Aspergillus ibericus, and purified by Saccharomyces cerevisiae YIL162 W, was evaluated. Their chemical structure and functionality were compared to a non-microbial commercial FOS sample. Prebiotics were fermented in vitro by fecal microbiota of five healthy volunteers. Microbial-FOS significantly stimulated the growth of Bifidobacterium probiotic strains, triggering a beneficial effect on gut microbiota composition. A higher amount of total short-chain fatty acids (SCFA) was produced by microbial-FOS fermentation as compared to commercial-FOS, particularly propionate and butyrate. Inulin neoseries oligosaccharides, with a degree of polymerization (DP) up to 5 , were identified only in the microbial-FOS mixture. More than 10% of the microbial-oligosaccharides showed a DP higher than 5. Differences identified in the structures of the FOS samples may explain their different functionalities. Results indicate that microbial-FOS exhibit promising potential as nutraceutical ingredients for positive gut microbiota modulation.The prebiotic potential of fructo-oligosaccharides  produced by a newly isolated  A growing interest in the consumption of prebiotics and in the understanding of their associated benefits to human health has been emerging in recent years. Prebiotics are food ingredients/components that can be selectively fermented by gut microbiota and, consequently, modify their composition, leading to an increased growth of bacteria beneficial to human health . To creaBatch fermentations are the most common and simple in vitro models for studying the microbiota of the human gut. Batch fermentation models have been used to study the prebiotic potential of dietary components such as inulin-type fructans, resistant starch, and other complex carbohydrates ,4,5,6. TAureobasidium pullulans (a yeast-like fungus) [Aspergillus spp. [Penicillium spp. [Fructo-oligosaccharides (FOS) are a recognized class of prebiotics and have been shown to promote a positive impact on gut microbiota composition and metabolic activity . FOS can fungus) , Aspergilus spp. , and Penium spp. . Structuium spp. .Because of their (β2→1)-fructose linkages, FOS reach the large intestine and the caecum intact, where they are fermented by the gut microbiota, resulting in changes in the bacterial communities’ composition and, consequently, in their metabolic activities . FOS conBifidobacterium and Lactobacillus are considered among the most important genera in the gut microbiota due to their probiotic activities and susceptibility to the action of prebiotics [Bifidobacterium by the influence of prebiotics are more likely to be seen when compared to Lactobacillus, which may be explained by the highest preference of Bifidobacterium for oligosaccharides and its higher prevalence in the human gut [Lactobacillus) and Bacteroidetes [Bifidobacterium can be found) [ebiotics . Changesuman gut . Prebiotroidetes . They ale found) , which ae found) .Bifidobacterium, and is rapidly absorbed in the proximal colon and further metabolized in the muscle, kidney, heart, or brain [Bacteroides via two main pathways: the fixation of carbon dioxide to form succinate and derived from lactate and acrylate. Although propionate is used as a minor source of energy for colonocytes, it is well known for its interaction with the immune system, which has an important anti-inflammatory effect [Clostridium [Among the most produced SCFA are acetate, propionate, and butyrate, whilst branched-chain fatty acids  and organic acids  are formed in a lower amount . The souor brain . Propiony effect . Butyratstridium .Aspergillus ibericus as a good FOS producer [A. ibericus proved to have a bifidogenic effect, to promote the growth of Lactobacillus, and to be hydrolytic resistant to the harsh conditions of the gastrointestinal tract [A. ibericus fermentation, new integrated fermentative strategies have been developed [A. ibericus in co-culture with a Saccharomyces cerevisiae YIL162 W, and were able to metabolize non-prebiotic sugars for simultaneous FOS production and purification [Recent studies identified a newly isolated strain of producer . FOS proal tract . To increveloped ,24. In aA. ibericus in co-culture with S. cerevisiae YIL162 W were characterized by gas chromatography-quadrupole mass spectrometry (GC-qMS), and their structure was further assessed by glycosidic linkage analyses. The microbial-FOS prebiotic potential was assessed by batch fermentation with human fecal inoculum, on the basis of (i) carbohydrate consumption, (ii) SCFA production, and (iii) gut microbiota composition modulation. Results were compared with a commercial non-microbial FOS sample (Raftilose® P95).In the present work, microbial-FOS produced by the A. ibericus with an S. cerevisiae YIL162 W was used, for simultaneous FOS production and purification by each strain, respectively. After fermentation, FOS mixture was desalted with activated charcoal in a shake flask, as described by Nobre et al. [Microbial-FOS samples were produced using an integrated fermentation strategy previously reported by Nobre et al. . A co-cue et al. , with fe® P95, a commercial FOS powder sample, was obtained from Beneo-Orafti Group .RaftiloseMicrobial-FOS samples were identified and quantified after derivatization in alditol acetates . The ald3I, hydrolyzed (TFA 0.5 M) and the resultant monosaccharides were reduced (NaBD4) and acetylated. The partially methylated alditol acetates (PMAAs) obtained were analyzed by GC-qMS, as previously described [The glycosidic linkages of the FOS sample were identified by methylation analysis. FOS were methylated using CHescribed . 1-Kesto2, 10% CO2, and 5% H2. The reagents cysteine-HCl and NaCl were obtained from Merck  and LabChem , respectively.Fresh feces were provided from three male and two female healthy donors, with ages between 23 and 39 years old. An informed consent form was distributed among the un-related anonymous donors to provide the participant’s information about the study and the consent certificate assigned for each. Participants had not received antibiotic treatment, nor probiotic or prebiotic supplementation for at least 6 months before the fecal sample donation and had no history of gastrointestinal diseases. The fecal samples were collected into sterile flasks, maintained under anaerobic conditions, and used within a maximum 2 h after collection. The fecal inoculum (FI) was prepared following the methodology previously described . Brieflyv/v) of a 0.5 g/L resazurin solution prepared in distilled water, 1.0% (v/v) of each trace minerals solution, and salt solution A. Salt solution B was added in the concentration of 0.2% (v/v). The salt solution A was constituted of 100.0 g/L NH4Cl, 10.0 g/L MgCl2·6H2O, 10.0 g/L CaCl2·2H2O while salt solution B was composed of 200.0 g/L K2HPO4·3H2O [Fecal fermentations were performed with a Nutrient Base Medium. This medium was constituted by trypticase soy broth (TSB) without dextrose, and bactopeptone, in a concentration of 5.0 g/L, respectively. Cysteine-HCl was added at a concentration of 0.5 g/L. This medium was also composed of 0.2% , ATCC , and Carlo Erba , respectively. The cysteine-HCl, NH4Cl, MgCl2·6H2O, and K2HPO4·3H2O were obtained from Merck . The final pH of the medium was adjusted to 6.8 with HCl (1 M) and then bubbled with N2 until a translucent/yellowish color. Next, 50 mL of basal medium was added to airtight anaerobic bottles, which were sealed with aluminum caps before sterilization by autoclave [TSB and bactopeptone were purchased from Fluka Analytical , and Becton Dickinson Biosciences , respectively. The reagents resazurin solution, trace minerals solution, and CaClutoclave .A. ibericus in co-culture with the S. cerevisiae, (ii) a commercial FOS sample derived from inulin-Raftilose® P95 , and (iii) a solution without any added carbon source—negative control. A gas combination of 85% N2, 10% CO2, and 5% H2 was then used to reflux the basal medium to assure anaerobic conditions [After sterilization, several carbon source solutions  were added to the basal medium at 20 g/L final concentration, namely (i) the microbial-FOS sample produced by the nditions .v/v) to the airtight anaerobic bottles previously prepared and incubated at 37 °C for 24 h without shaking. All manipulations were executed in an anaerobic workstation with an atmosphere composed of 85% N2, 10% CO2, and 5% H2. Samples were collected at 0, 12, and 24 h fermentation [Each fecal inoculum was added in a concentration of 2% ) was added and incubated at 37 °C for 1 h, with periodic shaking. Then, the samples were resuspended in 350 μL of NT1- buffer and incubated at 95 °C for 10 min. After this incubation step, samples were centrifuged at 11,000 g for 10 min at 4 °C, and 200 μL of supernatants were mixed with 25 μL of proteinase K and incubated at 56 °C for 1 h [Genomic DNA (gDNA) was extracted and purified from human fecal samples using a NZY Tissue gDNA Isolation kit, according to the manufacturer’s protocol with some alterations. Briefly, pellets were homogenized in Tris Ethylenediamine Tetra Acetic Acid (Tris-EDTA) buffer  and centrifuged at 4000  for 1 h . The folPurified bacterial gDNA was detected and amplified by quantitative real-time PCR (qPCR) using a CFX96 Touch™ Real-Time PCR Detection System , as previously described .® Green Supermix, 1 μL of gDNA sample (equilibrated to 20 ng/µL), 1 μL of forward and reverse primers (100 nM) targeting the 16S rRNA gene, and 2 μL of ultra-pure water. The primer sequences used in this study were obtained from STABvida  and are reported in qPCR reactions mixtures  included a volume of 5 μL of 2× iQTM SYBRThe amplification program consisted of one initial activation cycle of 95 °C for 10 min, followed by a denaturation step with 45 cycles at 95 °C for 10 s, an annealing step with 45 or 50 or 55 °C for 60 s according to each primer specific temperature, and an extension step at 72 °C for 15 s [A melting curve analysis was performed for each PCR to ensure the specificity of the amplification by monitoring SYBER Green fluorescence in the temperature interval from 60 to 97 °C, with an increase of 0.1 °C (per 0.01 min). Data were processed and analyzed using the LightCycler software obtained from Roche Applied Science .Clostridium leptum subgroup, Bacteroidetes, and Bacteroides), as well as recognized probiotics (Bifidobacterium and Lactobacillus), were selected as target groups. All analyses were carried out in quadruplicate, and standard curves were calculated using tenfold bacterial dilution gDNA standards of Clostridium leptum (ATCC 29065), Bacteroides vulgatus (ATCC 8482), Bifidobacterium longum subsp. infantis (ATCC 15697) , and Lactobacillus gasseri (ATCC 33323) .The most abundant phyla and genera in the healthy human gut microbiota  . In thisAs a first step, the fermentation supernatants were filtered using a 0.22 μm acetate cellulose syringe filter. Afterward, sugars and relevant SCFA  concentrations were calculated by high-performance liquid chromatography (HPLC).v/v)), and 0.04% of ammonium hydroxide  in water. The elution was carried out at a flow rate of 1 mL/min and 30 °C [2), nystose (GF3), and fructo-furanosylnystose (GF4)) were obtained from Wako (Japan). Sucrose and fructose standards were acquired from Merck and glucose from VWR (Belgium).For sugar analysis, an HPLC system from Jasco  was used. Briefly, this HPLC system was equipped with a Prevail Carbohydrate ES column  (Alltech) and a refractive index detector. The fecal samples were eluted with a mixture of acetonitrile  and pure water  coupled with a UV detector (wavelength: 210 nm) was used. Samples were eluted in a solution of sulfuric acid (5 mM) at a 0.7 mL/min flow rate and 60 °C [t-test, was used to determine significant differences in the quantification of bacteria populations by qPCR, at each time point. Repeated Measures ANOVA was used to evaluate the bacterial population over time. Differences were considered significant for p-values ≤ 0.05.The statistical analysis was carried out using GraphPad Prism 8.2.1 . The normality of the data’s distribution was evaluated through Shapiro–Wilk’s test. As the data proved to follow a normal distribution, One-way ANOVA, coupled with Tukey’s post hoc test or the Student’s S.cerevisiae YIL162 W strain is described in 3) was the fructo-oligosaccharide produced in higher amounts (495.8 mg/g of purified sample), followed by 1-kestose (204.6 mg/g). These FOS have an inulin structure, which consists of a linear chain of (β2→1)-fructose residues linked to a sucrose moiety. The identification of these structures is supported by glycosidic linkage analysis (t-Fru), (2→1)-Fru, and terminally linked glucose (t-Glc) were the residues found in higher proportions. In addition, the high amount of t-Fru and t-Glc  indicates the presence of fructo-oligosaccharides with a low polymerization degree. The FOS mixture composition showed that 87% of the identified carbohydrates had a degree of polymerization (DP) inferior or equal to 5 2-sucrose, which are classified as neoFOS inulin type. 1F-β-Fructofuranosylnystose (GF4) accounted for 76 mg/g, in equivalents of stachyose (DP 4). This could result in an underestimation of the GF4 quantity, as the GC-qMS sensitivity decreases inversely with increasing DP. The remaining residues found in smaller proportions  can be ascribed to other tri- and tetrasaccharides that were below the limit of quantification, such as levan and/or mixed-type FOS with a DP higher than 5, which accounted for about 13% of the sample (The residue (1→6)-Glc (3%) suggests the presence of oligosaccharides from the inulin neoseries type, such as neokestose (8.3 mg/g). Furthermore, the compound eluted after nystose , assignee sample .® P95, structurally characterized in a previous work, was richer in inulobiose and inulotriose and did not contain neoseries sugars  and after 24 h of fermentation with the microbiota from the five donors. At the beginning of fermentation, the microbial-FOS mixture contained in the basal medium included 23.9 ± 0.4 g/L oligosaccharides and 1.9 ± 0.1 g/L smaller carbohydrates, while Raftilose® P95 held 31 ± 0.9 g/L and 1 ± 1 g/L, respectively.Both microbial-FOS and Raftilose® P95 to 8 ± 5 g/L, representing a metabolization of 62 ± 10% and 76 ± 17% oligosaccharides, respectively.After 24 h of fermentation, the amount of oligosaccharides in the microbial-FOS decreased to 10 ± 3 g/L and in Raftilose4 were consumed, followed by 74 ± 14% GF3, and finally 68 ± 22% GF2 , Bacteroidetes (Bacteroides), and Actinobacteria (Bifidobacterium). To quantify changes in the bacterial population of the gut microbiota, the real-time polymerase chain reaction (qPCR) method with 16S rRNA-based specific primers  was usedBifidobacterium genus was lower than the other donors , and in similar proportion Bacteroides (0.28 ± 0.01 log 16S rRNA gene copies/ng of DNA) and Bifidobacterium (0.28 ± 0.08 log 16S rRNA gene copies/ng of DNA) (The human microbiota collected before fermentation was mainly composed by bacteria from the Firmicutes group (0.44 ± 0.01 log 16S rRNA gene copies/ng of DNA), followed by the  of DNA) .® P95, and without carbon source (negative control). Regarding the first 12 h of fermentation, Bacteroides and Bifidobacterium did not grow (p > 0.05) and there was a significant decrease of Clostridium leptum (p < 0.05) for all tested conditions, while Firmicutes phylum growth remained similar with Raftilose® P95 and significantly decreased with the microbial-FOS.Clostridium leptum, and Bacteroides did not grow (p > 0.05) for all tested conditions, regardless of the carbon source used. However, a significant growth for Bifidobacterium was observed between 12 h and 24 h of fermentation when using microbial-FOS (p < 0.05).After 12 h of fermentation and up to 24 h, Firmicutes, Clostridium leptum, and Bacteroides at 24 h (p < 0.05). Regarding Raftilose® P95, only the Firmicutes phylum had a lower population than the negative control (p < 0.05) at 24 h. Clostridium leptum and Bacteroides profiles were similar to the negative control. It was also possible to observe that the number of gene copies for Firmicutes in microbial-FOS fermentation was lower than Raftilose® P95 samples at 24 h (p < 0.05).Comparing the three different tested conditions, the fecal samples with microbial-FOS had lower microbial populations than the negative control for Firmicutes, ® P95 concerning the Firmicutes group, the Clostridium leptum subgroup and the Bacteroides group achieved a growing trend from 12 h to 24 h of fecal fermentation when compared to the negative control (p > 0.05). This might indicate that whereas Bifidobacterium initiates the fermentation process, the remaining bacterial communities have a delayed growing dynamic, which is consistent with their growth over the 12 to 24 h period.Interestingly, despite no growth effect being found for microbial-FOS nor Raftilose® P95, and without carbon source (negative control) is shown in The variation on the SCFA profile, namely acetate, propionate, and butyrate, and on the organic acids lactate, succinate, and formate during microbiota fermentation with microbial-FOS, Raftilose® P95 when compared to the negative control sample (p < 0.05). After 12 h of fermentation, microbial-FOS already had an increasing amount of acetate formed (p < 0.05). No significant differences were observed between fecal fermentations with microbial-FOS and Raftilose® P95.In this work, acetate, propionate, butyrate, lactate, succinate, and formate were produced by fermentation of the microbial-FOS. At 24 h of fermentation, there was a higher production of acetate for both microbial-FOS and Raftilose® P95 when compared to the negative control samples at both 12 h and 24 h (p < 0.05). Microbial-FOS fermentation led to a higher propionate production than Raftilose® P95 at 12 h (p < 0.05), but no significant difference was found at 24 h.Higher propionate production was also observed for fecal fermentations with microbial-FOS and Raftilose® P95 and negative samples at 24 h (p < 0.05 for both). In addition, higher butyrate production was observed for microbial-FOS samples when compared with Raftilose® P95 samples at 12 h (p < 0.05).Regarding butyrate, a strong butyrogenic effect was found in the fecal fermentations with microbial-FOS, as its production was higher than both Raftilose® P95 (p < 0.05 for both between 0 h and 24 h), although for the negative control samples the lactate concentration decreased over time (p < 0.05). Nonetheless, it did not show a statistically significant variation between 12 and 24 h for the microbial-FOS (p > 0.05), whereas it increased for Raftilose® P95 (p < 0.05). This was followed with a consistent trend in propionate and butyrate production, which was maintained with microbial-FOS between 12 and 24 h, while decreased with Raftilose® P95 (p < 0.05).Lactate concentration increased over the entire fecal fermentation for both microbial-FOS and Raftilose® P95 fermentations (4.04 ± 0.48 g/L at 12 h and 4.18 ± 0.76 g/L at 24 h), as compared to the negative control (2.50 ± 0.68 g/L at 12 h and 2.62 ± 0.50 g/L at 24 h) (p < 0.05). In addition, at 12 h of fermentation, the total amount of SCFA was statistically higher in samples from microbial-FOS than in Raftilose® P95 ones (p < 0.05). Regarding the SCFA concentration ratios, an acetate:propionate:butyrate ratio of 1.3:0.6:1.0 was obtained for the negative control, 2.1:1.5:1.0 for Raftilose® P95 and 1.1:1.1:1.0 for microbial-FOS.Considering the total amount of SCFAs, a higher production was observed in microbial-FOS (5.00 ± 0.37 g/L at 12 h and 5.29 ± 0.48 g/L at 24 h) and RaftiloseA. ibericus in mono-culture were structurally characterized in a previous work [w/w) of the total sugars at the optimal harvest point, increasing the caloric value of the mixture and, consequently, decreasing its prebiotic functionality [Microbial-FOS produced by ous work . In monoionality . The higA. ibericus with S. cerevisiae YIL162 W [Envisaging the production of FOS mixtures with higher purity and content, FOS were produced in a previous work using an integrated fermentation with co-culture of YIL162 W . The yeaA. ibericus and S. cerevisiae was assessed by fermentation with human gut microbiota, and compared with the prebiotic potential of a non-microbial commercial FOS sample. Raftilose® P95 was chosen among the available commercial FOS samples, since its structure is similar to the microbial-FOS in terms of DP, and at the same time different because it is derived from inulin and not from sucrose [® P95 and Inulin-S, Raftiline® HP, Fibrulose® F97, and Fibruline® Instant [n type), which are not found in microbial-FOS. Microbial-FOS are short oligomers synthesized from sucrose, with two to four fructose residues always linked to a terminal glucose unit (GFn type) [® P95 includes both types of FOS [® P95 had the lowest DP (2–7). On the other hand, both Inulin-S and Fibruline® Instant have a DP between 2–60; Raftiline® HP an average DP > 23; and Fibrulose® F97 a DP up to 20 [® P95 was chosen as the commercial sample for assessing prebiotic effect as compared to the microbial-FOS. The influence of the structures of each type of FOS in the microbial growth and function was evaluated.In the present study, the prebiotic potential of FOS produced in the co-culture with  sucrose . The maj Instant ,32. InulFn type) . Insteads of FOS . From thup to 20 ,32. For 3, followed by GF2 and GF4, as the main sugars composing the microbial-FOS, and in a smaller amount neokestose sucrose) 2sucrose) by other fungi strains have been reported, such as Penicillium citrinum [Xanthophyllomyces dendrorhous [A. ibericus in mono-culture [From the chemical characterization of the samples  it was psucrose) . The procitrinum  and Xantdrorhous . Neokest-culture .® P95 was richer in inulobiose and inulotriose and did not contain neoseries sugars (n type) in its composition, which were not found in microbial-FOS [® P95 sample by other authors [® P95 also showed a higher DP (2–7) than the microbial-FOS, and was mainly composed of tri-, tetra-, and penta-oligomers  present in the samples.Results on the carbohydrate consumption and SCFA production after 24 h of fermentation demonstrated that both microbial-FOS and Raftilose4 > GF3 > GF2) from the microbial-FOS sample seem to be preferentially consumed by the microbiota, even if no statistical difference was found (p > 0.05). Nevertheless, there was no sugar preference pattern in the Raftilose® P95 sample. It has been reported that each bacterial strain may exhibit distinct preferences for different chain lengths and types of FOS. For instance, Lactobacillus plantarum preferentially consumed GF2 and GF3 [Lactobacillus casei 1134 and Lactobacillus rhamnosus 1136 only utilized GF2 [. Furthermore, many Bacteroides species can ferment GF2 and GF3, but no information regarding GF4 consumption was provided [® P95).Long-chain oligosaccharides , including Raftilose® P95 [Bifidobacterium strains can utilize short-chain fructans, but not highly polymerized inulin [Lactobacillus in this matter. As perceived in this study, both microbial-FOS and Raftilose® P95 were capable of successfully modulating the gut microbiota composition. More than 70% of each fructo-oligomer was consumed after 24 h, with the exception of the GF2 of the microbial-FOS. This resulted in the metabolization of >60% of the total oligosaccharides in both prebiotic samples.Regardless, most of the bacteria are able to consume short-chain carbon sources, but only a few can utilize long-chain fructans ,39. Bifiose® P95 . It has d inulin . In addid inulin . On the Gut microbiota composition modulation by the carbohydrates was evaluated by qPCR method in the present study. The detection of groups, genera, or specific species within a complex bacterial population, such as human fecal samples, can be easily accomplished using this technique, which also has the advantage of being fast .Bifidobacterium genus present in the fecal sample of donor A  had similar Bifidobacterium growth as compared to Beneo P95 . In the present study, A. ibericus microbial-FOS stimulated a higher growth of Bifidobacterium strains, which may be related to the specific Bifidobacterium strains present in the gut microbiota under study, as well as on the microbiota consortium interaction.Nobre et al.  studied obtained . Neverthobtained . Results® P95, may also be related to their FOS structures, since only microbial-FOS has neokestose in its content [Clostridium and Bacteroides [F-FOS) since both have fructosyl units bound by a (β2→1)-linkages. However, (β2→6)-linkages between fructose and a glucosyl group (6G-FOS) are only present in the neoFOS series [Bifidobacterium strains have been shown to efficiently transport and metabolize (β2→6)-FOS [® FOS [Clostridium leptum, and Bacteroides. Other authors have reported that (β2→6)-linked oligosaccharides induce a decrease of the Clostridium group in fecal microbiota [The higher bifidogenic effect observed for the microbial-FOS samples, as compared to Raftilose content ,23. Neokteroides . The neoS series . Bifidob2→6)-FOS ,49. FurtS [® FOS . NeoFOS S [® FOS . Thus, bS [® FOS . Herein,crobiota . Hence, ® P95 have been found to lead to higher acetate production. This finding resonates with the underlying rationale that microbial-produced FOS can be used to stimulate the metabolic activity of the gut microbiota. Moreover, the higher acetate concentration in microbial-FOS samples could also be directly correlated with the strong bifidogenic effect found for the fecal samples with microbial-FOS  can also be used as a substrate by bacteria producers of butyrate and propionate [Clostridium spp. [Lactate concentration increased over the entire fecal fermentation for both microbial-FOS and Raftiloseopionate ,22, prevopionate , corroboium spp. .Moreover, the butyrate-producing bacteria are phylogenetically combined with non-butyrate producers, and consequently, its quantification with molecular techniques targeting the 16S rRNA gene becomes difficult . Nonethe® P95. Therefore, there is a very clear trend that supports the claim that microbial-FOS induces a stronger SCFA synthesis in the fecal sample fermentations undertaken in the present work. Regarding the SCFA concentration ratios, for microbial-FOS an acetate:propionate:butyrate ratio of 1.1:1.1:1.0 was obtained, and 2.1:1.5:1.0 for Raftilose® P95. Several authors have reported that polysaccharide fermentation frequently results in acetic, propionic, and butyric acids in a 3:1:1 ratio [Considering the total SCFAs, a higher production was observed in microbial-FOS as compared to the Raftilose:1 ratio , but the:1 ratio . In addi:1 ratio .Bifidobacterium population and in acetate production. This is a very interesting finding, which was not previously found on tests conducted with isolated strains, highlighting the importance of the strong interplay within gut microbiota and the effects of cross-feeding mechanisms. This also emphasizes the pitfalls of testing novel gut-modulating nutraceuticals on consortiums of isolated strains, as their complex metabolic interactions play a central role.The holistic analysis of the entire data acquired with this study puts in evidence how the differences in carbohydrate profiles from different FOS have a direct impact on the dynamics of the gut microbiota, namely distinct production of benefic SCFA. In particular, this underlying rationale is supported by the presence of a neoseries oligosaccharide, which has a bifidogenic effect demonstrated through an increase in the A. ibericus in co-culture with an S. cerevisiae YIL162 W using human fecal samples.The present study brings forward a first characterization of the prebiotic potential of a novel microbial-FOS produced by an ® P95. Neoseries-type FOS, such as neokestose (DP 3), possibly neonystose (DP 4), and neofructo-furanosylnystose (DP 5), were identified in the microbial-FOS but not found in Raftilose® P95. These structural differences may justify the strongest bifidogenic and butyrogenic effects found in fermentations run with the microbial-FOS. The microbial-FOS bifidogenic effect found in the present work, while fermented by human gut microbiota, reinforces the importance of cross-feeding mechanisms on the overall prebiotic evaluation of novel nutraceutical ingredients.Based on the structure analyses, there are significant differences in the structural composition of the microbial-FOS vs. RaftiloseA. ibericus were found to successfully modulate gut microbiota and stimulate the SCFA production, providing a significantly higher concentration of these beneficial components than Raftilose® P95. The further incorporation of the microbial-FOS in new formulations may result in excellent novel functional foods.FOS produced by


POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that  Macroautophagy, hereafter referred to as autophagy, represents a cellular recycling mechanism, which regulates degradation of misfolded, oxidised, aggregated, or simply unneeded proteins. Furthermore, autophagy can facilitate the degradation of entire organelles, such as mitochondria or peroxisomes . AutophaPhospholipid synthesis occurs predominantly at the ER and, to some extent, within mitochondria. Mitochondria can contribute to PE synthesis, which is used widely within the cell, as well as synthesis of those lipids specific to the organelle, such as cardiolipin (CL) and phosphatidylglycerol (PG). Phosphatidic acid (PA) represents the precursor for CL and PG synthesis , whereasThese lipid transport systems and the communication between the ER and mitochondria might represent important factors not only for co-ordinating lipid metabolism, but also in regulating autophagy. Such communication is facilitated by proteins or protein complexes, which bring the two organelles into close contact. These include membrane contact sites (MCS) between the ER and mitochondria, in yeast, namely ERMES ,27  obtained from Euroscarf. The single-deletion strain of POR1 was obtained from the Euroscarf knock-out collection. Yeast strains carrying pATG8–EGFP–ATG8 fusions were generated according to the method by Janke et al. [VPH1–yEGFP (introduced with a URA3 selection marker) were a kind gift from Sabrina Buettner. For expression of Por1, we used PCR amplification of POR1 using the following primers:All experiments were carried out in BY4741 2SO4, and 30 mg/L of all amino acids (except 80 mg/L histidine and 200 mg/L leucine), 30 mg/L adenine, and 320 mg/L uracil with 2% glucose. All media were prepared with ultrapure water (MilliQ) and subsequently autoclaved . Amino acid mixtures were sterilised separately as 10× stocks and added after autoclaving; this also applies to glucose and galactose stocks. All yeast cultures were inoculated from a stationary overnight culture to an OD600 = 0.1 or 0.15, and then grown at 30 °C and 145 rpm shaking for indicated time points. For Gal10-driven expression of Por1 or Psd1, we used SC medium with 1% D-glucose and 1% D-galactose (1:1 SCD/SCG) or, in the case of Psd1 expression, SC medium with 1.5% D-galactose and 0.5% D-glucose. These mixed-carbon source media were used to optimise growth conditions for por1∆, which has severe growth defects on galactose-only-containing growth media, while, at the same time, allowing for expression of the constructs. Cells were shifted to SCD/SCG medium 5 h after inoculation. Aliquots were harvested at indicated time points to study survival, vacuolar morphology or autophagy.A restriction digest of the PCR product was carried out with EagI (CGGCCG) and SacI (GAGCTC), which was then ligated into the similarly digested pESC-HIS plasmid (Stratagene). escribed . Transfoescribed . At leas4)2SO4 and amino acids after 8 h of growth, with two steps of washing with ultrapure water. Samples were adjusted to an OD600 of 1.All autophagy induction experiments were carried out on SC medium. For rapamycin-induced autophagy, rapamycin (dissolved in DMSO) was supplemented to a final concentration of 30 nM at 6 h after inoculation. Autophagy induction by nitrogen starvation was similarly performed as described before . In briepor1∆, and POR1- or PSD1-overexpression strains were inoculated from overnight cultures to an OD600 of 0.1  in 24-well plates (Sarstedt) in two or three independent experiments, each containing at least three biological replicates. The plate was automatically measured for 24–48 h using a BMG LabTech SPECTROstarNano plate reader with double orbital shaking at 400 rpm and 30 °C, with OD600 measurements every 30 min. Growth curves were plotted in GraphPad Prism.Wildtype, Autophagy was measured by monitoring the cytosol-to-vacuole translocation of Atg8 using fluorescence microscopy and immunoblotting (GFP liberation assay) of cells/cell extracts from strains carrying a GFP–Atg8 fusion protein expressed under its endogenous promoter and at its natural chromosomal locus ,42.Quantification of micrographs was performed from blinded pictures from 3 clones per replicate. Autophagic cells were defined as cells exhibiting clear vacuolar GFP fluorescence and expressed as the fraction of viable (PI-negative) cells. All micrographs were taken after 24 h, either on SCD/SCG (1:1) media or nitrogen starvation media. The quantification on SCD/SCG media contains 11 micrographs per strain, with 96–525 cells per micrograph; in total, 2871–4054 cells were evaluated. The quantification of GFP-positive vacuoles on nitrogen starvation includes 4 micrographs per replicate  per strain. In total, 1300–1550 cells were manually counted per genotype.600 of 3 were harvested at indicated time points, and cell extracts were obtained from chemical lysis as described in [For Western blot analysis, cell equivalents of an ODribed in . Proteinribed in . As seco600 = 8 equivalents of stationary yeast cell cultures were harvested and disrupted in 1.5 mL Eppendorf tubes with 100 µL of acid-washed glass beads using a Retsch mill Tissue Lyser with agitation for 3 min at 25 Hz. Subsequently, the protein concentration was measured applying a Bio-Rad protein assay. A total of 1.5 µg of total protein was used for the assay. The reaction was started by adding 50 µL α-naphthyl phosphate, and then incubated at 30 °C for 20 min. The reaction was stopped by adding 200 µL glycine–NaOH and fluorescence was measured using Tecan Infinite M Plex. To correct for intrinsic (background) ALP activity, the ALP activity of corresponding strains without Pho8∆60 manipulation was assessed and subtracted.Additional biochemical autophagy measurements were performed using the quantitative Pho8∆60 assay  for nonspecific autophagy, essentially as described earlier . In briePropidium iodide (PI) staining was used to determine loss of membrane integrity. Cells were harvested in 96-well plates at indicated time points and resuspended in 250 µL of 100 µg/L PI in PBS, and incubated in the dark for 10 min at room temperature. After incubation, cells were washed once with 250 µL PBS and analysed via flow cytometry (Beckmann Coulter Cytoflex). A total of 30,000 cells per strain and condition were measured and analysed with CytExpert software.S. cerevisiae strains (wildtype and por1∆) were inoculated to an OD600 = 0.1 from overnight cultures in SCD medium and shifted to SCD/SCG (1:1) medium after 5 h. OD600 equivalents of 1.0 were harvested at 24 h after shift and mounted on microscopy slides. Fluorescence was detected using a Nikon Eclipse Ni-U fluorescence microscope with a Hamamatsu Orca-Spark C11440-36U monochromatic camera and Nikon Intensilight C-HGFI illumination system. Images were captured, saved, and processed using NIS-Elements BR 4.13.05 64-bit and ImageJ software. For quantification, 416–687 cells per genotype and condition were manually counted.Vacuole morphology was determined using an endogenously GFP-tagged Vph1 strain. por1∆ cells were grown in the same manner as described for Vph1–GFP microscopy. After 24 h, cells were washed with 500 μL of YEPD containing 100 mM HEPES (pH 7.6). After centrifugation, the pellet was resuspended in 500 μL of YEPD with 100 mM HEPES (pH 7.6) and 200 μM quinacrine, and incubated for 10 min at 30 °C and 145 rpm. Afterwards, samples were transferred to ice and incubated for 5 min. After centrifugation, cells were washed twice in 500 μL ice-cold HEPES buffer supplemented with 2% D-glucose. The cells were pelleted by centrifugation, and then analysed using a Nikon Eclipse Ni-U epifluorescence microscope. For quantification, 1600–1930 cells per genotype were manually counted.Functional vacuole acidification was assessed using the fluorescent dye quinacrine. Vacuolar quinacrine internalisation depends on vacuolar acidification. Hence, quinacrine fluorescence inside the vacuole can be used as a measure of vacuole function ,46. Wild600 of 0.1, and shifted to SCD/SCG (1:1) media after 5 h. In total, 2 OD600 units were harvested after 24 h and homogenised with 0.5 mm zirconia beads in a cooled tissue lyser for 2 × 10 min at 30 Hz in 300 µL IPA. The whole homogenate was evaporated in a vacuum desiccator to complete dryness. Lipid extraction was performed according to [2O, samples were vortexed for another 15 min. Phase separation was induced by centrifugation at 13,400 rpm for 15 min. The organic phase was transferred to a glass vial and evaporated. Samples were reconstituted in 300 µL 1:2 MeOH/CHCl3. For lipidome, 5 µL of sample were diluted with 95 µL 4:2:1 IPA/MeOH/CHCl3 + 7.5 mM ammonium formate.Yeast cultures were inoculated from stationary overnight cultures in SCD to fresh SCD medium to an ODrding to ,48,49. Im/z 400–1000 from 0 min to 0.2 min in the positive mode, and within the range of m/z 350–1200 from 6.2 min to 6.4 min in the negative mode at a mass resolution of R m/z 200 = 140,000, automated gain control (AGC) of 3 × 106, and with a maximal injection time of 3000 ms. Ergosterol was determined by parallel reaction monitoring (PRM) FT-MS/MS between 0.2 and 1.7 min. For FT-MS/MS, micro-scans were set to 1, isolation window to 0.8 Da, normalised collision energy to 12.5%, AGC to 5 × 104, and maximum injection time to 3000 ms. t-SIM in positive (1.7 to 6 min) and negative (6.4 to 18 min) mode was acquired with R @ m/z 200 = 140,000; automated gain control of 5 × 104; maximum injection time of 650 ms; isolation window of 20 Th; and scan range of m/z 400 to 1000 in positive and m/z 350 to 1200 in negative mode, respectively. The inclusion list of masses targeted in t-SIM analyses started at m/z 355 in negative and m/z 405 in positive ion mode, and other masses were computed by adding 10 Th increment  up to m/z 1005 in positive mode and up to m/z 1205 in negative mode. All acquired spectra were filtered by PeakStrainer (https://git.mpi-cbg.de/labShevchenko/PeakStrainer/wikis/home (accessed on 18 June 2020)) [Mass spectrometric analysis was performed on a Q Exactive instrument  equipped with a robotic nanoflow ion source TriVersa NanoMate  using nanoelectrospray chips with a diameter of 4.1 µm. The ion source was controlled by the Chipsoft 8.3.1 software (Advion BioSciences). Ionisation voltage was +0.96 kV in the positive and −0.96 kV in the negative mode; back pressure was set at 1.25 psi in both modes. Samples were analysed by polarity switching . The teme 2020)) . Lipids e 2020)) . MoleculPOR1 or PSD1 was induced by shifting to SCD/SCG mixtures as described in the paragraph on growth conditions. In total, 80 OD600 units were harvested at 24 h after inoculation for experiments without additional overexpression, or at 24 h after shift for overexpression experiments. Total lipids were extracted with chloroform/methanol 2:1 (v/v) according to Folch et al. [v/v). A total of 30 µL of lipid extracts were sprayed on TLC plates using a CAMAG Linomat 5. PE (850757P), PS (840034P), and PC (850457P) were purchased from Sigma Aldrich and included as standards on each TLC plate. The PE standard was applied at 6 different dilutions to allow for calculation of a standard curve and determine absolute PE levels.Yeast cultures were inoculated from stationary overnight cultures in SCD to fresh SCD medium to an OD600 of 0.1. Expression of h et al. . The org3/MeOH/water (32.5:12.5:2) mixture as mobile phase [2SO4 and 0.5% MnCl2, followed by carbonization at 120 °C for 30 min. Developed TLC plates were imaged using a Bio-Rad Universal Hood II, and bands were quantified using image J.Neutral lipid separation and analysis was performed by thin-layer chromatography (TLC) on silica gel 60 plates (Merck), essentially as described before , using Cle phase ,56. TLC post hoc test or Welch’s ANOVA  with Dunett’s post hoc test was performed. A two-way ANOVA analysis was performed in Figure 2g. A Student’s t-test was applied when comparing two groups only. Information on individual statistical tests is given in the figure legends. Error bars indicate standard error of the mean (SEM) and asterisks in the figures indicate significant differences, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figures were prepared with GraphPad Prism and Adobe Illustrator CS6 (Adobe). Microscopic pictures were processed with Fiji [Statistical analysis was calculated in GraphPad Prism8. To disprove the null hypothesis (no difference between conditions) a one-way ANOVA corrected with a Tukey ith Fiji .POR1 deletion only mildly affects growth on fermentable carbon sources, whereas growth on non-fermentable carbon sources is largely affected by POR1 deletion  staining was used as a measure of cell death, which was significantly increased in por1∆, starting from day 3 of the starvation experiment  catalyses the decarboxylation of PS to form PE. Psd1 is dually targeted to mitochondria and the ER and, thus, can produce PE within both organelles depending on metabolic needs . Especia8 assays a–c. Succ8 assays . Psd1 unin por1∆ . We concVDACs are important proteins of the outer mitochondrial membrane with high abundance and involvement in multiple functions, which include metabolite shuttling ,67, mitoPOR1 deletion decreased basic autophagy levels and reduced the susceptibility to autophagy induction by rapamycin and nitrogen starvation. POR1 deletion further triggered vacuole dysfunction, which could causally link to autophagy impairment in por1∆, as vacuole function is absolutely required for autophagy. Vacuole dysfunction itself could be a direct cause of altered lipid metabolism, or due to changes in signalling. The yeast AMPK orthologue Snf1 kinase, for example, has been connected to porins in yeast [in yeast . Anotherin yeast . Whetherpor1∆ and wildtype (por1∆ (Our lipidomic analysis revealed significant changes in key phospholipids when comparing wildtype . In facte (por1∆ b, which POR1 deficiency reduces total PE levels (por1∆ (Our analysis further showed that E levels . A reducs (por1∆ . We reasInterestingly, Acc1, as well as Fas1, have been identified as crucial players in extending the autophagosomal membrane by de novo phospholipid synthesis during autophagy ,14. SchüPSD1 overexpression has been shown to increase the autophagic capacity [PSD1 and PSD2 resulted in reduced autophagy [PSD1 and PSD2 single mutants have been studied regarding their role in mitophagy, as discussed later [POR1 deletion has been associated with reduced efficiency in mitochondrial protein import [por1∆. Another recent study investigated the involvement of Psd1 during mitophagy in yeast [psd1∆ is due to impaired recruitment of Atg8 to mitochondria. Even though we did not investigate specific forms of autophagy, such as mitophagy, here, our findings of porin 1 involvement in autophagy and its relation to Psd1 function are compatible with the results from Vigié et al.Mitochondria and the ER exchange PS and PA via membrane contact sites ,28,75. Tcapacity , whereasutophagy . PSD1 aned later . Since Ped later , it can ed later . Inductin import ,69, Psd1in yeast . InteresFor future research, it remains to be investigated whether porin function is needed for the extension of autophagosomal membranes or, rather, at the step of autophagy initiation/PAS formation.


Phaseolus vulgaris is a grain cultivated in vast areas of different countries. It is an excellent alternative to the other legumes in the Venezuelan diet and is of great agronomic interest due to its resistance to soil acidity, drought, and high temperatures. Phaseolus establishes symbiosis primarily with Rhizobium and Ensifer species in most countries, and this rhizobia-legume interaction has been studied in Asia, Africa, and the Americas. However, there is currently no evidence to show that rhizobia nodulate the endemic cultivars of P. vulgaris in Venezuela. Therefore, we herein investigated the phylogenetic diversity of plant growth-promoting and N2-fixing nodulating bacteria isolated from the root nodules of P. vulgaris cultivars in a different agroecosystem in Venezuela. In comparisons with other countries, higher diversity was found in isolates from P. vulgaris nodules, ranging from α- and β-proteobacteria. Some isolates belonging to several new phylogenetic lineages within Bradyrhizobium, Ensifer, and Mesorhizobium species were also specifically isolated at some topographical regions. Additionally, some isolates exhibited tolerance to high temperature, acidity, alkaline pH, salinity stress, and high Al levels; some of these characteristics may be related to the origin of the isolates. Some isolates showed high tolerance to Al toxicity as well as strong plant growth-promoting and antifungal activities, thereby providing a promising agricultural resource for inoculating crops. Phaseolus vulgaris (common bean) is indigenous to America and is currently the most cultivated legume worldwide after soybean  may behave as short-lived perennials, bushes, or climbers (Phaseolus vulgaris L.), lima beans (P. lunatus L.), runner beans (P. coccineus L.), tepary beans (P. acutifolius A. Gray), and year-long beans (P. polyanthus Greenman) , which nodulates P. vulgaris, Campylotropis spp., Cassia spp.  and P. vulgaris ‘L2234-MGM’ (White cultivar) in Aragura and Lara and from the White cultivar in Guárico . No inoculants were used in these soil-collected areas; therefore, the strains obtained were considered to be indigenous to Venezuela. The ecosystems of the soil-collected areas and soil types are shown in Soil samples were collected from ten Venezuelan regions  and usedP. vulgaris ‘Tacarigua’ (Black cultivar) and ‘L2234-MGM’ (White cultivar) are used as trap hosts to isolate rhizobia strains. Seeds were surface-sterilized with 70% (v/v) ethanol for 1 min and 3% (v/v) sodium hypochlorite for 2 min, followed by washing four times with sterile distilled water  moistened with 5 mL of sterile Milli-Q water  in a sterile Petri dish. Filter paper and water were sterilized with an autoclave at 120°C and 0.2 MPa for 30 min /8-h dark photoperiod. After four weeks, root nodules were harvested and their surface was sterilized with 70% (v/v) ethanol for 30 s, washed with sterile distilled water, and then immersed for 1 min in 5% (v/v) sodium hypochlorite, followed by washing four times with sterile distilled water. Surface-sterilized root nodules were crushed in 500 μL of glycerol solution (50% [v/v]) to obtain bacterial suspensions. An aliquot (10 μL) of each suspension was streaked onto Yeast Mannitol agar (YMA) (30 min  was addear (YMA)  and incuar (YMA) .P. vulgaris ‘Tacarigua’ (Black cultivar) was used to examine the symbiotic activity and performance of isolated strains. One hundred and twenty isolates were selected  ethanol for 30 s and 3% (v/v) sodium hypochlorite for 2 min followed by washing four times with sterile distilled water. One milliliter of each rhizobial cell suspension containing 108 cells in TE buffer was then inoculated on each seed of ‘Black’ P. vulgaris. Inoculated seeds were sown in plant boxes (7.6×7.6×10.2 cm) with 200 g of vermiculite (Vermitech). Sterilized N-free nutrient solution  with a 16-h light/8-h dark photoperiod. Plants with no inoculation served as the treatment control (selected  and growsolution  was adde control .2 fixation activity based on acetylene reduction activity (ARA). ARA was detected using a Shimadzu GC-2014 gas chromatograph (Shimadzu) equipped with a Porapak N column (Agilent Technologies) with a 30-min incubation . Total genomic DNA was extracted from isolates using the CTAB method described by Sixty-three isolates were selected based on their nodulation ability. DNA was extracted from isolates grown in YM broth medium at 28°C for 4 days. Before genomic isolation, cells were collected and washed twice with equal volumes of PBS buffer , ATP synthase (atpD), and glutamate synthase (glnA) are described in α-proteobacteria and β-proteobacteria. The nifH primer set described in nodD gene primer set described in –1 ethidium bromide. Bands with the predicted sizes were then excised from gels, and DNAs were purified using a FastGene® agarose gel/PCR extraction kit (Nippon Genetics). According to the manufacturer’s protocols, PCR products were sequenced using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The sequences obtained were aligned using the ClustalW method and then compared in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) using the online software BLAST algorithm-based sequence alignment. Phylogenetic trees were constructed by Genetyx version 11 and MEGA version 12.0  under the following accession numbers: LC585433–LC585495 for 16S rRNA sequences, LC585496–LC585558 for the 8 cells L–1 was transferred onto YMA plates or broth followed by an incubation at 28°C for 5–10 days with different stress conditions, such as high temperature, alkalinity, acidic pH, high salinity, and a high concentration of Al at different pH levels, as described in 3·6H2O  under acidic (pH 4.5) or neutral (pH 6.8) conditions. After 5 days, colony-forming units (CFUs) were calculated by plate counting under stress conditions. The growth of isolates was estimated relative to the control treatment (non-stress) as follows: no growth; weak growth (10–20% of the control); good growth (30–60% of the control); and excellent growth (similar to/the same as the control) , spectinomycin , streptomycin , chloramphenicol , and nalidixic acid  , Rhizoctonia solani (MAFF No. 237699), Fusarium graminearum (MAFF No. 240353), Pyricularia oryzae (MAFF No. 101506), Colletotrichum gloeosporioides (MAFF No. 306534), Rosellinia necatrix (MAFF No. 328101), and Helicobasidium mompa (MAFF No. 328090). Two controls were set: a plate with an isolate without a pathogen and a plate with a pathogen without an isolate. The zone inhibition diameter and colony size were measured, and the percent inhibition of the growth of the test pathogen was calculated. The experiment was performed in triplicate for each rhizobia strain and pathogen.Bacterial isolates were grown in YM broth medium at 28°C for 5 days. Each cell suspension at 107 cells mL–1) of each culture was then spotted onto Pikovskaya’s medium to test for P solubilization or Aleksandrow’s agar to test for K solubilization. Pikovskaya’s medium contained 10 g glucose, 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.1 g MgSO4·7H2O, 0.2 g KCl, 0.002 g MnSO4·H2O, 0.002 g FeSO4·7H2O, and 0.5 g L–1 yeast extract supplemented with 5.0 g L–1 of inorganic phosphorus as tricalcium phosphate  as insoluble P and pH was adjusted to 7.0 2 supplemented with 2.0 g L–1 of Mica powder  as insoluble K and pH was adjusted to 7.2  and Floodplain (Apure) with acidic soils and high concentrations of exchangeable Al  and 512 nodules from ‘L2234-MGM’ (White cultivar), with 173 root nodules being collected from P. vulgaris grown in the four field locations and 1,039 nodules being harvested from pot cultivations inoculated with the sampled soils  showed the second highest nodulation production . 56 root nodules were obtained in the Amazonas (rainforest); this site is located in the Guiana Highlands, in which crop production has traditionally been performed . In Apure (Floodplain), where nutrients such as N and P are deficient, fewer root nodules were obtained than in most of the other sites. In Lara and DC, no nodule was obtained with the ‘Black’ cultivar in pot isolation. The Lara region is classified as a dried savanna with a xerophilic ecosystem in which soils are sandy with a low nutrient supply.A total of 1,212 root nodules were collected from two endemic ed soils . The AraP. vulgaris ‘Tacarigua’ (Black cultivar). Only 63 isolates produced nodules  nodule suspensions. Among them, 120 strains representative of each site based on their phenotypes were selected and inoculated into  nodules , and werα-proteobacteria (GI) and β-proteobacteria (GII) with two Pseudomonas species in the out-group (γ-proteobacteria). Most of the Venezuelan isolates  were identified as α-proteobacteria  and uncultured Bradyrhizobium (91.3%) in the Blast search.The GI cluster was divided into five sub-groups, GIA to GIE A. GIA con Falcón . Rhizobirtilizer  and 2. Gand Lara B. GIC com Falcon B. GID grultivars B. The ishizobium . GIE con strains B, with Visolates B. VGP6 aβ-proteobacteria, such as Burkholderia and Paraburkholderia /R. etli CFN42 (U80928.5), and four isolates not related to any reference strain. GIA isolates included VAFW5, which was in the Agrobacterium group based on MLST  was divided into two sub-groups  based on the nifH gene .Consequently, the detected . IneffecAll 63 genetically categorized isolates were phenotypically characterized and assessed under different abiotic stress conditions . Growth Bradyrhizobium in MLST, were fast growers (48 h). The majority of isolates utilized sucrose as a carbon source and exhibited the ability to live as free-living bacteria using Ashby medium, which is generally used for free-living diazotrophic bacteria (unpublished data).Strains showed different growth abilities under high temperature conditions. A total of 99% of isolates grew at 45°C, with weaker, better, or the same growth as the control. All isolates grew at 20°C, whereas those from hot regions  showed weak growth. VAFW14, VGP9, and VLaW3, classified as B. yuanmingense) and VTrP4 (Rhizobium sp.) from acidic soil (Ultisol), were not tolerant of NaCl at 3 or 4%. The growth of isolates from Apure, Guárico, and Falcon was not affected by NaCl, even at 4%. Four isolates obtained from Falcon (Aridisol), 14 out of 15 isolates from Guárico or Lara (Vertisol), and two from Andes (Ultisol) showed high salinity tolerance growing at 4% of NaCl  with better or the same growth as the control. Two isolates, VMiP5 from Alfisol ( of NaCl .Rhizobium sp.) did not grow even at pH 5.0. In contrast, all isolates survived under alkaline conditions at pH 10, with only 2% showing weak growth , 18% did not grow at pH 4.5, and VTrP4 , streptomycin (Str), kanamycin (Kan), nalidixic acid , and chloramphenicol (Cp). Some isolates exhibited multi-tolerance, and VAFP4, VAFW1, VAW10, VAW6, VMP3, and VLaW3 exhibited resistance to all antibiotics . A totalP≥0.05). VTrP4 (Rhizobium sp.) was susceptible to all antibiotics. Furthermore, it did not survive under low pH and high Al. In contrast, VGP6 and VGP9, classified as Bradyrhizobium sp., showed high tolerance to almost all abiotic stress conditions. Other isolates with high performance under abiotic stress were VAP4, VApP10, VApP8, VMP23 (classified as Rhizobium), and VGW2 (Mesorhizobium sp.) (No relationship was observed between antibiotic resistance and pH or Al tolerance (ium sp.) .–1, and were classified as Rhizobium , except for VAmP8, which was classified as Burkholderia in MLST. Auxin hormone production was dominated by Rhizobium  and Mesorhizobium (VGP2) isolates with more than 90 μg mL–1. Fourteen isolates did not produce detectable levels of IAA, but exhibited strong antifungal activity (Rhizobium sp.) for IAA (198.99 μg mL–1), VFP6 (Ensifer sp.) for P solubilization (1.67 index), and VMP23 (R. phaseoli) for K solubilization (1.22 index). Among all isolates, only VGP4 (Rhizobium sp.) and VFP6 (Ensifer sp.) showed Fe chelation .A summary of symbiotic performance, physiological profiles as plant partners, antifungal profiles, and phylogenetic groups based on MLST are shown in activity . IsolateP. aphanidermatum, whereas only ten isolates exhibited activity against C. gloeosporioides. VTrP29 (Rhizobium sp.) showed good responses against all fungal pathogens, while VGP9 (Bradyrhizobium sp.) exhibited very high potential against all tested pathogens, mainly P. aphanidermatum, R. necatrix, and H. mompa. VTrP4 (Rhizobium sp.) exhibited activity against four pathogens . These isolates with high antifungal performance exhibited low physiological activities for plant growth promotion and symbiotic performance.Furthermore, 46% of isolates exhibited antifungal activity against at least one pathogen at different levels . Twenty-P. vulgaris has been examined worldwide with various techniques and criteria. Collectively, the present results indicate that many species nodulate Venezuelan common beans; these cultivars were promiscuous hosts. Bacterial cells were only obtained from 19% of the 1,212 nodules collected in the present study  with low nutrients, such as N and P, which was similar to the distribution of Mesorhizobium in soils with low nutrients in Brazil , the nodulation of Bradyrhizobium species in P. vulgaris may be ascribed to the adaptation of legumes in Venezuela , Alfisol (Miranda and D.C), Inceptisol (Aragua), and Oxisol (Amazonas). However, several isolates belonging to Burkholderia were also found in Mérida and Amazonas   Genetic Diversity and Characterization of Symbiotic Bacteria Isolated from Endemic https://doi.org/10.1264/jsme2.ME20157Supplementary Material


Ligularia stenocephala Matsum. & Koidz. (LSE) and Secale cereale L. sprout (SCSE) (TEES-10®) were prepared. The purposes of the study were to evaluate anti-inflammatory activities of TEES-10® in vitro and to observe resolution of gingivitis in human with oral administration of TEES-10®. The effects of TEES-10® on normal periodontal ligament (PDL) cell viability, lipopolysaccharide (LPS) induced PDL cell viability and the changes of inflammatory mediator expression were evaluated in vitro. In the clinical trial, 150 mg of TEES-10® powder containing capsule was administered twice daily to the test group, while the control group administered placebos in a total 100 participants with gingivitis. Probing depth (PD), bleeding on probing (BOP), clinical attachment loss, gingival index (GI) and plaque index (PI) were measured at baseline and 4 weeks. Administering TEES-10® showed significant increase in PDL cell viability compared to administering LSE or SCSE alone. In addition, treating TEES-10® to LPS induced PDL cell significantly increased PDL cell viability compared to control. TEES-10® suppressed expression of NF-κB, p-ERK, ERK, COX-2, c-Fos and p-STAT and promoted expression of PPARγ in LPS induced PDL cells. In the clinical trial, significant improvement of GI and BOP was observed in the test group at 4 weeks. In addition, the number of patients diagnosed with gingivitis was significantly reduced in the test group at 4 weeks. Salivary MMP-8 and MMP-9 was also significantly decreased compared to placebo group. Within the limitations of this study, the TEES-10® would have an anti-inflammatory potential clinically in the chronic gingivitis patients.Complex extracts of  Periodontal disease is the result of host immune response against biofilm which induces destruction of alveolar bone and periodontal tissue . In healAmplified host responses result in the destruction of periodontal tissue through degradation of cell membrane by reactive oxygen species (ROS) from phagocytosis, inactivation of proteolytic enzyme inhibitor and activation of proteolytic enzymes such as MMP ,6. In diHost modulation therapy such as intake of functional foods or antioxidative nutrients like vitamin was suggested to enhance host defense system and down-regulate periodontal inflammation ,9. By suLigularia stenocephala Matsum. & Koidz. (LSE) and Secale cereale L. sprout (SCSE) showed its anti-oxidative and anti-inflammatory activities through in vitro studies [L. stenocephala improved inflammatory bowel diseases which is a chronic inflammation [L. stenocephala and S. cereale sprout (TEES-10®).With increasing interests to host modulation therapy with dietary foods, numerous traditional medicinal plants were tested. Among them, extracts of  studies ,14. In aammation . In the ®. Thus, the main objectives of this study were to evaluate anti-inflammatory activities of TEES-10® in vitro and to confirm improvement in chronic gingivitis in human with oral administration.Previous studies about host modulation therapy for periodontal disease had been mostly focused on nutrients and medications ,16. Howe®, a complex extract of LSE and SCSE, was provided by Famenity . Leaves of L. stenocephala and the sprout of S. cereale were selected, crushed and extracted twice with 50% aqueous ethanol then filtered. After second filtration, each filtrate was concentrated until brix was 20–25%. According to the ratio of solid content, dextrin added 1:1 in weight and dried to powder forms. LSE and SCSE were mixed in a ratio of 6:4 in weight to be TEES-10®. For clinical trials, 150 mg of TEES-10® powder were filled in hard capsules and used as TEES-10®-administered group (test group), while dextrin was filled in the same capsules and used as a placebo (placebo group).TEES-10®) were precisely weighed and dissolved into 50% aqueous methanol. In addition, thereafter, the test sample (5 μL) and standard samples  were subjected to high performance liquid chromatography  using a Supelco Discovery C18 column  filled with octadecylsilyl silica gel  at 0.7 mL/min of flow rate. Optimum HPLC separation was achieved at 40 °C. UV wave lengths were 330 nm for chlorogenic acid and 350 nm for tricin. The phosphoric acid  was mixed with distilled water and acetonitrile , respectively, and these solvents were used as a mobile phase. The different conditions for each standard were using mobile phases A (0.5% phosphoric acid in water) and B (0.5% phosphoric acid in acetonitrile). The mobile phase condition of chlorogenic acid was as follows: 0–7 min (10% B), 7–27 min (10–30% B), 27–28 min (30–70% B), 28–30 min (70% B), 30–31 min (70–10% B) and 31–40 min (10% B). The mobile phase condition of tricin was as follows: 0–10 min (10–30% B), 10–25 min (30% B), 25–27 min (30–90% B), 27–30 min (90% B), 30–31 min (90–10% B) and 31–35 min (10% B).The chlorogenic acid  and the tricin  as standard samples and the test sample  cell  was cultured according to the manufacturer’s protocol . In brie®, which is a combination of LSE and SCSE, at varying concentrations  was treated to the PDL cells. To evaluate the toxicity on PDL cells, the cells were seeded onto 96-well plates at a density of 8 × 103 cells/well. To compare cell viability, 10 µL of 250 µg/mL, 500 µg/mL and 1000 µg/mL of TEES-10® and, 500 µg/mL and 1000 µg/mL of LSE and SCSE were applied to 90 μL of medium. Cell viability was evaluated after 24 h.LSE alone, SCSE alone and TEES-10® 50 μg/mL + LPS treated (Tc50) group and TEES-10® 100 μg/mL + LPS treated (Tc100) group. In LPS treated, Tc50 and Tc100 groups, 1 μg/mL of LPS  was added onto plates to induce inflammation to the PDL cells. Then, 50 and 100 μg/mL TEES-10® were added to each Tc50 and Tc100 groups.Lipopolysaccharide (LPS) was applied to induce inflammation of the cells . PDL celAfter 24 h of incubation, change of PDL cell viability was observed using commercially available cell viability assay kit . According to manufacturer’s method, tetrazolium salt solution was treated to each well for 2 h. Then, absorbance was measured at 450 nm using microplate reader  .6 cells/dish and stabilized in a 5% CO2 incubator for 24 h. Dishes were divided into four study groups; sham control, LPS treated group, TEES-10® 50 μg/mL + LPS treated (Ti50) group and TEES-10® 100 μg/mL + LPS treated (Ti100) group. In LPS treated group, 1 μg/mL of LPS was treated to the dishes. Ti50 and Ti100 groups were treated with 1 μg/mL of LPS, then 50 and 100 μg/mL of TEES-10® were applied as allocated. The culture medium was removed after 24 h and the cells were washed in phosphate buffered saline. Intracellular protein was dissolved using 1 mM of phenylmethylsulfonylfluoride (PMSF), 1% protease inhibitor cocktail and NP40 cell lysis buffer  and centrifuged at 13,000 rpm at 4 °C for 5 min. After separating the supernatant, protein concentration was quantified using the Pierce™ BCA protein assay kit . After 30 μg of protein was electrophoresed using Bolt™ 4–12% Bis-Tris Plus Gels, proteins were transferred onto a nitrocellulose membrane using iBlot® Transfer Stack  and iBlot® Gel Transfer Device . Each membrane was blocked at room temperature for 1 h using 5% skim milk  and washed three times using 0.1% TBST buffer . Primary antibodies against NF-κB , p-ERK , ERK , COX-2 , c-Fos , p-STAT1 , PPARγ  and β-actin  were incubated with membranes overnight at 4 °C. The membranes were then incubated with the secondary antibody, HRP-conjugated IgG , at room temperature for 1 h. After washing three times, protein expression was measured using SuperSignal® West Pico Chemiluminescent Substrate  [Each PDL cell was seeded onto 12 dishes at a density of 1 × 10https://cris.nih.go.kr, accessed on 26 January 2017, registration number: KCT0004448). Verbal and written information about the study were given to each participant. Each participant signed an informed consent form before being enrolled in the study. The study design is depicted in ® capsule was provided twice a day for 4 weeks (300 mg per day). The control group received a placebo, which was delivered in the same shape and color as the TEES-10® capsule for 4 weeks. After 4 weeks, saliva and GCF collection and clinical evaluation was conducted.One hundred voluntary participants were recruited at the clinic of the Department of Periodontology, Yonsei University Dental Hospital. The study protocol was in accordance with the World Medical Association Declaration of Helsinki (Version 2008) and approved by the Institutional Review Board of Yonsei University Dental Hospital  and registered with the Clinical Research Information Service  aged between 19 and 80 years in good general health, (2) having a minimum of 18 teeth, (3) having less than 5 mm of CAL at the first visit and (4) being diagnosed as chronic gingivitis or periodontal health. Criteria for gingivitis were followed to the consensus report of the 2017 World Workshop . PatientExclusion criteria were as follows: (1) not agreed to informed consent, (2) being pregnant or lactating, (3) having a severe systemic disease, (4) taking antiplatelet agents or anticoagulants or having a history of hemorrhage or disease, (5) history of taking an antibiotic more than 3 days within the previous month, (6) having an oral mucosal inflammatory condition , or (7) judged as being inappropriate by the clinician for some other reason.The sample size was calculated based on previous randomized controlled trials, which evaluated the anti-inflammatory action of nutrient combination using sample size-calculating software (G *Power (Ver. 3.1.9.2), Kiel, Germany) . It was Six representative teeth  from each participant were selected for clinical evaluation. Probing depth (PD) and BOP were measured at the mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual and distolingual sites for each representative tooth. Gingival index (GI) and plaque index (PI) scores were also measured at the mesial, buccal, distal and lingual for each representative tooth . All meag at 4 °C for 10 min after thawing, to obtain the supernatant. Respectively, each concentration was measured after dilution at 1:5 and 1:100 with buffer solution. Human MMP-8 ELISA kit  and MMP-9 Quantikine human MMP-9 Immunoassay ELISA kit  were used according to the manufacturer’s instructions. The detection limit of MMP-8 was 0.156 ng/mL to 10 ng/mL and the detection limit of MMP-9 was 0.313 ng/mL to 20 ng/mL. Saliva collection and measurement were conducted at both baseline and 4 weeks.For collecting whole saliva, cotton roll was used. After soaking the sterilized cotton roll for 5 min, it was put into a saliva collection tube and stored at −80 °C. To measure the concentrations of matrix metalloproteinase (MMP)-8 and MMP-9, the saliva samples were centrifuged at 10,000× p < 0.05 were considered statistically significant. All data are presented as mean ± standard error of the mean (SEM). At in vitro test, significant differences were determined using analysis of variance and least-significant differences multi-comparison (LSD) test. At clinical trial, examination variables for effectiveness and safety were treated with an intent-to-treat (ITT) analysis. A paired t-test was used to compare the changes from baseline to 4 weeks between the groups. The incidence rate of gingivitis was calculated for each group and chi-square and McNemar’s test were used for comparative analysis.Statistical analysis was performed using commercially available software ; results with ®  or control groups (n = 50). After random selection, one person withdrew consent and two were excluded because they did not meet the timeline. Finally, 97 individuals  were selected as participants  while, that of the placebo group in baseline was sustained until week 4. There was no significant difference between placebo and TEES-10® intake group in baseline; however, at 4 weeks after intake, there was significant difference between placebo and TEES-10® intake group (p < 0.05). There was no significant difference between placebo and TEES-10® intake group in PI at either baseline or 4 weeks. There was a slight increase of PI during 4 weeks in both groups.Changes of clinical index including GI, PI, BOP and PD are summarized in ® intake group was 8.63 ± 0.77 at baseline; it significantly decreased to 6.44 ± 0.69 at week 4 (p < 0.01). At 4 weeks after intake, the BOP sum of control and TEES-10® intake group showed a significant difference between the groups (p < 0.01), while there was no significant difference in baseline.The BOP sum of the TEES-10® intake group was 3.11 ± 0.04 at baseline; it significantly decreased to 3.05 ± 0.04 at week 4 (p < 0.05).The PD of the TEES-10® intake group showed a statistically significant decrease from 42 subjects to 30 subjects out of total 48 subjects (p < 0.01). When comparing between the groups, there was no statistically significant difference at baseline. However, after 4 weeks, the TEES-10® intake group showed a significant decrease in the prevalence of gingivitis compared to the placebo group (p < 0.01).The prevalence of gingivitis in each group is summarized in ® intake group showed a statistically significant decrease in MMP-8 levels, from 72.11 ± 14.78 ng/mL to 24.41 ± 4.06 ng/mL, compared to placebo group (p < 0.05). MMP-9 level was decreased from 249.93 ± 86.36 ng/mL to 142.97 ± 38.87 ng/mL in the TEES-10® intake group, it showed statistically significant decrease compared to placebo group  and matrix metalloproteinase-9 in saliva are summarized in No adverse events were reported during the clinical trial period and there was no statistically significant change in weight and blood pressure 4 weeks after administration. The results of clinical laboratory tests such as alanine aminotransferase, aspartate transaminase also showed that the changes were not significant.®, complex extracts of L. stenocephala and S. cereale sprout, showed its anti-inflammatory potential to gingivitis at in vitro and clinical levels. The main findings were: (i) TEES-10® significantly increase PDL cell viability in LPS induced inflammatory condition; (ii) TEES-10® significantly decreased expression of inflammatory mediators such as NF-κB, p-ERK, COX-2, c-Fos and p-STAT and increased expression of anti-inflammatory mediators such as PPARγ; (iii) daily intake of TEES-10® would improve gingival health with significantly decreased number of gingivitis patients and reduced MMP-8 concentration.In the present study, TEES-10® treatment. In 100 μg/mL of TEES-10® treatment, all tested inflammatory mediators except c-Fos showed lower concentration even compared to sham control. It might be regarded that high concentration of TEES-10® facilitates complete resolution of LPS induced inflammation in cellular level (There were several studies reported medicinal effects of LSE. By applying LSE, MMP-2, MMP-8 and collagenase, which are related to tissue degradation, were suppressed in cellular level . In addiar level .L. stenocephala contains phenolic substance represented by flavonoid [S. cereale sprout also contains phenolic substance and it has been reported that antioxidant effects increase in proportion to the content of phenolic substance [® consisted of chlorogenic acid, which is a dietary phenolic compound, and tricin, which is dietary flavonoids. Flavonoids and other phenolic compounds are found in many medicinal plants and are in the spotlight as they have beneficial effects, such as antioxidant effects, antibacterial effects, anti-inflammation and immune system promotion [Extracts of lavonoid ,24. S. cubstance . In the romotion . The preromotion .L. stenocephala and S. cereal supports the host defense system with its antioxidant ability and reduces periodontal tissue destruction with anti-inflammatory effect such as suppressing nuclear factor κβ, interleukin-6, -10 and -1β [Since chronic gingivitis is the result of failure in immune system against dysbiotic change, enhancing the host defense system, which is called host modulation therapy, can reduce gingival inflammation in the same dental plaque accumulated condition . Various and -1β ,27. The  and -1β . Future ®, a combination of LSE and SCSE, induced more effective increase in PDL cell viability proliferation compared to each of LSE or SCSE alone at the same dose. This may imply that there is a synergistic relation between flavonoids in LSE and SCSE. There are cases where signaling-related mechanisms do not show simple dose-dependent effects and in this regard, it can be inferred that the flavonoids of LSE and SCSE may have mutually amplifying signaling relationships.Since dietary flavonoids are absorbed through the gastrointestinal tract but are metabolized rapidly, the plasma concentration of flavonoids are usually less than 1 μmol/L . However® powder twice a day showed significant improvement in BOP, GI and gingivitis status. Gingivitis is a condition in which the balance of local factors such as bacterial loads from plaque accumulation and the host immune response is disrupted [®. Therefore, it can be considered that dietary flavonoids from TEES-10® effectively improved host defense of the periodontal tissue.In the present clinical study, taking a capsule containing 150 mg of TEES-10isrupted . Since pisrupted . In the ® showed improvement of inflammation in vitro and clinically. This may imply that anti-inflammatory mechanism of TEES-10® on periodontal tissue is applicable even with low plasma concentration.There were some previous studies that evaluated efficacy of medicinal plants origin phenolic compounds on periodontal disease. The phenolic compounds from medicinal plants showed its effect on periodontal health by inhibiting the inflammatory mediators, as well as the periodontal pathogens . HoweverIn our study, change of MMP-8 and MMP-9 in saliva were measured to confirm improvement of gingivitis. Among various biomarkers, MMP-8 is one of the most sensitive for diagnosing gingivitis . Convent® powder twice daily without any complications. A large number of natural medicinal plant extracts have abundant beneficial phenolic compounds [®.Participants of the present study had intake of the capsule containing 150 mg of TEES-10ompounds . Howeverompounds . Further®. It is considered that flavonoids take significant role in anti-inflammatory effects of medicinal plants extracts; however, the exact cell signaling pathway is quite imprecise yet. In addition, there are various flavonoids in a single medicinal plant; therefore, the interactions between various flavonoids need to be studied. With a deeper understanding about the mechanisms and interactions of medicinal plants flavonoid, more effective host modulating medicines using natural plants for chronic gingivitis might be developed.For future studies, it is needed to confirm exact metabolic mechanism of TEES-10® showed its anti-inflammatory potential with suppression of inflammatory mediators in cell and improvement of gingivitis in human. Natural derived phenolic substances in TEES-10® are thought to account for anti-inflammatory characteristics. It was noteworthy that even with oral administration, which has limitation of low plasma concentration level, TEES-10® would show an improvement of gingivitis in human. TEES-10® may applied for host modulation therapy for gingivitis with its high anti-inflammatory effects. Future studies to examinppe the mechanisms of medicinal plants derived flavonoid may develop applying medicinal plants to host modulation therapy for chronic gingivitis.In summary, TEES-10


Staphylococcus aureus (MRSA). Herein, by fusing the CHAP  catalytic domain from the Ply187 lysin with the non-SH3b cell-wall binding domain from the LysSA97 lysin, a new chimeric lysin ClyC was constructed with Ca2+-enhanced bactericidal activity against all S. aureus strains tested, including MRSA. Notably, treating S. aureus with 50 μg/mL of ClyC in the presence of 100 μM Ca2+ lead to a reduction of 9 Log10 (CFU/mL) in viable bacterial number, which was the first time to observe a lysin showing such a high activity. In addition, the effective concentration of ClyC could be decreased dramatically from 12 to 1 μg/mL by combination with 0.3 μg/mL of penicillin G. In a mouse model of S. aureus bacteremia, a single intraperitoneal administration of 0.1 mg/mouse of ClyC significantly improved the survival rates and reduced 2 Log10 (CFU/mL) of the bacterial burdens in the organs of the infected mice. ClyC was also found stable after lyophilization without cryoprotectants. Based on the above observations, ClyC could be a promising candidate against S. aureus infections.Lysins, including chimeric lysins, have recently been explored as novel promising alternatives to failing antibiotics in treating multi-drug resistant (MDR) pathogens, including methicillin-resistant  Staphylococcus aureus is a common Gram-positive pathogen that colonizes the human surface skin  × 100%.The cytotoxicity of ClyC was studied using MTT -2, 5-diphenyltetrazolium bromide) assay. Briefly, 100 µL of CHO-K1 cells (Chinese hamster ovary cells) were seeded into a 96-well plate at a final concentration of 5 × 10S. aureus T23 and 190 µL of TSBG (TSB + 1% glucose) were dispensed into wells of a 96-well polystyrene plate , and incubated at 37 °C for 24 h to allow biofilm formation. After removing the supernatant and washing three times with sterile PBS, biofilms were treated with 200 µL of different concentration of ClyC or PBS (control) for 1 h. After washing twice with PBS, wells were dried and stained with 100 μL of 4% CV/well for 5 min at 37 °C. Following that, wells were washed with sterile PBS twice carefully, and resuspended in 200 µL of absolute ethanol. Finally, OD600 of each well was read by a microplate reader. The biofilm removal efficacy was presented as the difference in OD600 between ClyC and PBS treated wells.Crystal violet (CV) staining assay was used to determine the biofilm removing capability of ClyC. Briefly, 10 µL of overnight cultures of S. aureus T23 and divided into three groups (6 each). Three hours post infection (h.p.i), groups were administrated intraperitoneally with either 0.1 mg/mouse of ClyC, 0.1 mg/mouse of ClyC containing 100 μM Ca2+, or an equal volume of PBS. The survival rates for all groups were recorded for 15 days. Meanwhile, bacterial loads in blood and organs were also tested at different times . Briefly, blood was collected and serially diluted for CFU count on LB agar. Kidney, liver, and lungs were aseptically removed from each mouse, infiltrated in 1 mL PBS with 0.1% Triton X-100, and prepared to tissue homogenates using a QIAGEN Tissue Lyse II homogenizer at 1/20 frequency for 90 s. Finally, serial dilutions of each organ were plated on Baird-Parker agar for CFU count. Interleukin 8 (IL-8) level in blood was also measured using a commercial ELISA kit . Here, sera were separated from the mice blood by centrifugation at 3000 g for 30 min, and IL8 was detected following the instructions of the kit.To test the protective efficacy of ClyC, female BALB/c mice, 6–8 weeks old, were injected intraperitoneally with a lethal dose of S. aureus in vitro and in vivo without foreseeable harmful effects. Its Ca2+ enhanced activity means that a much smaller amount of ClyC could be used in vivo, which would reduce possible side effects and improve its therapeutic index.In conclusion, we report here a novel chimeric lysin, ClyC, that possess robust and stable bactericidal activity against