f58a28bc-ce15-41c6-9b80-19930169fba3","order_by":7,"name":"Alessandro Saba","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alessandro","middleName":"","lastName":"Saba","suffix":""},{"id":141671525,"identity":"fc288617-5bc2-4f0a-ac1c-aa48a94f92d0","order_by":8,"name":"Emilia Ghelardi","email":"data:image/png;base64,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","orcid":"","institution":"University of Pisa","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Emilia","middleName":"","lastName":"Ghelardi","suffix":""}],"badges":[],"createdAt":"2022-10-03 14:44:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2128764/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2128764/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":27430174,"identity":"9923a739-2a4a-4255-a88f-e391bd6a0d60","added_by":"auto","created_at":"2022-10-06 15:42:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124603,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative chromatograms of the analytes in a standard mixture (1000 ng/mL acetic acid, 200 ng/mL propionic and butyric acids) (A) and in one of the real samples (B). For each analyte, three MRM transitions were monitored: the one with the higher signal/noise ratio, which usually correspond to the most intense trace, was used for the quantification of the analyte (Q), while the other two transitions confirmed that the peak is attributable to the analyte (q). All MRM transitions are summarized in \u003cstrong\u003eTable S1\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-2128764/v1/183c360e495a0e479f083292.png"},{"id":27430791,"identity":"0c35e50c-eb3f-4577-80f3-7a0cb8d2edce","added_by":"auto","created_at":"2022-10-06 15:47:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":224925,"visible":true,"origin":"","legend":"\u003cp\u003eA) Concentration of acetic acid (ng/mL) in the culture supernatants of B. clausii (NR, OC, SIN, T), B. coagulans, B. breve, L. reuteri, L. rhamnosus, and S. boulardii. NR = B. clausii NR; OC = B. clausii OC; SIN = B. clausii SIN; T = B. clausii T; BC = B. coagulans; BB = B. breve; LRe = L. reuteri; LRh = L. rhamnosus; SB = S. boulardii. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001. NR vs. BC **, NR vs. LRh ***, NR vs. SB ***, OC vs. BC *, OC vs. LRh **, OC vs. SB **, SIN vs. BC *, SIN vs. LRh **, SIN vs. SB **, T vs. BC ***, T vs. LRh ****, T vs. SB ****, BC vs. BB **, BC vs. LRe ****, BB vs. LRh ***, BB vs. SB ***, LRe vs. LRh ****, LRe vs. SB ****. B) Concentration of propionic acid (ng/mL) in the culture supernatants of B. clausii (NR, OC, SIN, T), B. coagulans, B. breve, L. reuteri, L. rhamnosus, and S. boulardii. NR vs. T *, OC vs. BC *, OC vs. BB *, OC vs. LRe *, OC vs. LRh *, SIN vs. T *, T vs. BC ****, T vs. BB ****, T vs. LRe ****, T vs. LRh ****, T vs. SB ***. C) Concentration of butyric acid (ng/mL) in the culture supernatants of B. clausii (NR, OC, SIN, T), B. coagulans, B. breve, L. reuteri, L. rhamnosus, and S. boulardii. NR vs. BC ***, NR vs. BB ***, NR vs. LRe *, NR vs. LRh ***, NR vs. SB **, OC vs. BC ****, OC vs. BB ****, OC vs. LRe ***, OC vs. LRh ****, OC vs. SB ****, SIN vs. BC ****, SIN vs. BB ****, SIN vs. LRe **, SIN vs. LRh ****, SIN vs. SB ***, T vs. BC ****, T vs. BB ****, T vs. LRe ***, T vs. LRh ****, T vs. SB ****.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-2128764/v1/76907755bb420a5301920332.png"},{"id":29105358,"identity":"78af791b-722c-4daa-953c-d35310f09cd7","added_by":"auto","created_at":"2022-11-15 21:29:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":573236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2128764/v1/0dd68cbb-eccb-424a-b104-dad2b82e5564.pdf"},{"id":27430173,"identity":"1f0e0eb9-3fe4-4137-90b6-520e2d11df25","added_by":"auto","created_at":"2022-10-06 15:42:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15938,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-2128764/v1/2bbb147ee4ed84e3cb2bab8b.docx"}],"financialInterests":"Competing interest reported. This work was sponsored and funded by Sanofi S.p.A. M.C., A.B., S.C., D.M., A.P., F.C., R.Z., and A.S. have no conflict of interest to declare. E.G. has been a lecturer for Sanofi S.p.A.","formattedTitle":"HPLC-MS-MS quantification of short-chain fatty acids secreted by probiotic strains","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe human gut microbiota actively cooperates in maintaining physiological balance, in nutrient catabolism and absorption, as well as in the production of short-chain fatty acids (SCFAs) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. SCFAs are small carboxylic organic acids with a backbone of at most six carbon atoms. They represent the largest group of metabolic products obtained by microbial fermentation of dietary complex carbohydrates, otherwise undegradable by the human digestive system [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e, \u003cem\u003eEubacterium rectale\u003c/em\u003e, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e spp., \u003cem\u003eBifidobacterium\u003c/em\u003e spp., \u003cem\u003eLactobacillus\u003c/em\u003e spp., and members belonging to families \u003cem\u003eRuminococcaceae\u003c/em\u003e, \u003cem\u003eLachnospiraceae\u003c/em\u003e, and \u003cem\u003eBacteroidaceae\u003c/em\u003e are the main actors able to produce SCFAs in the human gut as result of bacterial metabolism [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe highest intestinal SCFA concentrations are reached by acetic, propionic, and butyric acids, which are known to exert countless beneficial effects and orchestrate several physiological functions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Acetic acid is the structurally simplest fatty acid and can, therefore, be used as metabolic substrate in fatty acid biosynthesis and Krebs\u0026rsquo; cycle [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Besides being a major energy source for skeletal muscle [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], acetic acid is also involved in lipid metabolism of the liver and adipose tissue [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The administration of acetate was reported to decrease food intake, body weight gain, blood cholesterol, and triglyceride levels [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Propionic acid improves the barrier function and epithelial integrity in the gut, and impacts on glucose and lipid liver homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies reported the ability of propionic acid to indirectly modulate gene expression and cell metabolism through immunomodulatory mechanisms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Butyric acid is probably the most multifunctional SCFA, acting as energy source for colonic epithelial cells and promoting beta oxidation rather than glycolysis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Enhancing the expression of tight-junction proteins, butyric acid facilitates the maintenance of the intestinal barrier integrity, thus hampering epithelial invasion by pathogenic microorganisms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It has also been revealed to inhibit tumoral cell expansion and release of pro-inflammatory cytokines [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], thus demonstrating anti-carcinogenic and anti-inflammatory properties. All the main SCFAs were found able to differently modulate appetite and energy intake by increasing colonic glucagon-like peptide-1 secretion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], thus reducing food craving [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, they are also directly involved in the microbiota-gut-brain axis neuroendocrine signaling, brain physiology, and neuronal damage prevention [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA correlation between lower intestinal SCFA concentrations and the exacerbation of different pathological conditions, such as inflammatory bowel diseases and neurological and neuropsychiatric disorders (\u003cem\u003ee.g\u003c/em\u003e., autism, depression, Alzheimer and Parkinson\u0026rsquo;s diseases, multiple sclerosis), has been confirmed [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The administration of properly selected probiotic microorganisms able to counteract SCFA deficiencies appeared as a promising novel approach to co-adjuvate the management of these conditions in humans and improve pathology-associated symptoms. For instance, psychobiotics producing SCFAs, neurotransmitters, and neuroendocrine hormones were revealed to provide wide health benefits to patients suffering from mental and neurological disorders [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Several studies demonstrated the ability of some probiotics to shift the gut microbiota composition towards higher abundances of SCFA-producing species [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Acetic, propionic, and butyric acids were also scarcely demonstrated to be secreted by probiotic microorganisms themselves in different amounts and in a species- or even strain-specific modality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], thus pointing out SCFA production by probiotics as beneficial additional feature for certain clinical conditions.\u003c/p\u003e \u003cp\u003eIn this study, the amount of acetic acid, propionic acid, and butyric acid actively secreted by nine probiotic strains was quantified by using an optimized and sensitive protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS).\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and chemicals\u003c/h2\u003e \u003cp\u003eAcetic acid (purity\u0026thinsp;\u0026ge;\u0026thinsp;99.8%), propionic acid (\u0026ge;\u0026thinsp;99.5%), propionic acid-2,3-\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e (99 atom % \u003csup\u003e13\u003c/sup\u003eC) and butyric acid (\u0026ge;\u0026thinsp;99.5%) analytical standards, water LC-MS grade, acetonitrile LC-MS grade (ACN), formic acid (FA, ≧ 98%), hydrochloric acid (ACS reagent, 37% w/w), NaOH solution 1 M, diethyl-ether (\u0026ge;\u0026thinsp;99%), 3-nitrophenylhydrazine (3NPH) hydrochloride, N-(3-dimethylaminopropyl)-N\u0026prime;-ethyl carbodiimide (EDC) hydrochloride, Bifidus Selective agar (BSM), BSM supplement, and Sabouraud-2% dextrose agar (SDA) were bought from Merck KGaA (Darmstadt, Germany). Acetic acid \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e (99 atom % \u003csup\u003e13\u003c/sup\u003eC) analytic standard was purchased from Cambridge Isotope Laboratories (Tewksbury, USA). Brain Heart Infusion agar (BHI) was obtained from Biolife (Monza, Italy), while Trypticase Soy agar (TSA) supplemented with 5% horse blood (TSH) was from bioM\u0026eacute;rieux (Paris, France). De Man, Rogosa, and Sharpe (MRS) agar was acquired from Thermo Fisher Scientific (Waltham, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial strains and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBacillus clausii\u003c/em\u003e NR, OC, SIN, and T (isolated from Enterogermina, Sanofi, Paris, France, and declared on the product label), \u003cem\u003eBacillus coagulans\u003c/em\u003e ATCC 7050 (isolated from Lact\u0026ograve; Pi\u0026ugrave;, Recordati, Milan, Italy, and declared on the product label), \u003cem\u003eBifidobacterium breve\u003c/em\u003e DSM 16604 (isolated from Neovaxitiol, IBSA Farmaceutici, Lodi, Italy, and declared on the product label), \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e DSM 17938 (isolated from Reuflor, Italchimici, Brescia, Italy, and declared on the product label with the old nomenclature \u003cem\u003eLactobacillus reuteri\u003c/em\u003e DSM 17938), \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e ATCC 53103 (isolated from Dicoflor, Dicofarm, Roma, Italy, and declared on the product label with the old nomenclature \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e ATCC 53103), and \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e CNCM I-745 (isolated from Codex, Zambon, Bresso, Italy, and declared on the product label) were tested in the present investigation. \u003cem\u003eB. clausii\u003c/em\u003e NR, OC, SIN, and T were isolated from Enterogermina as described in a previous work [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Stock solutions of NR, OC, SIN, and T kept at -80\u0026deg;C were thawed and streaked on BHI agar plates containing 1% (w/v) of glucose (BHIG). Plates were incubated at 37\u0026deg;C for 48h. \u003cem\u003eB. coagulans\u003c/em\u003e was seeded on TSH and plates were incubated at 37\u0026deg;C for 48-72h. \u003cem\u003eB. breve\u003c/em\u003e was propagated on BSM containing 0.116 g/L of BSM supplement and grown at 37\u0026deg;C for 48-72h in anaerobic atmosphere, generated by using Thermo Scientific\u0026trade; AnaeroGen\u0026trade; Compact (Thermo Fisher Scientific). \u003cem\u003eL. reuteri\u003c/em\u003e and \u003cem\u003eL. rhamnosus\u003c/em\u003e were streaked on MRS agar and plates were incubated at 37\u0026deg;C for 48h in 5% CO\u003csub\u003e2\u003c/sub\u003e-enriched atmosphere, generated by using Thermo Scientific\u0026trade; CO\u003csub\u003e2\u003c/sub\u003eGen\u0026trade; Compact (Thermo Fisher Scientific). \u003cem\u003eS. boulardii\u003c/em\u003e was seeded on Sabouraud-2% dextrose agar and plates were incubated at 30\u0026deg;C for 48-72h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of culture supernatants\u003c/h2\u003e \u003cp\u003eDifferent liquid culture media (\u003cem\u003ei.e.\u003c/em\u003e, Luria Bertani broth, BHI and BHIG broths, RPMI 1640 medium) were tested for microbial propagation. BHIG broth was selected as the most suitable culture medium, as it allowed an optimal uniform replication of all microorganisms and contained amino acids and glucose, which are important substrates for SCFA biosynthesis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Thus, for each strain, a well-isolated colony was inoculated in 5 mL of BHIG broth. Suspensions were incubated overnight at 37\u0026deg;C in constant shaking. Subsequently, 100 \u0026micro;L of microbial cultures were inoculated in 25 mL of fresh BHIG medium. Cultures were incubated at 37\u0026deg;C up to an optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 1.8 and then centrifuged at 3,870 rcf for 20 min at 4\u0026deg;C. Supernatants were collected and filtered using 0.22 \u0026micro;m filters to completely remove microbial cells. Supernatants were produced three times in separate days and stored at -80\u0026deg;C until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation\u003c/h2\u003e \u003cp\u003eMicrobial supernatants underwent a liquid-liquid extraction procedure before HPLC-MS-MS analysis, as previously described by De Baere and colleagues [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] with protocol modifications. Briefly, 200 \u0026micro;L of each sample were placed in a 2 mL tube and added with 10 \u0026micro;L of a 10 \u0026micro;g/mL internal standard mixture made of \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e-acetic acid and \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e-propionic acid. The former was used as the internal standard (IS) for the quantification of acetic acid, while the latter for both propionic and butyric acids. Samples were mixed and equilibrated at room temperature for 5 min. Thereafter, 20 \u0026micro;L of HCl 37% w/w were added, samples were mixed for 15 sec, and extracted for 20 min by gently shaking in an orbital shaker, using 1 mL of diethyl-ether. After a centrifugation step of 5 min at 1,230 rcf, the organic phase was transferred to new tubes and 100 \u0026micro;L of NaOH 1 M were added. Samples were shaked again for 20 min and then centrifuged. The organic phase was removed and the aqueous phase containing SCFAs was added with 10 \u0026micro;L of HCl 37% in order to obtain a pH value in the range 4\u0026ndash;7. Actually, pH is one of the limiting conditions of the derivatization process that was carried out to change the analytes\u0026rsquo; structure, thus improving chromatographic separation and enhancing instrumental sensitivity. It can be achieved as follows: 50 \u0026micro;L of each sample was added with 50 \u0026micro;L of HPLC-MS water and then derivatization was performed adding 50 \u0026micro;L of 3-nitrophenylhydrazine (3NPH) hydrochloride 200 mM and 50 \u0026micro;L of N-(3-dimethylaminopropyl)-N\u0026prime;-ethyl carbodiimide hydrochloride 120 mM. 3NPH was employed to convert SCFAs to their 3-nitrophenylhydrazone form, which had showed an excellent in-solution chemical stability [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Solutions were incubated at 40\u0026deg;C for 30 min in constant shaking. Afterwards, the derivatization reaction was quenched adding 200 \u0026micro;L of 0.1% formic acid, and derivatized samples were ready to be injected into the HPLC-MS-MS system for analysis. Quantification made use of calibration curves, prepared by serial dilution with water of stock standard solutions at the concentration of 1 \u0026micro;g/mL (for propionic and butyric acids) and 5 \u0026micro;g/mL (for acetic acid), to obtain concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.90 and 1.95 ng/mL for acetic acid, and 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 ng/mL for propionic and butyric acids. Each calibration point (50 \u0026micro;L) was diluted with 50 \u0026micro;L of sterile BHIG, previously extracted according to the procedure used for samples, and added with a proportional amount of the IS mixture in order to achieve, in all calibrators, the same initial concentration of IS in the samples. The use of the sample matrix to build the calibration curves makes calibrators similar to the samples, providing more reliable and reproducible results. Derivatization and injection of calibration points were then performed as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHPLC-MS-MS analysis\u003c/h2\u003e \u003cp\u003eThe instrumental layout consisted in a 1290 UHPLC Infinity II system (Agilent, Santa Clara, USA), including a binary pump, a column oven set at 40\u0026deg;C, and a thermostated autosampler, coupled to a QTRAP 6500\u0026thinsp;+\u0026thinsp;LC-MS-MS (Sciex, Ontario, Canada), working as a triple quadrupole and equipped with an IonDrive\u0026trade; Turbo V source (Sciex). Chromatographic separation was achieved by using a 110 \u0026Aring;, 2\u0026times;50 mm, 3\u0026micro;m particle size, Gemini C18 HPLC column (Phenomenex, Torrance, USA) protected by a C18 SecurityGuard\u0026trade; cartridge (Phenomenex) and using acetonitrile containing 0.1% formic acid (solvent A, A) and water with 0.1% formic acid (solvent B, B) as mobile phases. Gradient elution, with a 500 \u0026micro;L/min flow rate, was performed as follows: 0.0-0.3 min (A) 10%, 2.5\u0026ndash;3.5 min (A) 20%, 3.6\u0026ndash;4.5 min (A) 90%, 4.6\u0026ndash;5.5 (A) 10%. Injection volume was set at 5 \u0026micro;L. System control, data acquisition, and data processing were performed using Sciex Analyst\u0026reg; software (version 1.7.2). A mass spectrometry selected reaction monitoring method was operated in negative ion mode. For each compound, after the optimization of declustering potential (DP, -80 V), collision energy, and collision exit potential (\u003cb\u003eTable S1\u003c/b\u003e, Supplementary Information), three transitions were considered in the analysis. Based on the highest signal/noise ratios, one of them was used as quantifier (Q) and the other two as qualifiers (q) (\u003cb\u003eTable S1\u003c/b\u003e). Further operative parameters were set as follows: gas source 1, 45 arbitrary units; gas source 2, 30 arbitrary units; ion spray voltage, -4.5 kV; source temperature, 500\u0026deg;C; curtain gas, 35 arbitrary units; collision gas, N\u003csub\u003e2\u003c/sub\u003e; operative pressure with collision gas on, 3 mPa; entrance potential, -10V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cp\u003eTo evaluate the method ability to differentiate the molecules of interest from other possible components and interferents present in samples, specificity was checked by repeated injections of analytes into the system and their retention times were monitored. Linearity was evaluated within the calibration curve range built as aforementioned, while instrumental sensitivity was assessed by evaluating limits of analyte detection (LOD) and quantification (LOQ). Using the S-to-N Script tool of Sciex Analyst\u0026reg; software, concentrations providing a S/N ratio close to 3 and 10 were assumed as LOD and LOQ, respectively. Recovery and matrix effect were calculated as reported by Matuszewski and colleagues [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Recovery was evaluated by comparing the peak areas of analytes added to blank BHIG broth before and after the extraction procedure, while the estimation of matrix effect was performed comparing the peak areas of the analytes added to water (A) and blank BHIG broth (B) previously subjected to the extraction process [(B/A) x 100]. Precision (%), expressed as relative standard deviation (RSD%), intra-day and inter-day accuracies, calculated with the formula [(measured concentration/nominal concentration spiked) x 100], were measured in blank samples spiked with three different concentration levels of analytes (5 ng/mL, 50 ng/mL and 250 ng/mL for acetic acid, 1 ng/mL, 10 ng/mL, and 50 ng/mL for propionic and butyric acids). Stability of analytes as a result of a freeze-thaw cycle was evaluated, as well. Aliquots of freshly prepared samples, spiked at low, medium, and high concentrations (already used for the calculation of accuracies) were immediately injected and results were compared to those from a second aliquot of the same concentration frozen at -20\u0026deg;C and thawed at room temperature before the assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Three biological replicates with three technical replicates each were performed. All statistical analyses were performed with GraphPad Prism 8 (GraphPad Software Inc., USA). Statistical significance was set at a P-value of \u0026lt;\u0026thinsp;0.05. A one-way ANOVA followed by Tukey\u0026rsquo;s test for multiple comparisons was applied to infer statistically significant differences in the production of acetic, propionic, and butyric acids, separately.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMethod validation\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTraces and specific retention times of acetic, propionic, and butyric acids from standard solutions and samples were achieved as shown in \u003cstrong\u003eFigure 1\u003c/strong\u003e. Each compound exhibited three traces, each corresponding to a specific MRM transition: the one possessing the higher signal to noise ratio was used for quantification (quantifier, Q) of the analyte, while the others, which are representative of the specific analyte structure, confirmed its identity (qualifier, q). Retention time further confirmed the peak correspondence. These features demonstrated an excellent specificity and sensitivity (LOD and LOQ values) of the HPLC-MS-MS method for the quantification of SCFAs. Their values are reported in \u003cstrong\u003eTable 1\u003c/strong\u003e together with the recovery of the extraction process and contribution of matrix effect. Linearity resulted \u0026ge; 0.9997 for each analyte. Methodological inter-day and intra-day accuracy was in the optimal range of 85-115%, which is in compliance with the European Medicines Agency guidelines [28]. \u0026nbsp;Precision, representing the closeness among repeated individual measurements of the analytes, was always \u0026lt; 7%. Re-analysis of samples after storage showed no degradation of analytes or their relative internal standards, thus confirming the stability of our protocol.\u003c/p\u003e\n\u003cp\u003eTable 1\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.113744075829384%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalyte\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.696682464454977%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRetention time (min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.906793048973142%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLOD/LOQ (pg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.429699842022117%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.85308056872038%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMatrix Effect (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.113744075829384%\"\u003e\n \u003cp\u003eAcetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.696682464454977%\"\u003e\n \u003cp\u003e1.82 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.906793048973142%\"\u003e\n \u003cp\u003e4.90/9.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.429699842022117%\"\u003e\n \u003cp\u003e91.93 \u0026plusmn; 9.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.85308056872038%\"\u003e\n \u003cp\u003e91.17 \u0026plusmn; 7.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.113744075829384%\"\u003e\n \u003cp\u003ePropionic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.696682464454977%\"\u003e\n \u003cp\u003e2.62 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.906793048973142%\"\u003e\n \u003cp\u003e1.95/3.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.429699842022117%\"\u003e\n \u003cp\u003e117.75 \u0026plusmn; 13.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.85308056872038%\"\u003e\n \u003cp\u003e95.96 \u0026plusmn; 14.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.113744075829384%\"\u003e\n \u003cp\u003eButyric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.696682464454977%\"\u003e\n \u003cp\u003e3.76 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.906793048973142%\"\u003e\n \u003cp\u003e1.95/3.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.429699842022117%\"\u003e\n \u003cp\u003e104.40 \u0026plusmn; 4.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.85308056872038%\"\u003e\n \u003cp\u003e117.25 \u0026plusmn; 3.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantification of the SCFA amount in culture supernatants\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB. clausii\u003c/em\u003e, \u003cem\u003eB. coagulans\u003c/em\u003e, \u003cem\u003eB. breve\u003c/em\u003e, \u003cem\u003eL. reuteri\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, and \u003cem\u003eS. boulardii\u003c/em\u003e supernatants collected from actively replicating cells were subjected to a HPLC-MS-MS analysis to determine the amount of acetic, propionic, and butyric acids secreted in the culture medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding acetic acid (\u003cstrong\u003eFigure 2A\u003c/strong\u003e), \u003cem\u003eB. clausii\u003c/em\u003e T and \u003cem\u003eL. reuteri\u003c/em\u003e resulted the highest producers among the tested strains, secreting 602.00 \u0026plusmn; 54.15 ng/mL and 644.33 \u0026plusmn; 7.15 ng/mL, respectively. No differences were highlighted among the four \u003cem\u003eB. clausii\u003c/em\u003e strains. Secretion of acetic acid from \u003cem\u003eB. coagulans\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, and \u003cem\u003eS. boulardii\u003c/em\u003e was significantly lower compared to \u003cem\u003eB. clausii\u003c/em\u003e NR (BC: P = 0.0023; LRh and SB: P = 0.0003), OC (BC: P = 0.0215; LRh: P = 0.0031; SB: P = 0.0029), SIN (BC: P = 0.0132; LRh: P = 0.0019; SB: P = 0.0018), T (BC: P = 0.0002; LRh and SB: P \u0026lt; 0.0001), \u003cem\u003eB. breve\u0026nbsp;\u003c/em\u003e(BC: P = 0.0042; LRh and SB: P = 0.0006), and \u003cem\u003eL. reuteri\u003c/em\u003e (P \u0026lt; 0.0001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePropionic acid concentrations were found to be three orders of magnitude lower than acetic acid in the culture supernatants (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). \u003cem\u003eB. coagulans\u003c/em\u003e, \u003cem\u003eB. breve\u003c/em\u003e, \u003cem\u003eL. reuteri\u003c/em\u003e, and \u003cem\u003eL. rhamnosus\u003c/em\u003e did not secrete propionic acid at all in our conditions. \u003cem\u003eB. clausii\u003c/em\u003e T was able to produce the highest levels of propionic acid (1.21 \u0026plusmn; 0.38 ng/mL), resulting significantly different from NR (P = 0.0374), SIN (P = 0.0112),\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eS. boulardii\u003c/em\u003e (P = 0.0007).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe levels of butyric acid were found to be slightly higher than those of propionic acid (\u003cstrong\u003eFigure 2C\u003c/strong\u003e). \u003cem\u003eB. coagulans\u003c/em\u003e, \u003cem\u003eB. breve\u003c/em\u003e, and \u003cem\u003eL. rhamnosus\u003c/em\u003e were unable to secrete butyric acid, while all \u003cem\u003eB. clausii\u003c/em\u003e strains showed a comparable secretion (NR: 2.72 \u0026plusmn; 0.47 ng/mL; OC: 2.70 \u0026plusmn; 0.31 ng/mL; SIN: 2.70 \u0026plusmn; 0.06 ng/mL; T: 3.04 \u0026plusmn; 0.25 ng/mL). A higher concentration of butyric acid was found in \u003cem\u003eB. clausii\u003c/em\u003e NR, OC, SIN, and T culture supernatants compared to \u003cem\u003eL. reuteri\u0026nbsp;\u003c/em\u003e(NR: P = 0.0380; OC and T: P = 0.0002; SIN: P = 0.0012) and \u003cem\u003eS. boulardii\u003c/em\u003e (NR: P = 0.0029; OC: P = 0.0001; SIN and T: P \u0026lt; 0.0001).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDifferent HPLC technologies have been used over years to quantify SCFAs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], especially in studies where the intestinal microbiota was altered in association with various pathological conditions [\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In our study, coupling tandem mass spectrometry to HPLC (HPLC-MS-MS) to quantify acetic, propionic, and butyric acids in microbial culture supernatants led to a very specific and sensitive detection, which is properly required when low analyte concentrations are present, as in the case of SCFAs [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe evaluation of SCFA secretion by probiotic strains can provide dissimilar results, even when the same microbial strain is tested, probably due to the culture conditions and media used for microbial growth [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In fact, experimental protocols and environmental conditions influence the outcomes of analyses in terms of both quality and quantity of secreted SCFAs. For this reason, to guarantee a uniform microbial growth and obtain comparable results from the different tested strains, in this study a unique culture medium (\u003cem\u003ei.e\u003c/em\u003e., BHIG) containing substantial concentrations of glucose and amino acids, which are the main essential substrates for SCFA synthesis, was selected and the same culture conditions were applied.\u003c/p\u003e \u003cp\u003eAmong members of the \u003cem\u003eBacillus\u003c/em\u003e genus, \u003cem\u003eB. clausii\u003c/em\u003e and \u003cem\u003eB. coagulans\u003c/em\u003e have been used as probiotics for years considering the beneficial effects exerted in several gastrointestinal disorders [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Since no evidence is present in the literature regarding their direct production of SCFAs, the present study highlights new metabolic features of these species. The ability of \u003cem\u003eB. clausii\u003c/em\u003e NR, OC, SIN, and T to secrete acetic, propionic, and butyric acids in our \u003cem\u003ein vitro\u003c/em\u003e model suggests their potential to produce SCFA also \u003cem\u003ein vivo\u003c/em\u003e. These compounds could contribute to the properties these strains have demonstrated as adjuvant treatment in several gastrointestinal dysfunctions [\u003cspan additionalcitationids=\"CR42 CR43 CR44 CR45\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. \u003cem\u003eB. coagulans\u003c/em\u003e strains are worldwide recognized as effective probiotics, and the role of \u003cem\u003eB. coagulans\u003c/em\u003e SANK 70258 in ameliorating ulcerative colitis and leading the gut microbiota composition towards the enrichment in butyrate-producing bacteria has recently been shown [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Herein, \u003cem\u003eB. coagulans\u003c/em\u003e ATCC 7050 was proven to be able to secrete acetic acid, while propionic and butyric acids were not detected in its culture supernatant.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBifidobacterium\u003c/em\u003e spp. have been demonstrated to confer many benefits to the human health, mainly when administered for pediatric pathologies, such as allergies, obesity, diarrhea, colic, and celiac disease [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Different strains of \u003cem\u003eB. breve\u003c/em\u003e are widely effective in preventing or ameliorating symptoms of several diseases, including Alzheimer\u0026rsquo;s disease (\u003cem\u003ei.e\u003c/em\u003e., \u003cem\u003eB. breve\u003c/em\u003e A1) and obesity-associated insulin sensitivity (\u003cem\u003ei.e\u003c/em\u003e., \u003cem\u003eB. breve\u003c/em\u003e BR03 and B632) by directly or indirectly modulating the local concentration of SCFAs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In the present investigation, high concentrations of acetic acid were found in the culture supernatant of \u003cem\u003eB. breve\u003c/em\u003e DSM 16604. As expected, propionic and butyric acids were not detected, since the biosynthetic pathways for propionate and butyrate are not present in \u003cem\u003eBifidobacterium\u003c/em\u003e species [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous lactobacilli are commonly administered as probiotics due to their beneficial properties [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Among lactobacilli, \u003cem\u003eL. reuteri\u003c/em\u003e DSM 17938 is a well-characterized and largely commercialized probiotic microorganism, found to be suitable for the prevention and co-treatment of chronic constipation, colic, diarrhea, and gastroenteritis, especially in children [\u003cspan additionalcitationids=\"CR54 CR55\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The ability of \u003cem\u003eL. reuteri\u003c/em\u003e to produce SCFAs is a strain-dependent feature, as previously evidenced for \u003cem\u003eL. reuteri\u003c/em\u003e NCIMB 11951, 701359, 701089, 702655, and 702656 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Herein, we demonstrated the ability of \u003cem\u003eL. reuteri\u003c/em\u003e DSM 17938 to secrete large amounts of acetic acid and butyric acid to a lesser extent, thus suggesting a possible mechanism of action for reaching the health benefits associated to its administration. \u003cem\u003eL. rhamnosus\u003c/em\u003e, whose persistence on the international market has lasted for more than 30 years due to its efficacy in managing several clinical conditions, is another bacterial species considered to have excellent probiotic properties [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. \u003cem\u003eL. rhamnosus\u003c/em\u003e strains were often demonstrated to be able to promote butyrogenesis and shape the gut microbiota with increased abundances of butyrate-producing bacteria [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. \u003cem\u003eL. rhamnosus\u003c/em\u003e GG turned out to secrete acetic, propionic, and butyric acids in skim milk supplemented with prebiotics, as reported by Asarat and colleagues [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], while another study showed the release of only propionic acid by this strain in MRS [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In this study, \u003cem\u003eL. rhamnosus\u003c/em\u003e ATCC 53103 was able to secrete acetic acid in BHIG, but propionic and butyric acids were not detected in its culture supernatant. Our findings on \u003cem\u003eB. breve, L. reuteri\u003c/em\u003e, and \u003cem\u003eL. rhamnosus\u003c/em\u003e are in line with previous observations reporting \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e species as mainly acetate producers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral species of \u003cem\u003eSaccharomyces\u003c/em\u003e are intrinsically able to determine an enrichment of SCFA-producing bacteria in the gut microbiota [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], but only a few acidify the intestinal environment through the secretion of high levels of acetic acid themselves [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Up to date, no information about secretion of propionic and butyric acid by \u003cem\u003eS. boulardii\u003c/em\u003e is available in the literature. Although many efforts have been made on \u003cem\u003eS. cerevisiae\u003c/em\u003e strains for enhancing the production of SCFAs by genetic engineering [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], a clear characterization of \u003cem\u003eS. boulardii\u003c/em\u003e ability to secrete SCFAs is still lacking. \u003cem\u003eS. boulardii\u003c/em\u003e CNCM I-745 was shown to release both acetic, propionic, and butyric acids in its culture supernatant, confirming its potency as regards SCFA metabolism.\u003c/p\u003e \u003cp\u003eIn conclusion, the application of a novel sensitive HPLC-MS-MS protocol for the detection and quantification of SCFAs allowed us to establish that all the tested probiotic strains are able to actively secrete acetic acid \u003cem\u003ein vitro\u003c/em\u003e and a part of them all the three main short-chain fatty acids. Although our study cannot exclude a different microbial behavior \u003cem\u003ein vivo\u003c/em\u003e, we believe that the \u003cem\u003ein vitro\u003c/em\u003e production of SCFAs should be taken into consideration as key feature when next generation probiotics and psychobiotics are evaluated for their potential clinical effectiveness. An in-depth characterization of strains contained in probiotic formulations as regards SCFA secretion could be a novel aspect to consider in the probiotic research and contribute to the spread of more targeted and personalized bacteriotherapy strategies to promote human health and manage diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Centre for Instrumentation Sharing of University of Pisa (CISUP) is kindly acknowledged for providing the Sciex QTrap 6500+ mass spectrometer used for the spectrometric assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding and conflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was sponsored and funded by Sanofi S.p.A. MC, AB, SC, DM, AP, FC, RZ, and AS have no conflict of interest to declare. EG has been a lecturer for Sanofi S.p.A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDatasets generated during the current study will be made available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eLeBlanc JG, Chain F, Mart\u0026iacute;n R, Berm\u0026uacute;dez-Humar\u0026aacute;n LG, Courau S, Langella P (2017) Beneficial effects on host energy metabolism of short-chain fatty acids and vitamins produced by commensal and probiotic bacteria. 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Metab Eng 34:36\u0026ndash;43. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ymben.2015.12.005\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n\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":"short-chain fatty acids, probiotics, HPLC-MS-MS, acetic acid, propionic acid, butyric acid, secretion","lastPublishedDoi":"10.21203/rs.3.rs-2128764/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2128764/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eShort-chain fatty acids (SCFAs) are the main by-products of microbial fermentations occurring in the human intestine and are directly involved in the host’s physiological balance. As impaired gut concentrations of acetic, propionic, and butyric acids are often associated with systemic disorders, the administration of SCFA-producing microorganisms has been suggested as attractive approach to solve symptoms related to SCFAs deficiencies. In this research, nine probiotic strains (\u003cem\u003eBacillus clausii \u003c/em\u003eNR, OC, SIN, and T\u003cem\u003e, Bacillus coagulans \u003c/em\u003eATCC 7050\u003cem\u003e, Bifidobacterium breve \u003c/em\u003eDSM 16604\u003cem\u003e, Limosilactobacillus\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ereuteri \u003c/em\u003eDSM 17938,\u003cem\u003e Lacticaseibacillus rhamnosus \u003c/em\u003eATCC 53103,\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Saccharomyces boulardii \u003c/em\u003eCNCM I-745) commonly included in commercial formulations were tested for their ability to secrete SCFAs by using an improved and sensitive protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS). All tested microorganisms were shown to secrete acetic acid, with only \u003cem\u003eB. clausii\u003c/em\u003e and \u003cem\u003eS. boulardii \u003c/em\u003eadditionally able to produce propionic and butyric acids. Quantitative differences in the secretion of SCFAs were also evidenced. The application of HPLC-MS-MS may help in the analysis of SCFA production by probiotics, especially for their administration as targeted bacteriotherapy to improve SCFAs deficiencies.\u003c/p\u003e","manuscriptTitle":"HPLC-MS-MS quantification of short-chain fatty acids secreted by probiotic strains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-06 15:42:05","doi":"10.21203/rs.3.rs-2128764/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":"b2ccd524-04f7-4ef8-95b0-53db49ea27b2","owner":[],"postedDate":"October 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-11-15T21:29:13+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-06 15:42:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2128764","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2128764","identity":"rs-2128764","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}