Production,Purification, and Antibiofilm Activity of a Novel Exopolysaccharide from Arthrobacter sp. B4

A novel exopolysaccharide (EPS), namely, B4-EPS, is produced by Arthrobacter sp. B4. Response surface methodology (RSM) was employed to optimize the fermentation medium for increasing B4-EPS production. Based on Plackett–Burman design (PBD), glucose, yeast extract, and KH₂PO₄ were selected as significant variables, which were further optimized by a central composite design (CCD). According to response surface and canonical analysis, the optimal medium was composed of 16.94 g/L glucose, 2.33 g/L yeast extract, and 5.32 g/L KH₂PO₄. Under this condition, the maximum yield of B4-EPS reached about 8.54 g/L after 72 hr of batch fermentation, which was pretty close to the predicted value (8.52 g/L). Furthermore, B4-EPS was refined by column chromatography. The main homogeneous fraction (B4-EPS1) was collected and applied to assay of antibiofilm activity. B4-EPS1 exhibited a dose-dependent inhibitory effect on biofilm formation of Pseudomonas aeruginosa PAO1 without antibacterial activity. About 86.1% of biofilm formation of P. aeruginosa PAO1 was inhibited in the presence of 50 µg/mL B4-EPS1, which was more effective than the peer published data. Moreover, B4-EPS1 could prevent biofilm formation of other strains. These data suggest B4-EPS may represent a promising strategy to combat bacterial biofilms in the future.     

INVESTIGATION OF THE GIBBERELLIC ACID OPTIMIZATION WITH A STATISTICAL TOOL FROM Penicillium variable IN BATCH REACTOR

The culture conditions for gibberellic acid (GA₃) production by the fungus Penicillium variable (P. variable) was optimized using a statistical tool, response surface methodology (RSM). Interactions of culture conditions and optimization of the system were studied using Box–Behnken design (BBD) with three levels of three variables in a batch flask reactor. Experimentation showed that the model developed based on RSM and BBD had predicted GA₃ production with R ²  = 0.987. The predicted GA₃ production was optimum (31.57 mg GA₃/kg substrate) when the culture conditions were at 7 days of incubation period, 21% v/w of inoculum size, and 2% v/w of olive oil concentration as a natural precursor. The results indicated that RSM and BBD methods were effective for optimizing the culture conditions of GA₃ production by P. variable mycelia.     

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens,Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48–72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N_{21 days} of Serratia to be ca 3 Log₁₀(CFU cm^?2) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.     

Cellular hydrophobicity of Listeria monocytogenes involves initial attachment and biofilm formation on the surface of polyvinyl chloride

Aims: To clarify the cellular properties of Listeria monocytogenes involved in adhesion to and biofilm formation on polyvinyl chloride, a widely used material in the food manufacturing process. Methods and Results: A significant correlation between the ability of initial adherence to and biofilm formation on PVC was observed for 24 L. monocytogenes strains (Spearman rank‐correlation coefficient, r_s = 0·89). The swimming motility assay revealed no relationship between initial adherence and motility of L. monocytogenes. The microbial adhesion to solvent assay revealed an interaction of L. monocytogenes cells with nonpolar solvents, and a significant correlation was also observed between the degree of interaction with nonpolar solvents and initial adherence to PVC (r_s = 0·87 and r_s = 0·84, between initial adherence and affinities to decane and hexadecane, respectively). Conclusions: Results indicate that cellular hydrophobicity of L. monocytogenes is an important property involved in the initial adherence to and biofilm formation on PVC. Significance and Impact of Study: This study clarified the factors involved in the adherence to and biofilm formation ability of L. monocytogenes strains with PVC.     

Changes in biofilm structure during the colonization of chalcopyrite by Acidithiobacillus thiooxidans

Biofilms of Acidithiobacillus thiooxidans were grown on the surface of massive chalcopyrite electrodes (MCE) where different secondary sulfur phases were previously formed by potentiostatic oxidation of MCE at 0.780?≤?E _an?≤?0.965 V (electrooxidized MCE, eMCE). The formation of mainly S⁰ and minor amounts of CuS and S _n ^2? were detected on eMCEs. The eMCEs were incubated with A. thiooxidans cells for 1, 12, 24, 48, and 120 h in order to temporally monitor changes in eMCE's secondary phases, biofilm structure, and extracellular polymeric substance (EPS) composition (lipids, proteins, and polysaccharides) using microscopic, spectroscopic, electrochemical, and biochemical techniques. The results show significant cell attachments with stratified biofilm structure since the first hour of incubation and EPS composition changes, the most important being production after 48–120 h when the highest amount of lipids and proteins were registered. During 120 h, periodic oxidation/formation of S⁰/S _n ^2? was recorded on biooxidized eMCEs, until a stable CuS composition was formed. In contrast, no evidence of CuS formation was observed on the eMCEs of the abiotic control, confirming that CuS formation results from microbial activity. The surface transformation of eMCE induces a structural transformation of the biofilm, evolving directly to a multilayered biofilm with more hydrophobic EPS and proteins after 120 h. Our results suggest that A. thiooxidans responded to the spatial and temporal distribution and chemical reactivity of the S _n ^2?/S⁰/CuS phases throughout 120 h. These results suggested a strong correlation between surface speciation, hydrophobic domains in EPS, and biofilm organization during chalcopyrite biooxidation by A. thiooxidans.     

Biofilms isolated from washing machines from three continents and their tolerance to a standard detergent

The goal of this comparative study was to investigate biofilm forming microorganisms living in washing machines (WMs). Biofilms were sampled from 11 washing machines from four countries and three continents. Among the 94 isolated strains, 30% were potential human pathogens. Representative strains were selected and biofilm formation was evaluated with the crystal violet (CV) assay. The majority of the WM isolates formed more biofilm than their reference strains. Biofilms of P. putida WM (the largest biofilm producer) were exposed to different concentrations (0.0007–7 g l^?1) of the standard detergent IEC-A* at 30°C for 30 min and observed with confocal laser scanning microscopy. Using quantitative CVA, P. putida WM biofilm removal required higher detergent concentrations than the type strain. However, for both strains the recommended detergent concentration (7 g l^?1) was insufficient to completely clean surfaces from cell debris and exopolymeric substances.     

Hydrolytic Enzyme Production is Associated with Candida Albicans Biofilm Formation from Patients with Type 1 Diabetes

Oral candidosis is common in patients with diabetes mellitus, as yeasts, particularly Candida albicans, have the propensity to colonise, form biofilms and release hydrolytic enzymes which cause inflammation. This study aimed to investigate these characteristics in isolates from three groups of patients with type 1 diabetes: individuals with better controlled diabetes (BCD; ≥6 <8%), individuals with poorly controlled diabetes (PCD; ≥8%) and non-diabetics (ND; HbA_1c <5.9%). The biomass (Bm), phospholipase (P_z), haemolysin (H_z) and proteinase (Pr_z) were assessed using a microtitre biofilm assay and agar-based hydrolytic enzyme assays. Biofilm formation was significantly increased in the PCD group compared to ND and BCD groups (P < 0.05). No significant differences in P_z levels were observed between groups, whereas both H_z and Pr_z were significantly greater in the diabetes groups than in the healthy control group (P < 0.05). Statistically significant correlations were found to exist between the HbA_1c levels of the patients and the Bm (R = 0.384; P = 0.033), haemolysin activity (R = ?0.455; P = 0.010) and proteinase activity (R =  ?0.531; P = 0.002). There was no apparent correlation between the Bm and P_z activity (R = ?0.305; P = 0.053) or H_z activity (R = ?0.100; P = 0.296). However, a negative correlation was found between Bm and Pr_z values (R = ?0.343; P = 0.030). These data suggest that biofilm formation is likely to play a role in the pathogenicity of oral candidosis, and in patients with diabetes, this may be due to the ability of C. albicans to adapt to the altered physiological environment. The production of hydrolytic enzymes is independently associated with this growth modality.     

Screening of lipid high producing mutant from rhodotorula glutinis by low ion implantation and study on optimization of fermentation medium

In order to obtain lipid producing strain with high-yield, the wild type stain Rhodotorula glutinis was treated by low ion implantation, and optimization of fermentation medium for higher lipid yield was carried out using mutant strain. It was found that the strain had a higher positive mutation rate when the output power was 10 keV and the dose of N⁺ implantation was 80 × 2.6 × 10¹³ ions/cm². Then a high-yield mutant strain D30 was obtained through cid-heating coupling ultrasonic method and lipid yield was 3.10 g/L. Additionally, the surface response method was used to optimize fermentation medium. The three significant factors (glucose, peptone, KH₂PO₄) were optimized using response surface methodology (RSM), and the optimized parameters of fermentation medium were as follows: glucose 73.40 g/L, peptone 1.06 g/L and KH₂PO₄ 3.56 g/L. Finally the fermentation characteristic of high-yield mutation strain D30 was studied, when fermentation time was 10 days, which lipid yield increased to 7.81 g/L. Fatty acid composition of the lipid was determined by GC, and the most represented fatty acids of mutant D30 were C16:0 (11.4 %), C16:1 (5.66 %), C18:1 (49.3 %), and C18:2 (27.0 %).     

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.     

Optimization of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> (KU923381) for diesel oil degradation using response surface methodology (RSM)

Efficiency of Enterobacter cloacae KU923381 isolated from petroleum hydrocarbon contaminated soil was evaluated in batch culture and bioreactor mode. The isolate were screened for biofilm formation using qualitative and quantitative assays. Response surface methodology (RSM) was used to study the effect of pH, temperature, glucose concentration, and sodium chloride on diesel degradation. The predicted values for diesel oil degradation efficiency by the statistical designs are in a close agreement with experimental data (R ² = 99.66%). Degradation efficiency is increased by 36.78% at pH = 7, temperature = 35°C, glucose = 5%, and sodium chloride concentration = 5%. Under the optimized conditions, the experiments were performed for diesel oil degradation by gas chromatographic mass spectrometric analysis (GC-MS). GC-MS analysis confirmed that E. cloacae had highly degrade hexadecane, heptadecane, tridecane, and docosane by 99.71%, 99.23%, 99.66%, and 98.34% respectively. This study shows that rapid bioremoval of hydrocarbons in diesel oil is acheived by E. cloacae with abet of biofilm formation. The potential use of the biofilms for preparing trickling filters (gravel particles) for the degradation of hydrocarbons from petroleum wastes before their disposal in the open environment is highly suggested. This is the first successful attempt for artificially establishing petroleum hydrocarbon degrading bacterial biofilm on solid substrates in bioreactor.     

Screening of bromotyramine analogues as antifouling compounds against marine bacteria

Rapid and efficient synthesis of 23 analogues inspired by bromotyramine derivatives, marine natural products, by means of CuSO₄-catalysed [3+2] alkyne–azide cycloaddition is described. The final target was then assayed for anti-biofilm activity against three Gram-negative marine bacteria, Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8) and Paracoccus sp. (4M6). Most of the synthesised bromotyramine/triazole derivatives are more active than the parent natural products Moloka’iamine (A) and 3,5-dibromo-4-methoxy-β-phenethylamine (B) against biofilm formation by the three bacterial strains. Some of these compounds were shown to act as non-toxic inhibitors of biofilm development with EC₅₀ < 200 μM without any effect on bacterial growth even at high concentrations (200 μM).     

Conditioning film and initial biofilm formation on electrochemical CaCO3 deposition on a metallic net in the marine environment

Electrochemical deposition of minerals is a unique technology for artificial reef constructions, relying on calcium carbonate (CaCO₃) build-up over metallic structures through electrolysis of seawater. The present study traces the first 72 h following electric current termination on bacterial biofilm build-up on a metallic net covered with CaCO₃. 16S rRNA clone libraries indicated a dynamic succession. Proteobacteria and Bacteroidetes were evident at all sampling times while Cyanobacteria appeared only within the first 8 h. A significant increase in total organic carbon (TOC) and total protein was observed after 48 h with a significant correlation (R ² = 0.74), indicating TOC is a good tool for characterizing initial biofilm formation. 18S rRNA gene sequences obtained 72 h following current termination indicated a significant presence of Cnidarians (51%). Understanding the dynamics among primary bacterial settlers is important because they play a crucial role in driving the colonization of sessile invertebrate communities on artificial, as well as natural surfaces.     

Inhibitory effects of single-walled carbon nanotubes on biofilm formation from Bacillus anthracis spores

This study reports the inhibitory effect of single walled carbon nanotubes (SWCNTs) on biofilm formation from Bacillus anthracis spores. Although the presence of 50 to 100 μg ml^?1 of SWCNTs in the suspension increased spore attachment in the wells of 96-well plates, the presence of 200 μg ml^?1 of SWCNTs in the germination solution decreased the germination percentage of the attached spores by 93.14%, completely inhibiting subsequent biofilm formation. The inhibition kinetics of 50 μg ml^?1 SWCNTs on biofilm formation showed that this concentration inhibited biofilm formation by 81.2% after incubation for 48 h. SWCNT treatment in the earlier stages of biofilm formation was more effective compared to treatment at later stages. Mature biofilms were highly resistant to SWCNT treatment.     

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Optimization of the extraction process of phenolics from Bryophyllum pinnatum was carried out using response-surface methodology (RSM). The effect of different variables such as ratio of solvents, plant material/solvent ratio, extraction time, and temperature were investigated. An optimal phenolics yield of 7.952 mg/g gallic acid equivalence (GAE) was achieved at reduced levels of methanol/water ratio (1:1, v/v). During optimization, the product yield was enhanced by ～2-fold at reduced extraction solvent (methanol/water) up to 37%. Validation of the RSM model for extraction of total phenolic content (TPC) was confirmed by high-performance liquid chromatography (HPLC) analysis. The obtained experimental values were in good agreement with the predicted values, thereby indicating the appropriateness of the model generated. Phenolic extracts from B. pinnatum were further examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods for determining the radical scavenging activities. EC₅₀ values of B. pinnatum extracts (BPEs) obtained by these methods were in accordance with the amount of phenolics present in the extract. Significant correlation was found between total phenolic content and antioxidant activities (p < 0.05).     

Factors Affecting Formation of Large Calcite Crystals (≥1 mm) in Bacillus subtilis 168 Biofilm

B4 is the most common medium used in general organomineralization studies and has been used to assay or to characterize mineral precipitation potential. In an exercise for the optimization of the laboratory conditions of crystal precipitation in vitro, we used Bacillus subtilis 168 as a type strain and its isogenic mutants. While literature is mainly focused on observing generic precipitation, we investigated the requirement to obtain large crystals (≥1 mm), which could be advantageous in wide-ranging implications for bioconsolidation of soil, sand, stone, and cementitious materials. Calcite crystals are visible on B4 agar plates within 7 days at 37°C after inoculum of B. subtilis 168 strain. In this study, we show that to form large crystals with a diameter ≥1 mm several conditions must be met: i) Reduced amount of B4 medium into the Petri plate improve crystal formation. 55 mm Petri plates contained only 4 ml of B4 agar medium reached a plateau in 6 days at 37°C. High moisture and presence of water condense would decrease crystal formation. ii) Inoculation of cells using a rod instead of a circular shaped spot. When the same number of B. subtilis cells was streaked, rod-shape biofilm significantly fostered crystal precipitation, while spot-shape prevented precipitation. iii) When more than one biofilm is present within the same plate, mutual interactions can affect precipitation in each biofilm. iv) Spherical nucleation sites are identified as initial step during the formation of large calcite crystal.     

Manipulation of pyrite colonization and leaching by iron-oxidizing <Emphasis Type="Italic">Acidithiobacillus</Emphasis> species

In this study, the process of pyrite colonization and leaching by three iron-oxidizing Acidithiobacillus species was investigated by fluorescence microscopy, bacterial attachment, and leaching assays. Within the first 4–5 days, only the biofilm subpopulation was responsible for pyrite dissolution. Pyrite-grown cells, in contrast to iron-grown cells, were able to oxidize iron(II) ions or pyrite after 24 h iron starvation and incubation with 1 mM H₂O₂, indicating that these cells were adapted to the presence of enhanced levels of reactive oxygen species (ROS), which are generated on metal sulfide surfaces. Acidithiobacillus ferrivorans SS3 and Acidithiobacillus ferrooxidans R1 showed enhanced pyrite colonization and biofilm formation compared to A. ferrooxidans ^T. A broad range of factors influencing the biofilm formation on pyrite were also identified, some of them were strain-specific. Cultivation at non-optimum growth temperatures or increased ionic strength led to a decreased colonization of pyrite. The presence of iron(III) ions increased pyrite colonization, especially when pyrite-grown cells were used, while the addition of 20 mM copper(II) ions resulted in reduced biofilm formation on pyrite. This observation correlated with a different extracellular polymeric substance (EPS) composition of copper-exposed cells. Interestingly, the addition of 1 mM sodium glucuronate in combination with iron(III) ions led to a 5-fold and 7-fold increased cell attachment after 1 and 8 days of incubation, respectively, in A. ferrooxidans ^T. In addition, sodium glucuronate addition enhanced pyrite dissolution by 25 %.     

Lack of O‐polysaccharide enhances biofilm formation by Bradyrhizobium japonicum

Aims: To reveal the effects of the O‐polysaccharide antigen of Bradyrhizobium japonicum LPS on biofilm formation and motility. Methods and Results: Wild type and O‐antigen‐deficient mutant strains of B. japonicum were tested for biofilm formation on polyvinyl chloride (PVC) surfaces and motility on semi‐solid (0·3%) agar media. After 7 days of incubation, the amount of biofilms formed by the mutant was c. 3·5‐fold greater than that of the wild type. Unlike biofilm formation, the motility assay revealed that the mutant strain was less motile than the wild type. Conclusions: This study shows enhanced biofilm formation and decreased motility by the O‐antigen‐deficient mutant, suggesting that the lack of the O‐polysaccharide of the rhizobial LPS is associated with biofilm‐forming ability and movement. Significance and Impact of the Study: LPS plays an important role in both pathogenic and beneficial bacteria. It has also been reported that LPS deficiency negatively affects biofilm formation. However, our results demonstrate that the O‐antigen‐deficient mutant enhances biofilm formation, presumably through a significant increase in hydrophobicity. It is notable that the hydrophobicity of cell walls might be a key regulator in controlling biofilm development in B. japonicum.     

Effects of mineral and rapeseed phosphorus supplementation on phytate degradation in dairy cows

The objective of this study was to evaluate the effects of diet composition on phytate (InsP₆) degradation in dairy cows. In Experiment 1, four diets that differed in the amount and source of phosphorus (P) were fed to 24 lactating cows in a 4 × 4 Latin Square design. The control diet (Diet C) contained 4.18 g P/kg dry matter (DM). Diet MP contained additional mineral P (5.11 g P/kg DM), Diet RS contained rapeseed and rapeseed meal as organic P sources (5.26 g P/kg DM) and Diet RSM contained rapeseed meal and rapeseed oil (5.04 g P/kg DM). Total P (tP) and InsP₆ excretion in faeces were measured. In Experiment 2, we used a rumen simulation technique (Rusitec) to estimate ruminal disappearance of tP and InsP₆ from Diets C, MP and RSM. In Experiment 1, tP concentration in faeces increased with tP intake and was highest for Diets RS and RSM. The source of supplemented P had no influence on tP digestibility, but tP digestibility was reduced for Diets MP, RS and RSM in comparison to that for Diet C. InsP₆ disappearance decreased in Diet MP (85.0%) and increased in Diets RS (92.7%) and RSM (94.0%) compared to that in Diet C (90.0%). In Experiment 2, P source influenced ruminal tP disappearance (Diet MP, 78.6%; Diet RSM, 75.3%). InsP₆ disappearance for Diet C (98.1%) was higher than that for Diets MP (95.6%) and RSM (94.9%). The results confirmed the high potential of ruminants to degrade InsP₆, but differences in diet composition influenced InsP₆ disappearance. Further studies of the site of InsP₆ degradation are required to understand the relevance of InsP₆ degradation for the absorption of P.     

Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p < 0.05). TA, GN, BP, and RT significantly inhibited the expression of the sea gene in S. aureus C-29 (p < 0.05), while suppression attempts by PP and POP proved unsuccessful. After result analysis, it can be derived that TA, GN, BP, and RT inhibit the production of SEA. Of the six samples, each one significantly inhibited biofilm formation (p < 0.05). Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.     

The effects of rapeseed meal and legume seeds as substitutes for soybean meal on productivity and gastrointestinal function in rabbits

The aim of this study was to determine the effects of soybean meal (SBM) substitution by a mixture of rapeseed meal (RSM), white lupine seeds (WLS) and pea seeds (PS) on productivity, nutrient digestibility, nitrogen retention and gastrointestinal function in Hyplus rabbits. The Control diet (SBM₁₅) contained 15% SBM, whereas Diet SBM_7.5 contained 7.5% SBM, 5% RSM, 4% WLS and 3% PS. In Diet SBM_0, SBM was completely replaced by RSM, WLS and PS (10%, 8% and 6%, respectively). A production trial was performed on 90 Hyplus rabbits aged from 35 to 84 d (45 each sex; 953 ± 4.6 g). A digestion and balance trial was conducted on 30 rabbits. Additionally, several parameters of the gastrointestinal tracts from eight animals from each group were analysed, where special attention was paid to the enzymatic activity of microbiota and the short-chain fatty acids concentration in caecum and colon. The experimental diets did not cause significant differences regarding performance parameters evaluated in vivo and post-mortem, and in the nutrient and energy digestibility or nitrogen retention. The observed changes in the enzymatic activity of large gut microbiota, including the selective increase in secretion of glycoside hydrolases by bacterial cells, seem to be responsible for the unchanged growth performance of rabbits fed diets where SBM was substituted by a mixture of RSM, WLS and PS. The obtained results indicate that in rabbit diets SBM may be, partially or completely, successfully replaced by a feed mixture of RSM, WLS and PS.