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.