Mild Acid Hydrolysis Research Articles

Novel adjuvants derived from attenuated lipopolysaccharides and lipid As of purple non-sulfur photosynthetic bacteria

Adjuvants are substances that enhance adaptive immune response to antigen. Development of a safe and effective immunostimulant adjuvant is essential for the efficacy of a vaccine to protect against infectious pathogens. Purple non-sulfur photosynthetic bacteria exhibited nontoxic natural lipid A variants that are distinct in their chemical structures from that of the Escherichia coli-type lipid A. In this study, the adjuvant efficacy of attenuated lipid A variants and their corresponding lipopolysaccharides (LPSs), derived from purple photosynthetic bacteria (Rhodocyclus tenuis and Rhodobacter sphaeroides) were evaluated. LPS was extracted using modified phenol-chloroform-petroleum ether method and lipid A was separated by mild acid hydrolysis. Trinitrophenol (TNP) was conjugated to hen egg albumin (TNP-HEA) and used as haptenic antigen. The LPS and lipid A adjuvant candidates were formulated in oil-in-water emulsion (OIWE) and evaluated to elicit anti-TNP IgG against TNP-HEA conjugate in BALB/c female mice. The anti-TNP IgG titers were measured using ELISA. The intact LPS-based adjuvants present in OIWE formulation showed significantly higher efficacy to elicit anti-TNP IgG titers against TNP-HEA conjugate compared to their corresponding lipid A-based adjuvants. As expected, the OIWE formulations of all LPS- and lipid A-based adjuvant candidates showed higher activities compared to the aqueous formulations. Slow reduction in the levels of anti-TNP IgG antibodies in the serum was observed over 4 months after immunization using the LPS- and lipid A-based adjuvant candidates which may provide a long protection against pathogens. The attenuated LPSs and lipid A’s from the photosynthetic bacteria showed promising results to develop novel safe and effective adjuvants that can evoke the immune response. The most promising adjuvant candidate was the LPS-based adjuvant from R. tenuis.

Structure of the O-specific polysaccharide of Azospirillum doebereinerae type strain GSF71T

O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of plant-growth-promoting rhizobacteria Azospirillum doebereinerae GSF71T and studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. It was established that the polysaccharide is linear and consists of tetrasaccharide repeating units with the following structure:

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6.

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1→3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→O-PS2→2)-α-l-Rhap4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides.

Conjugation of Meningococcal Lipooligosaccharides Through Their Non-Reducing Terminus Results in Improved Induction a Protective Immune Response

The present studies prove that conjugation of meningococcal lipooligosaccharides through their non-reducing terminus conserves their inner epitopes resulting in conjugates potent to induce a protective immune response. Four different oligosaccharides were obtained by specific degradations of the same L7 lipooligosaccharide (L7-LOS), and each was linked to tetanus toxoid by direct reductive amination. Two were truncated oligosaccharides with incomplete inner epitopes and were obtained by mild acid hydrolysis of lipooligosaccharide. The terminal galactose of one oligosaccharide was additionally enzymatically oxidized. These oligosaccharides were conjugated through a newly exposed terminal Kdo in reducing end or through oxidized galactose localized at non-reducing end of the core, respectively. The third was a full-length oligosaccharide obtained by O-deacylation of the L7-LOS and subsequent enzymatic removal of phosphate substituents from its lipid A moiety. The fourth one was also a full-length O-deacylated lipooligosaccharide, but treated with galactose oxidase. This allowed direct conjugation to tetanus toxoid through terminal 2-N-acyl-2-deoxy-d-glucopyranose or through oxidized galactose, respectively. Comparison of the immune performance of four conjugates in mice revealed, that while each was able to induce significant level of L7-LOS-specific IgG antibody, the conjugates made with the full-length saccharides were able to induce antibodies with increased bactericidal activity against homologous meningococci. Only full-length oligosaccharides were good inhibitors of the binding of L7-LOS to the bactericidal antiserum. Moreover, induction of the significant level of the L7-LOS-specific antibody by full-length lipooligosaccharide conjugated from non-reducing end, provided also the direct evidence that internal core epitopes are fully responsible for the immunorecognition and immunoreactivity.

The K90 capsular polysaccharide produced by Acinetobacter baumannii LUH5553 contains di-N-acetylpseudaminic acid and is structurally related to the K7 polysaccharide from A. baumannii LUH5533

Acinetobacter baumannii isolate LUH5553 carries the KL90 capsule gene cluster, which includes genes for three glycosyltransferases (Gtrs) and the ItrA3 initiating transferase, as well as a set of genes for synthesis of a higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid (Pse5Ac7Ac). The K90 capsular polysaccharide (CPS) has a tetrasaccharide repeat (K90 unit), which begins with d-GlcpNAc and contains Pse5Ac7Ac. The higher sugar was cleaved by mild acid hydrolysis of the CPS, and structures of the initial and modified polysaccharides were established by 1D and 2D 1H and 13C NMR spectroscopy. K90 contains α-d-Galp-(1 → 6)-d-GlcpNAc and α-d-GlcpNAc-(1 → 3)-d-GlcpNAc fragments, and formation of these glycosidic linkages is catalysed respectively by Gtr14 and Gtr15. The gtr14 and gtr15 genes occur in several A. baumannii KL gene clusters, including KL5 and KL7 that carry itrA2 rather than itrA3. As ItrA2 introduces d-GalpNAc rather than d-GlcpNAc as the first monosaccharide, Gtr15 can transfer d-GlcpNAc to either of these amino sugars, suggesting that this enzyme has relaxed specificity. Consequently, the third, novel glycosyltransferase, Gtr163, forms the β-(2 → 3) linkage between Pse5Ac7Ac and d-Galp. Wzy polymerases encoded by KL90 and KL7 are 54% identical and form the same linkage between the K units to give branched polysaccharides with the same main chain but different disaccharide side chains, β-Pse5Ac7Ac-(2 → 3)-d-Galp in K90 and α-Leg5Ac7Ac-(2 → 6)-d-Galp in K7.

Self-sustainable azolla-biorefinery platform for valorization of biobased products with circular-cascading design

As the circular bioeconomy is gaining momentum around the globe, biorefinery will play a key role in the framework of sustainability as integral component. A self-sustainable biorefinery model using azolla cultivation was designed at the focal point of a circular loop. This model is aimed to enable a cascading valorization of bio-based products through a series of sequentially integrated biological/thermal unit operations. Azolla pinnata with an inherent ability to sequester CO2 along with simultaneous nitrogen fixation capability will serves as a potential and renewable carbon resource (feedstock) for the integrated unit operations viz., acidogenesis, photosynthesis, hydrolysis, and pyrolysis. The cascading loop is initiated by acidogenesis of spent wash (distillery wastewater-DSW) in a semi pilot scale bioreactor for the production of biohydrogen and volatile fatty acids (VFA). Treated spent wash after acidogenesis (TSW) was used as feed for Azolla pinnata cultivation, which eventually depicted good COD and nitrates removal efficiency. Harvested A. pinnata biomass (AB) showed presence of good amount of carbohydrates (237 mg/g) and proteins (160 mg/g) along with 11% of lipids (composed of 22% omega fatty acids) thus making it a good nutritional feed for livestock. AB after lipid extraction was subjected to mild acid-hydrolysis and the hydrolysate was used as substrate for acidogenesis to facilitate circular/closed loop operation. The techno-economics of biorefinery process evaluated using SuperPro Designer software depicted the feasibility of the integrated strategy. This study extends the scope of biobased platform with self-sustainability as core objective.

Fermentation of Microalgae Biomass through Mild Acid Pretreatment for Bioethanol Production

Conversion of microalgae biomass to bioethanol is actively being researched to establish a cost effective and sustainable production technology. The main challenge is to break down the carbohydrates content in the biomass to obtain fermentable sugar for subsequent fermentation process. This study focuses on the effectiveness of the usage phosphoric acid pretreatment and capability of Saccharomyces diastaticus as the fermentation microbe to produce ethanol. Scenedesmus dimorphus microalgae biomass was used as the feedstock due to its high carbohydrate content. Mild acid hydrolysis at various conditions were carried out on biomass and the hydrolysates were subjected to fermentation. The optimum condition of acid pre-treatment using phosphoric acid was determined in this study. Based on the results, bioethanol yield of 94% was obtained at optimum condition of 2.5% v/v phosphoric acid at temperature of 120 °C for 30 min. This study proved that combination of phosphoric acid pre-treatment process with Saccharomyces diastaticus yeast provides a practicable method for the production of bioethanol from microalgae.

Structural studies of O-specific polysaccharide(s) and biological activity toward plants of the lipopolysaccharide from Azospirillum brasilense SR8

Lipopolysaccharide (LPS) was extracted from dry bacterial cells of plant-growth-promoting bacterium Azospirillum brasilense SR8 (IBPPM 5). The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis, 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments, computational NMR-based structure analysis, and Smith degradation. The OPS was shown to contain two types of repeating units of the following structure: [Display omitted] Both OPS structures are present in A. brasilense 54, from which structure 1 has been reported earlier (Fedonenko et al., 2011), whereas to our knowledge structure 2 has not been hitherto found in bacterial saccharides. Treatment of wheat seedling roots with LPS of A. brasilense SR8 increased the number of root hair deformations as compared to seedlings grown without LPS, but had no effect on adsorption of the bacteria to the root surface. A. brasilense SR8 was able to utilize LPS of several structurally related Azospirillum strains.

Structure of the O-specific polysaccharide from a halophilic bacterium Halomonas ventosae RU5S2EL

Halomonas ventosae RU5S2EL, a halophilic Gram-negative bacterium isolated from salt sediments of lake Elton (Russia), was cultivated and the lipopolysaccharide was extracted by the Westphal procedure. The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments as well as Smith degradation. The OPS was found to consist of branched pentasaccharide repeating units of the following structure:▪

Structure and gene cluster of the O antigen of Escherichia coli F17, a candidate for a new O-serogroup

Escherichia coli F17 isolated from horse feces was studied in respect to the O antigen (O polysaccharide) structure and genetics. The lipopolysaccharide was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the O polysaccharide, which was studied by sugar analysis and selective solvolysis with CF3CO2H along with one- and two-dimensional 1H and 13C NMR spectroscopy. The O polysaccharide was found to have a branched pentasaccharide repeat (O-unit) containing one residue each of d-galactose, d-mannose, l-rhamnose, d-glucuronic acid, and N-acetyl-d-glucosamine; about 2/3 units bear a side-chain glucose residue. To our knowledge, the F17 O-polysaccharide structure established is unique among known bacterial polysaccharide structures. The O-antigen gene cluster of E. coli F17 between the conserved genes galF and gnd was sequenced and found to be 99% identical to that of E. coli 102,755 assigned to a novel OgN8 genotype (A. Iguchi, S. Iyoda, K. Seto, H. Nishii, M. Ohnishi, H. Mekata, Y. Ogura, T. Hayashi, Front. Microbiol. 7 (2016) 765). Genes in the cluster were annotated taking into account the F17 O-polysaccharide structure. The data obtained confirm that E. coli F17 and E. coli strains belonging to the OgN8 genotype can be considered as a candidate to a new E. coli O-serogroup. The O antigen of this novel type was demonstrated to make for an effective shield protecting the intimate outer membrane surface of bacteria from direct interaction with bacteriophages.

Influence of acid depolymerization parameters on levan molar mass distribution and its utilization by bacteria

Levan is a fructan composed of β -(2, 6) linkages in its main chain. Its health properties, especially its prebiotic potential can be partially modified by changing its molar mass distribution. Given that native levan is rarely fermented by probiotic bacteria, especially lactic acid bacteria (LAB), levanoligosaccharides (LOS) were produced by mild acid hydrolysis. The response surface methodology (RSM) was applied to determine the optimum parameters for depolymerization. Gel permeation chromatography (GPC) was used to characterize the LOS produced and to show the differences between inulin and levan. The prebiotic potential of four fractions of LOS with different molar mass distributions was investigated. MRS (Mann Rogosa Sharpe) medium supplemented with the LOS were inoculated with bacterial strains and growth was monitored by measuring the turbidity of the cultures. The utilization of oligofructans was also confirmed by RP-UHPLC-UV-ESI-MS (liquid chromatography coupled with mass spectrometry) measurements of LOS derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP). It was observed that the degree of polymerization of LOS has an influence on the growth of the tested bacteria.

Ultrasonic Irradiation Coupled with Microwave Treatment for Eco-friendly Process of Isolating Bacterial Cellulose Nanocrystals.

The isolation of crystalline regions from fibers cellulose via the hydrolysis route generally requires corrosive chemicals, high-energy demands, and long reaction times, resulting in high economic costs and environmental impact. From this basis, this work seeks to develop environment-friendly processes for the production of Bacterial Cellulose Nanocrystals (BC-NC). To overcome the aforementioned issues, this study proposes a fast, highly-efficient and eco-friendly method for the isolation of cellulose nanocrystals from Bacterial Cellulose, BC. A two-step processes is considered: (1) partial depolymerization of Bacterial Cellulose (DP-BC) under ultrasonic conditions; (2) extraction of crystalline regions (BC-NC) by treatment with diluted HCl catalyzed by metal chlorides (MnCl2 and FeCl3.6H2O) under microwave irradiation. The effect of ultrasonic time and reactant and catalyst concentrations on the index crystallinity (CrI), chemical structure, thermal properties, and surface morphology of DP-BC and BC-NC were evaluated. The results indicated that the ultrasonic treatment induced depolymerization of BC characterized by an increase of the CrI. The microwave assisted by MnCl2-catalyzed mild acid hydrolysis enhanced the removal of the amorphous regions, yielding BC-NC. A chemical structure analysis demonstrated that the chemical structures of DP-BC and BC-NC remained unchanged after the ultrasonic treatment and MnCl2-catalyzed acid hydrolysis process.

Mild Acid Hydrolysis of Rice Straw for Highly Efficient Methane Generation with Hydrolysates

Glucose and xylose are the dominant hydrolysis products of lignocellulose, and they can be used as a potential substrate for biogas generation. To reveal the acid hydrolysis metabolism and enhance the methane production, mild acid hydrolysis of rice straw was conducted to obtain hydrolysates with different glucose to xylose ratios. The methane production and microbial metabolism process using mixed sugars and hydrolysates were investigated. The results showed that the cellulose and hemicellulose in the rice straw were converted to the desired sugars by controlling the acid hydrolysis conditions. The glucose to xylose ratios tested were 70:30, 60:40, 50:50, 40:60, and 30:70. The anaerobic digestion process had the highest methane yield with the 40:60 glucose to xylose ratio in the mixed sugars and hydrolysate, which were 334.1 mL/g and 318.2 mL/g based on the chemical oxygen demand (COD), respectively. The metabolic process with the 40:60 glucose to xylose ratio had good butyrate type fermentation and a high COD removal rate. The corresponding acid hydrolysis conditions were 1% HCl and a 7.5% solid loading at 60 °C for 48 h. Therefore, the mild acid hydrolysis conditions can adjust the glucose to xylose ratio in the hydrolysate for high methane production.

Use of Algae Biomass Obtained by Single-Step Mild Acid Hydrolysis in Hydrogen Production by the β-Glucosidase-Producing Clostridium beijerinckii Br21

Macroalgae biomass is a potential feedstock for fermentative H2 production: it has high carbohydrate concentration and is lignin-free. Here, we optimize a mild acid treatment of Kappaphycus alvarezii biomass by an experimental design 24. The optimal acid treatment conditions were 90 °C, HCl concentration of 55.9 mmol/L, 0.375 g of algae mass, and 8 h of treatment. Under these conditions, the hydrolysate presented mono-, but also di- and oligosaccharides. We used this hydrolysate as substrate for fermentative hydrogen (H2) production by the Clostridium beijerinckii Br21 grown in two different conditions: (1) in medium containing galactose and no β-glucosidase activity; (2) in medium with cellobiose to stimulate β-glucosidase activity, which was 0.33U/mL. The fermentative assay conducted in the presence of the C. beijerinckii Br21 with β-glucosidase activity provided higher H2 concentration and yield as compared to the assay accomplished in the presence of the inoculum with no β-glucosidase activity—171.76 ± 2.10 and 140.95 ± 10.92 mL of H2/L, and 70.3 ± 0.9 and 62.7 ± 4.9 mL of H2/g of dry algae, respectively. Therefore, a fermentative β-glucosidase-producing organism such as C. beijerinckii Br21 can potentially complete biomass saccharification for later renewable H2 production.

Lipid A structural characterization from the LPS of the Siberian psychro-tolerant Psychrobacter arcticus 273-4 grown at low temperature.

Psychrobacter arcticus 273-4 is a Gram-negative bacterium isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in the Kolyma region in Siberia. The survival strategies adopted to live at subzero temperatures include all the outer membrane molecules. A strategic involvement in the well-known enhancement of cellular membrane fluidity is attributable to the lipopolysaccharides (LPSs). These molecules covering about the 75% of cellular surface contribute to cold adaptation through structural modifications in their portions. In this work, we elucidated the exact structure of lipid A moiety obtained from the lipopolysaccharide of P. arcticus grown at 4°C, to mimic the response to the real environment temperatures. The lipid A was obtained from the LPS by mild acid hydrolysis. The lipid A and its partially deacylated derivatives were exhaustively characterized by chemical analysis and by means of ESI Q-Orbitrap mass spectrometry. Moreover, biological assays indicated that P. arcticus 273-4 lipid A may behave as a weak TLR4 agonist.

Phosphoglycolipid Profiling of Bacterial Endotoxins

Abstract Much interest is at present focused on bacterial endotoxins, also known as lipopolysaccharides (LPS), as they are responsible for the development of clinical symptoms of Gram-negative sepsis which is the leading cause of death in intensive care units. Endotoxicity is associated with the special phosphoglycolipid part of LPS, termed lipid A. Main challenges in the structural elucidation of lipid A arise from its amphiphilic character and inherent heterogeneity. A mass spectrometrybased de novo method combined with reversed-phase liquid chromatography for the detailed structural characterization of complex lipid A mixtures (obtained by mild acid hydrolysis of LPS) from different bacterial sources has been developed. Tandem mass spectrometric measurements were performed with an electrospray-ionisation quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer in both negative- and positive-ionization modes in order to explore fragmentation pathways. It was found that characteristic product ions in the positive-ion mode could be used for the unambiguous assignment of the phosphorylation site, whereas the use of both ionization modes provided consistent and/or complementary information about the fatty acyl distribution between the two glucosamine moieties of lipid A. Since the immunostimulatory (advantageous) vs. proinflammatory (endotoxic) effect of the lipid A is closely related to the fine chemical structure, our relatively simple structural elucidation strategy could offer great potential in the bioanalysis of native lipid A samples and lipid A-based vaccines

Structure of the O-specific polysaccharide from Azospirillum fermentarium CC-LY743T

O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of nitrogen-fixing bacterium Azospirillum fermentarium CC-LY743T (IBPPM 578) and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments. The polysaccharide was found to be linear and to consist of alterating α-l-fucose and α-d-mannose residues in tetrasaccharide repeating units of the following structure: →2)-α-D-Manp-(1 → 3)-α-L-Fucp-(1 → 3)-α-D-Manp-(1 → 3)-α-L-Fucp-(1→

Isolation and Characterization of Nanocellulose Obtained from Industrial Crop Waste Resources by Using Mild Acid Hydrolysis

Cellulose, microcrystalline cellulose and nanocellulose were prepared from three agricultural waste resources: pineapple leaf (PALF), banana rachis (BR), and sugarcane bagasse (SCB). Each waste resource was first converted into microcrystalline cellulose which was subsequently converted into cellulose nanoparticles by using mild (30% w/v) and strong (60% w/v) sulfuric acid concentrations for extraction. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) were used to characterize each waste resource and extracted cellulosic materials. Furthermore, nanocelluloses were studied by zeta potential, size analysis, and transmission electron microscopy (TEM). Cellulose nanowhiskers were successfully obtained and isolated with a 33% average yield by applying a mild acid treatment. Substrates BR and SCB proved to be more promising agricultural waste resources in terms of their crystalline cellulosic content and properties.

Structural studies on the O-polysaccharide of Escherichia coli O57

Mild acid hydrolysis of the lipopolysaccharide of Escherichia coli O57 afforded an O-polysaccharide, which was isolated by gel permeation chromatography (GPC) and studied by sugar analysis, Smith degradation and solvolysis with trifluoroacetic acid, along with 2D 1H and 13C NMR spectroscopy. The O-polysaccharide was found to contain d-Glc, d-Gal, d-GalA, d-GlcNAc, and l-FucNAc, as well as O-acetyl groups. Smith degradation of the O-deacetylated polysaccharide destroyed side-branch β-Glсp and α-GalpA to give a modified linear polysaccharide. Solvolysis cleaved selectively the linkage of α-l-FucpNAc to give a pentasaccharide corresponding to the O-polysaccharide repeat. A comparison of the NMR spectra of the initial and O-deacetylated polysaccharides showed that α-GalpA is non-stoichiometrically O-acetylated at position either 2 (∼30%) or 3 (∼40%). The following structure of the O-polysaccharide was established, which is unique among known bacterial polysaccharide structures: [Display omitted]

Evaluation of Biochemical Differences and Immunostimulatory Properties of LPS and Lipid a Extracted From Brucella Strains

Background The intrinsic heterogeneity determination in Brucella Lipopolysaccharide (LPS) is important for explaining its chemical nature and biological behavior. This is significant for practical purposes, since LPS is the most relevant antigen during infection and vaccination. The purpose of the present study was to compare biochemical and immunological differences of LPS and lipid A in three strains of Brucella: B. melitensis (virulent strain), B. melitensis (vaccine strain, Rev1), and B. abortus (vaccine strain, S19). Materials and Methods LPSs were extracted from Brucella strains using hot phenol-water method, and lipid A was obtained through mild acid hydrolysis. Glycan, phosphate, KDO, and protein concentration were evaluated in both LPS and lipid A samples. Immunological effects of Brucella LPS and lipid A were investigated measuring mitogenesis, IL-6, and Nitric Oxide (NO) production. Results LPS and lipid A of B. melitensis have more glycan, KDO, protein, and phosphate compared with B. abortus. Different species of Brucella LPS and lipid A induced NO production in a time- and dose-dependent manner via J774A.1 cells. One μg/ml LPS extracted from different strains of Brucella can induce maximum NO production. However, lipid A from S19 cannot induce NO and lipid A from B. melitensis induces NO production in higher doses of KDO than its LPS. Maximal production of IL-6 and higher mitogenic index in human lymphocytes was observed by Rev1 LPS. Conclusion Regarding the diverse biochemical and immunostimulatory properties of LPS and lipid A, these strains of Brucella can be used potentially for different approaches, such as designing subunit brucellosis vaccines or effective adjuvants. For instance, LPS from B. abortus, as an effective and safe adjuvant due to its less toxicity, and Rev1 LPS, as subunit vaccines in developing anti-Brucella vaccines due to its high immunopotency, have been applied in several studies.