Presence Of Exopolysaccharides Research Articles

Bacteria inhabiting spider webs enhance host silk extensibility

Spider silk is a promising material with great potential in biomedical applications due to its incredible mechanical properties and resistance to degradation of commercially available bacterial strains. However, little is known about the bacterial communities that may inhabit spider webs and how these microorganisms interact with spider silk. In this study, we exposed two exopolysaccharide-secreting bacteria, isolated from webs of an orb spider, to major ampullate (MA) silk from host spiders. The naturally occurring lipid and glycoprotein surface layers of MA silk were experimentally removed to further probe the interaction between bacteria and silk. Extensibility of major ampullate silk produced by Triconephila clavata that was exposed to either Microbacterium sp. or Novosphigobium sp. was significantly higher than that of silk that was not exposed to bacteria (differed by 58.7%). This strain-enhancing effect was not observed when the lipid and glycoprotein surface layers of MA silks were removed. The presence of exopolysaccharides was detected through NMR from MA silks exposed to these two bacteria but not from those without exposure. Here we report for the first time that exopolysaccharide-secreting bacteria inhabiting spider webs can enhance extensibility of host MA silks and silk surface layers play a vital role in mediating such effects.

Assessment of microbial exopolysaccharides for improving soil fertility and crop yield in semi-arid India

Soil degradation and poor fertility are major concerns in semi-arid regions of India. Microbial exopolysaccharides (EPS) have demonstrated potential in mitigating abiotic and biotic stresses in these areas. However, the studies on the impact of EPS on crop performance and soil fertility under natural field conditions remain limited. To address this knowledge gap, a field experiment was conducted during the rainy season of 2019-20 and 2020-21 at ICAR-NIASM, Baramati, Maharashtra, to examine the effects of microbial EPS on average crop yields of soybean and maize, microbial colonization status in soybean and maize, and soil fertility parameters in basaltic soils of India. Seeds inoculated with Rhizobium or nitrogen fixers and 10 kg ha-1 EPS resulted in a significant increase in soybean and maize yields by 57.7% and 44.8%, respectively, compared to the control. Additionally, the Rhizobium treated seeds with EPS 10 kg ha-1 showed improvements in soil organic carbon, dehydrogenase activity, and nitrogen content by 15.5%, 214.5%, and 70.0%, respectively, in soybean compared to the control. Conversely, the application of microbial EPS did not cause any significant change in soil pH, E.C., or available P, K, Fe, Mn, and Zn contents in both crops. While the presence of EPS within the rhizosphere of soybean actively promoted nodule formation, it also enhanced microbial colonization within the rhizosphere of both soybean and maize. Moreover, the best subset regression analysis revealed that EPS altered the soil parameters for the yield of both crops. The findings of this study suggest that EPS treatment, in combination with Rhizobium or nitrogen fixers, can significantly enhance crop yield, promote nodule formation in soybean, and improve microbial colonization within the rhizosphere of both soybean and maize. These results highlight the potential of microbial EPS as a viable and sustainable solution to address soil degradation and poor fertility in semi-arid regions of India.

The role of probiotic exopolysaccharides in adhesion to mucin in different gastrointestinal conditions

The presence of exopolysaccharides (EPS), a type of biomacromolecules, on the surface of probiotics play an important role in mucoadhesion, and it can be severely influenced by environments during gastrointestinal transit. In this study, the impact of gastrointestinal factors on surface properties of two probiotics (Lactobacillus rhamnosus GG and Pediococcus pentosaceus LI05) was investigated. Probiotic suspensions had relatively high viscosities and exhibited pronounced shear-thinning behavior due to the presence of EPS. The ζ-potential of both probiotics was relatively low and was not believed to play an important role in mucoadhesion. Compared to the control, the adhesive forces tended to decrease in the presence of gastric acids but increase in the presence of bile salts, since bile salts led to a thicker more open EPS layer compared to gastric acids. Although the functional groups of EPS in both probiotics are similar according to the study by FT-IR spectroscopy, the molecular weight of purified EPS in LI05 was much higher, ranging from 10,112 Da to 477,763 Da, which may contribute to higher rupture length in LI05 group. These results suggest that probiotic-mucin interactions are governed by the compositions and changes in the EPS of the probiotics in different gastrointestinal conditions, which contribute to a better understanding of the mucoadhesive behavior of the probiotics in the GIT.

Understanding the fermentation factors affecting the separability of fermented milk: A model system study

The demand for high-protein fermented milk products is globally increasing. The effect of added starter culture, cutting pH and fermentation temperature on separability, i.e., how well curd and whey separate, were evaluated in a model system aiming to resemble Greek-style yoghurt. Skim milk was fermented with three commercial starter cultures at 39 °C or 43 °C to a pH of 4.45 or 4.60. Stirred fermented gels were concentrated to produce high-protein (~10%) fermented milk. The starter culture influenced the microstructure as well as the association (interactions) between exopolysaccharides (EPS) with the surface of the protein aggregates. The high association of EPS increased the viscosity and gel strength of the fermented model gels and decreased the zeta potential, the amount of whey removed as well as the protein and total solids concentration in the white mass. Increased fermentation temperature (from 39 °C to 43 °C) resulted in increased particle size and zeta potential values when EPS-producing starter cultures were applied. Cutting pH did not influence the separability of fermented stirred gels. The presence of EPS and pronounced association with protein increased the particle size, negative surface charge and structural rigidity, establishing a more stable system with reduced separability.

Observation of exopolysaccharides (EPS) from Lactobacillus helveticus SBT2171 using the Tokuyasu method.

Some species of lactic acid bacteria used for the production of natural cheese produce exopolysaccharides (EPS). Electron microscopy is useful for analyzing the microstructure of EPS produced by lactic acid bacteria. However, pretreatments used to observe the microstructure of EPS by electron microscopy, such as dehydration and resin embedding, can result in EPS flowing out easily from the cell. Therefore, in this study, the Tokuyasu method was conducted on cryosection to reduce EPS outflow. Two types of observation method, namely, using lectin and ruthenium red, were conducted in an attempt to observe EPS produced by Lactobacillus helveticus SBT2171. Observation using the lectin method confirmed that colloidal gold particles conjugated with a lectin recognizing β-galactoside were present in the capsule. Structures that appeared to be β-galactoside-containing slime polysaccharides that were released from the cell wall were also observed. Observation using ruthenium red showed that capsular polysaccharides (CPS) in the capsule were present as a net-like structure. Colloidal gold conjugation with an anti-β-lactoglobulin antibody, in addition to ruthenium red staining, allowed the identification of slime polysaccharides released from the cell wall in the milk protein network derived from the culture medium. Based on these results, the Tokuyasu method was considered to be a useful pretreatment method to clarify and observe the presence of EPS. In particular, both CPS in the capsule and slime exopolysaccharides released from the cell wall were visualized.

Terrestrial Microorganisms: Cell Factories of Bioactive Molecules with Skin Protecting Applications.

It is well known that terrestrial environments host an immense microbial biodiversity. Exposed to different types of stress, such as UV radiation, temperature fluctuations, water availability and the inter- / intra-specific competition for resources, terrestrial microorganisms have been evolved to produce a large spectrum of bioactive molecules. Bacteria, archaea, protists, fungi and algae have shown a high potential of producing biomolecules for pharmaceutical or other industrial purposes as they combine a sustainable, relatively low-cost and fast-production process. Herein, we provide an overview of the different bioactive molecules produced by terrestrial microorganisms with skin protecting applications. The high content in polyphenolic and carotenoid compounds produced by several strains, as well as the presence of exopolysaccharides, melanins, indole and pyrrole derivatives, mycosporines, carboxylic acids and other molecules, are discussed in the context of their antioxidant, photo-protective and skin-whitening activity. Relevant biotechnological tools developed for the enhanced production of high added value natural products, as well as the protecting effect of some antioxidant, hydrolytic and degrading enzymes are also discussed. Furthermore, we describe classes of microbial compounds that are used or have the potential to be used as antimicrobials, moisturizers, biosurfactants, pigments, flavorings and fragrances.

Effects of exopolysaccharides from Escherichia coli ATCC 35860 on the mechanical properties of bacterial cellulose nanocomposites

The effects of growing bacterial cellulose (BC) in the presence of exopolysaccharides (EPS) extracted from Escherichia coli ATCC 35860 on the mechanical properties of BC have been studied. After harvesting and purifying the EPS, its composition was analyzed by gas chromatography-mass spectrometry. When adding the purified EPS into the culture media, another kind of EPS, composed of fructose, was produced by Gluconacetobacter xylinus ATCC 23769 and a minor portion of the added EPS was incorporated into cellulose fibrillar network. The characteristics of BC nanocomposites synthesized in the presence of purified EPS was systematically studied through tensile testing, x-ray diffraction, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy. The results revealed that the EPS affected the cellulose-cellulose interactions during the physical aggregation of crystalline microfibrils, but did not impact the co-crystallization process during BC synthesis. The addition of 4 or 8 mg/L purified EPS into the culture media, was found to significantly improve the mechanical properties of BC nanocomposites while maintaining BC crystallinity and crystal size.

Microencapsulation of functional strains by high pressure homogenization for a potential use in fermented milk

This study was aimed to evaluate the potential of high pressure homogenization for the microencapsulation of two probiotic lactic acid bacteria, Lactobacillus paracasei A13 and Lactobacillus salivarius subsp. salivarius CET 4063 to produce functional fermented milks. Microcapsules of the considered functional microorganisms were obtained by HPH treatments at 50MPa in the presence of sodium alginate and vegetable oil. The microencapsulated microorganisms were then inoculated as adjuncts to produce fermented milks. As controls were used fermented milks in which the two probiotic lactobacilli were inoculated without encapsulation. The viability of the strains was monitored during almost 2months of refrigerated storage. The survival of lactic acid bacteria after the gastric-duodenal simulated test was determined. Fermented milk texture parameters, the presence of exo-polysaccharides and the production of volatile molecules were also evaluated over storage. The microcapsules, for both the considered probiotic strains, were homogeneous and with a size<100μM and therefore did not adversely affect the sensory properties of the fermented milks. The encapsulation decreased the hyperacidity phenomena generally related to the inclusion of probiotic microorganisms in fermented milks. The lower acidity of the products due to the microencapsulation was fundamental for the improvement of the viability of the starter culture and the sensory characteristics of the products. The microencapsulation conditions increased the resistance to the simulated digestion processes, although the strain Lb. paracasei A13 generally showed a higher resistance to the gastric barrier respect to Lb. salivarius CECT 4063. By contrast, the data obtained showed a reduction of EPS production by the microencapsulation. The volatile profiles showed specific profiles in relation to the probiotic strain used and microencapsulation process. In conclusion, the results of this study underlined the applicative potential of HPH microencapsulation of probiotic microorganisms to produce fermented milk with improved functionality and with enhanced sensory properties.

Methicillin-Resistant Staphylococcus aureus Biofilms and Their Influence on Bacterial Adhesion and Cohesion.

Twenty-five methicillin-resistant Staphylococcus aureus (MRSA) isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype was spa type t127 (22 out of 25 isolates); the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested.

CELL SURFACE PROPERTIES OF HALOTOLERANT PROBIOTIC LACTOBACILLI

Cell surface characteristics of two halotolerant commercial probiotics (Lactobacillus casei Shirota and L. plantarum 299v) and three halotolerant potential probiotics (L. plantarum, L. pentosus and L. acidipiscis) isolated from Chiapas cheese were evaluated as well as the presence of exopolysaccharides (EPS) in their cell surface. Results showed no differences between the commercial probiotics and the strains of potential probiotics analyzed. All lactobacilli had similar autoaggregation levels (between 19.22 to 21.08%) and were able to coaggregate with gram-positive and gram-negative pathogens. The hydrophobicity to non-polar solvents (xylene, octane and hexadecane) was low. L. casei Shirota and L. plantarum isolated from Chiapas cheese had high affinity to chloroform; furthermore, it was possible to demonstrate the presence of capsular exopolysaccharides (CPS) in these strains because of the presence of carboxyl groups. From the results obtained in this work, it may be concluded that low hydrophobicity affinity to chloroform in these strains is related to the presence of CPS and that the higher affinity to chloroform might be related to CPS in the surface of halotolerant lactobacilli.

Changes in the surface properties and stability of biocolloids of a sludge system upon extraction of heavy metals

The influence of chemical and biological extraction of heavy metals on the stability of natural sludge sol, as well as on the wetting and electrosurface properties of its particles, is studied. The biological extraction of metals is found to result in hydrophobization of sludge colloids and reduce their negative charge. Heavy metal extraction destabilizes the sludge sol, the level of destabilization being maximum upon bioleaching. The presence of exopolysaccharides considerably stabilizes the sludge system; however, it weakly affects the system’s stability after extraction of heavy metals.

Promising biotechnological applications of antibiofilm exopolysaccharides

Polysaccharides are polymers of carbohydrates with anenormous structural diversity, from long linear repetition ofthe same monomer to highly branched structures of dif-ferent sugars. This high structural diversity reflects thefunctional diversity of these molecules. There are twotypes of polysaccharides, storage polysaccharides (i.e.glycogen) and structural polysaccharides, which are nor-mally secreted by the cell and form different cell structures(i.e. cellulose, chitin). Extracellular polysaccharides orexopolysaccharides belong to this last group.Exopolysaccharides are produced not only by microor-ganisms, but also by algae, plants and animals (Suther-land, 2005). Bacterial exopolysaccharides are a majorcomponent of the extracellular polymeric substance(EPS) or matrix of biofilms, and mediate most of thecell-to-cell and cell-to-surface interactions required forbiofilm formation and stabilization (Flemming and Win-gender, 2010). The matrices of biofilms from natural envi-ronments, such as marine and fresh water, soil, or chronicinfections, contain a ubiquitous composition of polysac-charides. More than 30 different matrix polysaccharideshave been characterized so far. Several are homopoly-saccharides (i.e. glucans, fructans, cellulose), but most ofthese are heteropolysaccharides consisting on a mixtureof sugar residues. Exopolysaccharides can even differbetween strains of single species, as exemplified bystrains of

Exopolysaccharides (EPS) as anti-corrosive additives for coatings

Exopolysaccharides (EPS) are a class of renewable polymers that show interesting anti-corrosive properties and could potentially be used as an alternative for zinc phosphates. When combined with a waterborne styrene-acrylic polymer dispersion (SA-1), exopolysaccharides were shown to give an improvement in the anti-corrosive performance. Electrochemical studies show higher charge transfer resistance (Rct) values for the SA-1/EPS combinations as compared to the SA-1 itself suggesting that in the presence of EPS less corrosion took place. Outdoor exposure test shows that the presence of EPS188 gave good corrosion protection for up to a year when exposed under marine conditions. The distribution of EPS modification throughout the coating was made visible using Confocal Laser Scanning Microscopy and indicated that EPS was distributed rather homogeneously. A working mechanism was proposed in which the carboxylic acid groups on the oxidised EPS form a complex with iron ions formed by the anodic reaction and this insoluble complex forms a protective layer between the coating and metal. The results in this paper show that by using renewable polysaccharide additives the anti-corrosive coating performance can be improved.

Prokaryotic communities and operating metabolisms in the surface and the permafrost of Deception Island (Antarctica)

In this study we examined the microbial community composition and operating metabolisms on the surface and in the permafrost of Deception Island, (Antarctica) with an on site antibody microarray biosensor. Samples (down to a depth of 4.2 m) were analysed with LDChip300 (Life Detector Chip), an immunosensor containing more than 300 antibodies targeted to bacterial and archaeal antigens. The immunograms showed positive antigen-antibody reactions in all surface samples (lichens, pyroclasts) and the top layer of the permafrost. The results indicated the presence of exopolysaccharides, bacteria belonging to the Alpha-, Delta- and Gammaproteobacteria, Bacteroidetes, Gram-positive Actinobacteria and Firmicutes, as well as archaeal species, most probably Methanobacterium spp. Positive reactions with antibodies to proteins and peptides revealed the presence of nitrogen fixation (NifHD, GlnB, HscA), methanogenic (McrB), iron homeostasis and iron scavenging (ferritins and DPS proteins) proteins, as well as ABC transporters, which indicated that these processes were operating at the time of sampling. These results were validated with other molecular ecology techniques such as oligonucleotide microarrays, 16S bacterial rRNA gene sequence analysis, aerobic viable counts and microscopy. Molecular ecology results showed a differentiated pattern along the depth of the drill, being the top active layer the most diverse, with Acidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes and the phototrophs Cyanobacteria and Chloroflexi as dominant groups. Actinobacteria and Firmicutes were dominant in depths from 0.5 to 2 m, and Betaproteobacteria from 3 to 4.2 m. The geochemical analysis revealed the presence of low molecular weight organic acids (acetate, formate) which could be used by microorganisms as energy sources for sulfate, nitrate and metal reduction under anaerobic conditions.

Characterization of bio-synthesized nanoparticles produced by Klebsiella oxytoca

Structural and morphological properties of biogenic ferrihydrite nanoparticles produced by bacteria Klebsiella oxytoca are investigated. The stability of water dispersions of biomineral particles produced by Klebsiella oxytoca was monitored by UV-Vis spectroscopy. Their chemical composition was determined by FT-IR spectroscopy. The vibrational spectra of biogenic ferrihydrite nanoparticles revealed typical absorption peaks of exopolysaccharides. Morphological analysis based on Raman spectroscopy indicated the presence of exopolysaccharides on the surface as well as inside the pores of the ferrihydrite nanoparticles. Structural investigations of ultrasonic assisted samples of different concentration of water dispersed particles were performed using small angle X-ray scattering analysis. Model calculations and fitting procedures revealed scattering objects of an elongated shape with 6.73±0.16 nm radius of gyration.

The role of the exopolysaccharides in enhancing hydraulic conductivity of biological soil crusts

Biological soil crusts (BSCs) are highly specialized topsoil microbial communities commonly found in arid and semiarid environments, permeated by a polymeric matrix of polysaccharides. BSCs can in principle influence edaphic properties such as texture, pore formation and water retention, which in turn determine water distribution and biological activity in dry lands. This paper investigates the influence of biotic and abiotic factors on BSC hydraulic conductivity, a parameter gauging the ease with which water can move through the pore spaces. Texture, phototroph abundance, microbial composition, and extracellular carbohydrate content were considered as potentially relevant parameters in a correlational study of BSC samples that spanned 1.5 orders of magnitude in hydraulic conductivity. A newly developed, non-destructive extraction method enabled us to directly quantify the specific role of extracellular polysaccharides on soil permeability on a variety of samples. Hydraulic conductivity showed a strongest correlation with texture (positive with sand content, negative with silt and clay). A weaker negative correlation with carbohydrate content, especially with polysaccharides having a molecular weight < 100 kDa, was also detected. In multiple regression analyses texture (silt content) was sufficient to explain most of the variation in hydraulic conductivity However, experimental removal of polymeric carbohydrates, resulted invariably in a substantial decrease in hydraulic conductivity for any given sample (between 1.7 and 3.3 fold). Our results suggest that while soil texture determines overall hydraulic conductivity in BSCs, the presence of exopolysaccharides can significantly enhance it, likely by conferring a spongy structure to a BSC thus increasing the number of waterways within it.

Impact of exopolysaccharides on the stability of silver nanoparticles in water

The stability of commercial silver nanoparticles (SNPs) in aquatic environment plays a significant role in its toxicity to the environment and to human health. Here, we have studied the impact of bacterial exopolysaccharides (EPS) to the stability of engineered SNPs. When nanoparticles are present in neutral water, the nanoparticles exhibited low zeta potential and are least stable. However, in the presence of EPS (10–250 mg/L), the negative surface charge of nanoparticles increased and therefore the propensity of nanoparticles to aggregate is reduced. In UV–visible spectroscopic analysis a decrease in absorbance at plasmon peak of SNPs (425 nm) was observed till the addition of 50 mg/L of EPS, beyond that a blue shift towards 417 nm was observed. The adsorption of EPS was confirmed by Fourier-transform infrared spectroscopy. The EPS adsorbed SNPs were more stable and exhibited the zeta potential of higher than −30 mV.

Adsorption of copper on Pseudomonas aureofaciens: Protective role of surface exopolysaccharides

Adsorption of copper on exopolysaccharide (EPS)-rich and (EPS)-poor soil rhizospheric Pseudomonas aureofaciens cells was studied as a function of pH and copper concentration at different exposure time in order to assess the effect of cell exopolysaccharides on parameters of adsorption equilibria. The surface properties of bacteria were investigated as a function of pH and ionic strength using potentiometric acid–base titration and electrophoresis that permitted the assessment of the excess surface proton concentration and zeta-potential of the cells, respectively. For adsorption experiments, wide range of Cu concentration was investigated (0.1–375 μM) in order to probe both weak and strong binding sites at the surface. Experimental results were successively fitted using a Linear Programming Model approach. The groups with p K a of 4.2–4.8 and from 5.2 to 7.2, tentatively assigned as carboxylates and phosphoryl respectively, are the most abundant at the surface and thus essentially contribute to the metal binding. The presence of exopolysaccharides on the surface decreases the amount of copper adsorbed on the bacterial cell wall apparently via screening the underlining functional groups of the cell wall. At the same time, dissolved EPS substances do not contribute to Cu binding in aqueous solution. Results of this study allow quantification of the role played by the surface EPS matrix as a protective barrier for metal adsorption on bacterial cell walls.

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin ( Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin ( Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion, which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and R1 stimulated growth of Lactobacillus / Enterococcus, Bifidobacterium, and Bacteroides / Prevotella in a similar way to that seen with inulin. The EPS R1 also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10–24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS R1 and with the increases in Bacteroides in cultures with both microbial EPS (R1 and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids.

Rheological properties of fermented milk produced by a single exopolysaccharide producing Streptococcus thermophilus strain in the presence of added calcium and sucrose

The effect of calcium supplementation, presence of exopolysaccharides (EPS) and sucrose on the texture of low‐fat fermented milk was examined. Reconstituted skim milk (14%) was acidified by 2% glucono‐δ‐lactone (GDL) or a single strain, EPS‐producing Streptococcus thermophilus, and incubated at 42°C. The levels of calcium and sucrose addition ranged from 0 to 9 mm and 0 to 45 mm respectively. Compared with acidified milk (GDL), fermented milk containing EPS showed different rheological properties in the presence of added calcium and sucrose. Calcium addition reduced gel characteristics: G′, K, and the second maximum of derivative of shear rate‐shear stress; while sucrose addition also increased them but subsequently reduced syneresis. Added calcium or sucrose increased gel hysteresis and reduced apparent yield stress.