Production, purification, and functional characterization of glucan exopolysaccharide produced by Enterococcus hirae strain OL616073 of fermented food origin.

Microbial exopolysaccharides (EPS) are high molecular weight polymeric substances with great diversity and variety of applications in the food and pharma industry. In this study, we report the extraction of an EPS from Enterococcus hirae OL616073 strain originally isolated from Indian fermented food and its purification by ion exchange and size exclusion chromatography for physical-functional analyses. The EPS showed two prominent fractions (EPS F1 and EPS F2) with molecular mass 7.7 × 104 and 6.5 × 104 Da respectively by gel permeation chromatography. These fractions were further characterized by FTIR, HPTLC, GC-MS, and NMR as a homopolysaccharide of glucose linked with α-(1 â†’ 6) and α-(1 â†’ 3) glycosidic linkages. The porous, spongy, granular morphology of EPS was observed under scanning electron microscopy. EPS has revealed strong physico-functional properties like water solubility index (76.75 %), water contact angle (65.74°), water activity (0.35), hygroscopicity (3.05 %), water holding capacity (296.19 %), oil holding capacity (379.91 %), foaming capacity (19.58 %), and emulsifying activity (EA1-72.22 %). Rheological analysis showed that aqueous solution of EPS exhibited a non-Newtonian fluid behavior and shear-thinning characteristics. Overall, EPS exhibits techno functional properties with potential applications as a functional biopolymer in food and pharma industry.

Exopolysaccharides produced by Enterococcus genus - An overview.

Exopolysaccharide (EPS) biomolecules produced by lactic acid bacteria (LAB) are of prodigious interest due to their unique structural, physico-chemical, and functional characteristics. Several genera of LAB including Enterococcus spp. have been studied for EPS production by various research groups worldwide. EPS produced by various strains from Enterococcus spp. have shown a wide range of functional and technological properties with potential commercial applications. Numerous techniques are used in the characterization of Enterococcus EPS to reveal their structure, linkage, monosaccharide units, functional groups, morphology, and thermal properties. Bioactive potentials of Enterococcus EPS include antioxidant, antibacterial, antibiofilm, anticancer, immunological, prebiotic, and antidiabetic potentials which have been widely reported. These functional and biological properties make Enterococcus EPS a candidate of great importance for multiple applications in the area of food, pharmaceuticals, biomedical and environmental. This review is focused on EPS produced by various strains of the Enterococcus genus isolated from different sources. Several procedures and parameters involved in the production and purification of Enterococcus EPS are also deliberated along with the functional aspects and potential applications.

Bacterial diversity of traditional fermented food, Idli by high thorough-put sequencing.

The bacterial composition of naturally fermented Indian food, Idli was studied by high-throughput Illumina amplicon sequencing at different taxonomic levels. Metagenomic investigation revealed fold change with respect to some of the phylotypes in 06th to 12th h of fermentation, suggesting the synergistic mode of nutrition. After 12th h fermentation, bacterial populations were stabilized towards 15th h fermentation. The bacterial phyla found as Firmicutes, Fusobacteria, Proteobacteria, Actinobacteria, Bacteroidetes and others in various proportions with respective to fermentation time. Among these Firmicutes and Proteobacteria were the predominant bacterial associates in this product. Initially at 0th h time interval Firmicutes (7%) and Proteobacteria (93%) were present adequately in the product which has been changed to Firmicutes (68%), Proteobacteria (31%) at the end of the fermentation (15th h). Phylum Firmicutes represented various major genus such as Lactococcus, Weissella, Lactobacillus, Enterococcus, Bacillus and Macrococcus whereas Proteobacteria revealed the presence of Enterobacter, Erwinia, Serratia, Pseudoalteromonas, Vibrio and Klebsiella genus. Co-occurrence and Co-exclusion network were developed to ensure the positive and negative association in the eubacterial genus detected in entire batter fermentation event. Some genera like Weissella, Lactococcus and Enterococcus are showing increase in abundances in auxiliary succession events can be established for starter culture development. Supplementary material: The online version contains supplementary material available at 10.1007/s13197-022-05421-4.

Oxidative stress alleviating potential of galactan exopolysaccharide from Weissella confusa KR780676 in yeast model system.

In the present study, galactan exopolysaccharide (EPS) from Weissella confusa KR780676 was evaluated for its potential to alleviate oxidative stress using in vitro assays and in vivo studies in Saccharomyces cerevisiae (wild type) and its antioxidant (sod1∆, sod2∆, tsa1∆, cta2∆ and ctt1∆), anti-apoptotic (pep4∆ and fis1∆) and anti-aging (sod2∆, tsa1∆ and ctt1∆)) isogenic gene deletion mutants. Galactan exhibited strong DPPH and nitric oxide scavenging activity with an IC50 value of 450 and 138 µg/mL respectively. In the yeast mutant model, oxidative stress generated by H2O2 was extensively scavenged by galactan in the medium as confirmed using spot assays followed by fluorescencent DCF-DA staining and microscopic studies. Galactan treatment resulted in reduction in the ROS generated in the yeast mutant cells as demonstrated by decreased fluorescence intensity. Furthermore, galactan exhibited protection against oxidative damage through H2O2 -induced apoptosis inhibition in the yeast mutant strains (pep4∆ and fis1∆) leading to increased survival rate by neutralizing the oxidative stress. In the chronological life span assay, WT cells treated with galactan EPS showed 8% increase in viability whereas sod2∆ mutant showed 10-15% increase indicating pronounced anti-aging effects. Galactan from W. confusa KR780676 has immense potential to be used as a natural antioxidant for nutraceutical, pharmaceutical and food technological applications. As per our knowledge, this is the first report on in-depth assessment of in vivo antioxidant properties of a bacterial EPS in a yeast deletion model system.

Preferential growth stimulation of probiotic bacteria by galactan exopolysaccharide from Weissella confusa KR780676.

Many microbial exopolysaccharides (EPS) are reported to have prebiotic properties. This study reports the prebiotic properties of a galactan exopolysaccharide produced by Weissella confusa KR780676 of fermented food origin. We have reported potential techno-functional properties of this EPS earlier. We have studied the parameters such as enzymatic hydrolysis, growth proliferation effect on probiotic bacteria and in vitro fecal fermentation to demonstrate the prebiotic property of the galactan. The galactan showed strong resistance to simulated human digestive juices such as α-amylase (99.1%), gastric (98.4%) and intestinal fluid hydrolysis (98.8%). It also encouraged the growth of probiotic bacteria viz, Lactobacillus plantarum MTCC9510 (recently reclassified as Lactiplantibacillus plantarum) and L. fermentum MTCC903 (recently reclassified as Limosilactobacillus fermentum) from 6.69 to 9.45 log CFU/mL and 7.13 to 9.05 log CFU/mL respectively in 72 h of fermentation. Carbohydrate utilization also exhibited the same pattern. Fecal fermentation of galactan for 48 h through in vitro studies exhibited significant changes in microbial population and short chain fatty acids (SCFAs) production. Bifidobacterium sp. and Lactobacillus sp. showed significantly (p ≤ 0.05) higher growth when compared to Enterococcus sp. (9.62 log CFU/mL, 8.45 log CFU/mL and 8.31 log CFU/mL respectively from 6.54 log CFU/mL) at the end of fermentation. SCFAs (acetic acid, butyric acid and propionic acid production) increased significantly (p ≤ 0.05) from 4.53 to 69.12 mM/L, 1.27 to 5.47 mM/L and 0.68 to 42.11 mM/L respectively. These results clearly prove the potential prebiotic property of the galactan EPS and could be used as an effective prebiotic to positively modulate the gut microbiome homeostasis.

Bacterial exopolysaccharides for improvement of technological, functional and rheological properties of yoghurt.

Exopolysaccharides (EPS) are known to have technological and functional applications in food industry including dairy based products such as yoghurt. Yoghurt is a widely consumed dairy based product due to pleasant taste and texture, as well as a source of nutrients and bioactive compounds. At the same time, structural, rheological and sensorial properties are important in the production of good quality yoghurt. Various natural hydrocolloids including EPS with stabilizing and texture enhancing properties could be useful in enhancing these desirable properties. Apart from that, EPS may enhance various other functional properties of yoghurt such as antioxidant and prebiotic potential. Based on its prebiotic property, symbiotic products could be developed by combining EPS and probiotic bacterial strains. EPS has potential to provide physical and micro structural stability, thereby enhancing the protein distribution and viscoelastic properties. Main focus of the present review is to provide an insight on the action of EPS as a functional hydrocolloid on the technological, rheological and functional properties of yoghurt and related products.

Overview of exopolysaccharides produced by Weissella genus - A review.

Exopolysaccharides (EPS) from lactic acid bacteria (LAB) are much diversed in structure, composition and applications which also adding a great commercial potential due to its generally recognized as safe (GRAS) status. LAB genus such as Lactobacillus, Leuconostoc, Streptococcus, Weissella, Lactococcus are known to produce EPS. Among this genus, Weissella is enormously reported for diversity and high production of EPS with wide range of industrial applications and bio-functional properties. This review summarize in detail about the Weissella EPS from genus to functional application. Physico-chemical characterization from production, purification step to structural elucidation of Weissella EPS is comprehensively discussed along with their properties. Weissella genus has revealed various EPS with significant functional potentials, making massive application in food and pharma industries as viscosifiers, biothickener, emulsifiers and stabilizers. In addition to this, biological properties of these EPS revealed multiple health promoting properties which can be explored for further applications in food and pharmaceutical sectors.

Report on aflatoxin-binding activity of galactan exopolysaccharide produced by Weissella confusa KR780676.

Galactan exopolysaccharide (EPS) produced by Weissella confusa KR780676 isolated from an Indian traditional fermented food has been reported earlier. In this manuscript, we have studied aflatoxin-binding ability of this galactan EPS. Aflatoxin B1 (AFB1) binding ability of galactan EPS was observed in an increasing trend with increasing EPS concentration (20-100 mg/mL). At lower concentrations (< 20 mg/mL) of EPS, the binding activity was undetectable, while notable binding was seen from 30 mg/mL. Enhanced AFB1 binding (32.40%) was recorded at 50 mg/mL of EPS and it increased gradually up to 34.79% at 100 mg/mL concentrations of EPS. The intensity of bands in high-performance thin-layer chromatography (HPTLC) analysis confirms the AFB1 binding efficiency of galactan EPS, which shows its potential application for removal of toxins in food and feed industry. Galactan EPS binding activity to AFB1 is further studied with particle size analysis (PSA). This is the first study reporting the aflatoxin-binding activity of any kind of EPS from lactic acid bacteria.

Evaluation of oil-in-water (O/W) emulsifying properties of galactan exopolysaccharide from Weissella confusa KR780676.

Galactan exopolysaccharide (EPS) was extracted from Weissella confusa KR780676 isolated from idli batter. The present study reports the effect of galactan EPS concentration, pH, ionic strength, temperature, salinity, monovalent salts on the emulsion formed with vegetable oils (virgin coconut oil, groundnut oil, olive oil and mustard oil). Emulsion determination test revealed the oil-in-water type of emulsion. Microscopic observation showed the phase of oil droplet distribution of emulsions. Groundnut and olive oil showed significant emulsifying activity (50-60%) and stability (90%) irrespective of the EPS concentration. One percent of EPS also showed good emulsifying activity (50-70%) and stability (100%) with groundnut and olive oil in wide range of temperature (- 20, 4, 30, 40, 50 and 60 °C), pH (3-8), salinity (1, 5, 10, 20 and 30% of NaCl) and monovalent salt (1, 5, 10, 20 and 30% of KCl). Galactan could be used as a prominent emulsifier for applications in the food industry as it possesses significant activity with most of the influential factors in wide range and also obtained from lactic acid bacteria that are regarded as Generally Regarded as Safe (GRAS).

Interface between food grade flavour and water soluble galactan biopolymer to form a stable water-in-oil-in-water emulsion.

In the present investigation, emulsifying potential of galactan exopolysaccharide (EPS) extracted from Weissella confusa KR780676 has been evaluated with various food grade flavours (vanilla, cardamom and pineapple). Concentration of EPS was optimized as 1% with these flavours, in addition to the effect of salinity (NaCl), monovalent ion (KCl) and temperature on emulsion activity (EA), and emulsion stability (ES) was also inspected. Filter paper wetting test exhibited water-in-oil-in-water (w/o/w) and oil-in-water (o/w) type emulsions. The extent in granule disintegration and the retrogradation process of flavour emulsions were studied with pasting properties. Electron micrography and particle size analysis revealed the morphology and the size of emulsion droplets. Thermal stability of emulsions has found 100% at various temperatures (-20 to 60 °C) for vanilla and pineapple flavour, whereas, it was varying for cardamom as per the temperature disparity. Emulsion stability of vanilla and pineapple flavour was retained as such for various concentrations of NaCl whereas decreased for cardamom in direct proportion. In case of KCl all the three flavours showed greater stability. These emulsifying properties indicate that galactan EPS can be a prospective alternative to commercial biopolymers in food and pharmaceuticals industries.

Physical and functional characterization of succinoglycan exopolysaccharide produced by Rhizobium radiobacter CAS from curd sample.

Identification and rheological behaviour of succinoglycan exopolysaccharide (EPS) from Rhizobium radiobacter CAS isolated from curd was published in earlier reports. In current investigation physicochemical and functional properties of CAS EPS has been studied. SEC-MALLS revealed molecular weight (Mw), number molecular weight (Mn) and polydispersity index (PDI) of CAS EPS as 4.05×106g·mol-1, 3.82×106g·mol-1 and, 1.06 respectively. Thermogravimetric analysis showed the high thermal stability of CAS polymer where approximately 77% of CAS hydrocolloid maintain physical integrity and stability with temperature degradation (Td) at 290°C. Scanning electron microscopy and particle size analysis confirmed the porous and hygroscopic nature and 2.049µm size of CAS EPS respectively. Equally important functional properties such as water activity (0.55), water solubility (95%), water contact angle (54.83°), water binding capacity (101g/g), water holding capacity (68.19g/g), oil binding ability (soybean and groundnut oils were found 4.35g/g and 3.68g/g) and swelling index (17.5mL/g) were examined for CAS EPS. Prevention of syneresis has been studied at 1% CAS EPS concentration which significantly prohibited the degree of syneresis of starch. These physico-functional properties make CAS EPS a prominent candidate for food processing and product development sector.

Effect of γ-irradiation on physico-chemical and antioxidant properties of galactan exopolysaccharide from Weissella confusa KR780676.

Exopolysaccharide (EPS) produced by Weissella confusa KR780676 from an Indian fermented food (Idli) has been characterized as galactan in earlier report. In this study, the galactan EPS was irradiated at different doses (0, 4, 8, and 16 kGy) using 60Co as γ-source and effect of γ-irradiation on physico-chemical and antioxidant potential of galactan EPS was studied. Color and flow properties showed moderate change after the γ-irradiation treatment. Particle size analysis and scanning electron microscopy exhibited significant breakdown of the EPS particles into smaller average hydrodynamic diameter size from 495.54 to 198.44 nm with increasing irradiation doses. Overall, pH showed decrease, while the water activity showed an increasing trend with the increasing irradiation dose. No significant effect was observed in the functional groups of EPS as revealed in the FT-IR spectroscopic analysis. After the treatment, semi-crystalline nature of the EPS changed to amorphous as shown in XRD analysis due to the breakdown. Antioxidant activities of galactan EPS increased marginally after the treatment. However, hydroxyl radical scavenging activity increased significantly from 48 to 67%. Changes in physico-chemical and antioxidant properties of galactan EPS with γ-irradiation treatment altered the functional properties of EPS positively, making it more suitable for wider application in the food industry.

Production, partial characterization and antioxidant properties of exopolysaccharide α-d-glucan produced by Leuconostoc lactis KC117496 isolated from an idli batter.

Exopolysaccharide (EPS) produced from Leuconostoc lactis KC117496 isolated from the naturally fermented idli batter has been reported earlier. Here, study aimed to optimize the carbon source to enhance the yield of EPS production, partial characterization and antioxidant activity of α-d-glucan EPS. Among different disaccharides (sucrose, maltose, lactose) and monosaccharides (glucose, galactose and fructose), combination of sucrose and glucose at 2% showed highest EPS production of 4.55 g/L in MRS medium. The molecular weight was identified as ~ 4.428 × 103 kDa with MALDI-TOF mass spectroscopy. 1D and 2D NMR results exhibited the presence of α-1-6 and α-1-3 linked glucose revealed EPS as a glucan. Antioxidant properties of glucan (500 µg/mL) revealed the significant oxidation alleviation potential such as DPPH (74%) and Hydroxyl radical activity (97.8%), whereas metal chelating activity (70%) was lower as compare to control standard. These characteristics of glucan EPS reveals its potential application in food and pharmaceutical industry.

Characterization of α-D-glucan produced by a probiont Enterococcus hirae KX577639 from feces of south Indian Irula tribals.

Exopolysaccharide (EPS) producing probiotic strain Enterococcus hirae KX577639 were isolated from the feces of South Indian Irula tribes. EPS yield was 18.57 g/L (dry weight) at 48 h in 2% sucrose supplemented MRS medium. TLC and GC-MS analysis confirmed the presence of predominant glucose monomer indicating the homopolysaccharide nature of EPS. FTIR and NMR studies revealed that the EPS were branched α-D-glucan polymer with α-(1 → 6) and α-(1 → 3) linkages. SEM analysis of glucan-EPS revealed porous and starch like cracked granules of aggregation. AFM studies proved spherical lumps and dense, grainy like network. The thermal behavior of glucan-EPS showed degradation temperature of 315.98 °C and melting point of 296.67 °C. The XRD analysis confirmed the amorphous nature of EPS with a crystalline index of 0.48. The water solubility index and water holding capacity of glucan-EPS showed 46.5% and 202.04%. These distinctive features of the glucan EPS could find its potential application in functional food products as the α-(1 → 3) linkage are resistant to human digestive enzymes and can serve as a nutrient to gut bacteria. This the first study reporting the EPS production by Enterococcus hirae.

Synthesis of silver nanoparticles using bacterial exopolysaccharide and its application for degradation of azo-dyes.

In this study, the synthesis and characterization of exopolysaccharide-stabilized sliver nanoparticles (AgNPs) was carried out for the degradation of industrial textile dyes. Characterization of AgNPs was done using surface plasmon spectra using UV-Vis spectroscopy, X-ray diffraction (XRD) and Raman spectroscopy. The morphological nature of AgNPs was determined through transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM), which indicated that the AgNPs were spherical in shape, with an average size of 35 nm. The thermal behaviour of AgNPs revealed that it is stable up to 437.1 °C and the required energy is 808.2J/g in TGA-DTA analysis. Ability of EPS stabilized AgNPs for degradation of azo dyes such as Methyl orange (MO) and Congo red (CR) showed that EPS stabilized AgNPs were found to be efficient in facilitating the degradation process of industrial textile dyes. The electron transfer takes place from reducing agent to dye molecule via nanoparticles, resulting in the destruction of the dye chromophore structure. This makes EPS-AgNPs a suitable, cheap and environment friendly candidate for biodegradation of harmful textile dyes.

Physico-chemical characterization of galactan exopolysaccharide produced by Weissella confusa KR780676.

Physico-chemical properties of a galactan exopolysaccharide (EPS) produced by Weissella confusa KR780676 isolated from an idli batter were studied. In our previous paper, we had reported the isolation and characterization of a linear galactan (containing α-(1→6)-linked galactose units) isolated from an acidic fermented food. In this manuscript, we are reporting the physico-chemical properties viz., Colour evaluation (L* 81.79, a* 3.15, b* 15.04), flow properties (compressibility index 38.46%), syneresis, light transmittance (50.7% for 5days), pasting properties (temperature 64.6°C), texture (hardness 9.876N) and particle size (549.81dnm) for the galactan were carried out. This galactan also possessed high oil absorption capacity (5.09mL/g) with good emulsifying activity (69.4%) and emulsion stability up to 15days. Flocculating activity of 65.3-81.7% was observed with 0.2mg/mL EPS in a wide range of pH (2.0-12.0). These functional properties will make the galactan EPS a good candidate for use as thickening and gelling agent and emulsifier to form long-term emulsions in food, pharmaceutical and cosmetic products, as well as for bio-treatment of wastewater and hydrocarbon-polluted environments.

Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food.

An exopolysaccharide (EPS) producing strain PUFSTM055 isolated from Idli batter (an Indian traditional cereal-legume based fermented food) was identified as Weissella confusa KR780676. The strain was shown to produce 17.2g/L (dry weight) of EPS in 2% sucrose supplemented MRS broth and the EPS was characterized. HPTLC analysis confirmed the presence of galactose monomers, indicating the homopolysaccharide nature of EPS. Fourier-transform infrared spectroscopy and nuclear magnetic resonance analysis revealed that the EPS was found to be a novel linear galactan containing α-(1→6)-linked galactose units. Scanning electron microscopy of the EPS revealed the presence of porous and spongy starch-like granules. Topographical examination of EPS by atomic force microscopy revealed that the EPS formed densely packed mesh-like structure with irregular spherical lumps. The EPS also showed high thermal stability with a degradation temperature of 287.5 °C and melting point at 274.65 °C. EPS was semi-crystalline with crystallinity index of 0.23 and showed 100% water solubility index. These characteristics of the EPS would make it a promising hydrocolloid for food industries as bio-thickeners, stabilizers and also as an encapsulating material for delivery of food bioactive compounds. This is the first study reporting the galactan compose of the EPS from lactic acid bacteria.