It is verified that the work described in this thesis entitled "Production and characterization of dextran and prebiotic isomalto-oligosaccharides from Weissella cibaria RBA12 isolated from Pummelo (Citrus maxima) for functional food applications". by Rwivoo Baruah for the award of the degree of Doctor of Philosophy is an authentic record of the results obtained from research work carried out under my supervision mainly in the Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India. The present researches are conducted on "Production and characterization of dextran and prebiotic isomalto-oligosaccharides from Weissella cibaria RBA12 isolated from Pummelo (Citrus maxima) for functional food applications". The thesis consists of 6 chapters.
Weissella cibaria RBA12 was optimized and the dextran production was scaled up to 2.5 l in batch fermentation followed by studies in dextran production in fed-batch fermentation. The in situ production of dextran by Weissella cibaria RBA12 in whole wheat flour, wheat bran and rye bran was carried out to determine its application in the sourdough fermentation.
Further investigation into the nature and properties of both glucansucrase and glucan from Weissella cibaria RBA12 is needed to elucidate their potential applications. The purification of crude dextransucrase from Weissella cibaria RBA12 with a specific activity of 1 U/mg by 25% (v/v) PEG-400 and 15% (w/v) PEG-1500 fractionation resulted in a specific activity of 16.8 U/mg and 7.3 U/mg with 17- and 7-fold purification, respectively.
Chapter 4 describes the synthesis, purification and characterization of dextran produced by the Weissella cibaria RBA12. Dextran from Weissella cibaria RBA12
The structural and physicochemical characterization of dextran from Weissella cibaria RBA12 has demonstrated properties that enable it as an efficient food hydrocolloid that can be used in the bakery, dairy and confectionery industries. Therefore, the fed-batch mode can be used for large-scale production of dextran from Weissella cibaria RBA12.
Structural and physicochemical characterization of dextran produced from Weissella cibaria RBA12
Production and scale up of dextran from Weissella cibaria RBA12 in bioreactor
Functional food applications of dextransucrase and dextran from Weissella cibaria RBA12
General Introduction
Introduction
Lactic acid bacteria in food industry
Microbial diversity of India
Lactic acid bacteria have been demonstrated as part of the autochthonous microflora of tomatoes due to low pH and organic acids (Brackett, 1988; Sajur et al., 2007). The presence of different lactic acid bacteria (LAB) of different genera is also seen in different fruits (Naeem et al., 2012).
Bacteria from genus Weissella
Glucansucrase from LAB
The A domain includes a barrel (α/β)8 which contains three proposed catalytic residues (nucleophilic aspartate, acid/base glutamate and transition state stabilizing aspartate) at the end of a deep pocket (Vujicic-Zagar et al., 2010). Sanchez-Gonzalez et al., 1999 showed that the presence of different molecular forms of dextransucrase from L.
Homopolysaccharides: polymers composed of glucose or fructose units, such as glucans which contain repetitive glucose units joined by α-(1→6) glycosidic linkages
Dextransucrase from Weissella confusa Cab3 isolated from fermented cabbage was purified and the assay conditions were optimized to give a specific activity of 11.7 U/mg at 35°C, pH 5.4 and ionic strength of 20 mM (Shukla and Goyal, 2011) . Dextransucrase from Weissella cibaria JAG8 isolated from apple peel was purified and optimal assay conditions for dextransucrase were 35°C, pH 5.4 and 5.0% (w/v) sucrose concentration.
Heteropolysaccharides: polymers composed of a variety of sugar residues, mainly glucose, galactose, fructose and rhamnose, such as kefiran that contains equal
- Isomalto-oligosaccharides
- Functional foods and nutraceuticals
- Significance of Investigation
- Objectives of present study
- Specific Objectives of the present study
Low molecular weight dextrans (40, 60 and 70 kDa) are generally preferred in clinical applications (Naessens et al. 2005). The main reasons for the growth of the functional food market are the current population and health trends (Gul et al., 2016).
Screening, identification and characterization of exopolysaccharide producing Weissella cibaria RBA12
Introduction
The α-D-glucans are classified into dextrans, mutans, alternans and reuterans depending on their glycosidic bond composition and organization (Leemhuis et al., 2013). The potential health benefits of α-D-glucan make it a product of great interest in food applications (Patel et al., 2012). It serves as a substitute for non-bacterial hydrocolloids such as guar gum and hydroxypropylmethylcellulose for the production of gluten-free soft bread with good texture, shelf life, and therefore has potential application in the baking industry for patients suffering from celiac disease (Schwab et al. ., 2008; Galle et al., 2010).
Material and Methods .1 Chemicals and reagents
- Isolation and culturing of the isolate
- Enzyme production medium
- Screening and isolation of glucansucrase producing bacterial strain
- Glucansucrase enzyme assay
- Preparation of reagents for reducing sugar estimation
- Calculation of enzyme activity
- Phenotypic characterization of the isolate
- Antibiotic sensitivity of isolate RBA12
- Carbohydrate fermentation of isolate RBA12
- Molecular characterization of the isolate RBA12
- Effect of temperature and shaking on glucansucrase production
- Fermentation profile of the isolate RBA12
- Glucan estimation by Phenol-Sulfuric acid method
One percent of the culture was used to inoculate 100 ml of MRS medium containing 2% (w/v) sucrose and incubated for 24 hours at 28ºC. 1% (v/v) culture of the selected isolates was inoculated into 5 ml of enzyme production medium as described by Tsuchiya et al. The morphological characterization of the isolate RBA12 was performed using scanning electron microscopy (Zeiss, Sigma, Germany).
Results and Discussion
- Screening of glucansucrase producing isolates
- Antibiotic sensitivity analysis of isolate RBA12
- Sequence analysis of 16S rRNA gene of isolate RBA12
An indole lysine agar motility test showed that isolate RBA12 was non-motile and negative for tryptophanase. The ability of isolate RBA12 to utilize and ferment carbohydrates with acid formation was tested. Most of the carbohydrate fermentation profile of isolate RBA12 was similar to other Weissella strains as previously reported by Bjorkroth et al. (2002) and Shukla and Goyal (2011).
Sl no Carbohydrate RBA12
Effect of temperature and shaking on glucansucrase production from isolate RBA12
The effect of different shaking conditions from 80 to 200 rpm on glucansucharase production was studied and compared with static conditions at 20 °C (Figure 2.3.7). The maximum enzyme activity of 7.2 U/ml was observed at 180 revolutions per minute, which is 14% more than under static conditions (6.15 U/ml). The lag phase during shaking at 180 rpm was reduced to 4 hours compared to an extended lag phase of 6 hours under static conditions.
Fermentation profile of Weissella cibaria RBA12
The decrease in pH was due to the formation of lactic acid, which is a product associated with the growth of lactic acid bacteria and also responsible for enzyme inactivation. The concentration of glucan slowly decreased after 24 h, which may be due to the use of glucan as a secondary substrate by the bacterium to maintain it in stationary phase after depletion of the primary substrate sucrose. Sucrose utilization by the isolate can be correlated with growth, an exponential decrease in sucrose is observed in the log phase of the isolate.
Conclusions
This is the first report of the isolation of any lactic acid bacteria from Pummelo (Citrus maxima) or any citrus fruit. 2012) Isolation characterization and identification of lactic acid bacteria from fruit juices and their efficacy against antibiotics. A photometric adaptation of the Somoyogi method for the determination of glucose. 2012) Potentials of exopolysaccharides from lactic acid bacteria.
Purification, optimization of assay and stability studies of dextransucrase from Weissella cibaria RBA12
Introduction
Dextran produced by dextran sucrase has gained importance due to its commercial applications in clinical, pharmaceutical, food, photofilm, fine chemical and other industries (Majumder et al., 2007), while oligosaccharides are used as prebiotics that help intestinal microflora by the number of Bifidobacteria and lactic acid bacteria (Chen et al., 2000). The use of enzymes for industrial purposes usually depends on their stability during isolation, purification and storage (Joo et al., 2005). Weissella species have gained importance due to their increasing applications in the food industry and only few reports are available on the purification and stability studies of dextran sucrase from Weissella cibaria (Schwab et al., 2008; Galle et al., 2010; Rao and Goyal 2013 a ) .
Materials and Methods .1 Chemicals and reagents
- Maintenance of the isolate Weissella cibaria RBA12
- Enzyme activity assay
- Determination of protein
- Estimation of protein
- Purification of dextransucrase by gel filtration
- SDS-PAGE analysis of purified enzyme .1 Preparation of stock solutions
- Preparation of SDS-PAGE gels
- Preparation of acrylamide 30% (w/v) solution
- Silver staining of protein
- Identification of dextransucrase by Periodic Acid Schiff staining protocol The dextran synthesizing activity of dextransucrase was detected by conducting non-
- Optimization of reaction conditions and biochemical characterization of dextransucrase
- Effect of temperature, pH and ionic strength on dextransucrase activity The purified dextransucrase (specific activity 20.0 U/mg; 0.36 mg protein/ml) was used
- Effect of sucrose concentration on dextransucrase activity
- Effect of metal ions on dextransucrase activity
- Effect of denaturing agents on dextransucrase activity
- Thermal and pH stability of dextransucrase
- Effect of additives on stability of dextransucrase
The cell-free supernatant was analyzed for enzyme activity as described in Chapter 2, Section 2.2.5.1 and for protein concentration as described in Section 3.2.4. Enzyme activity was measured by quantifying the amount of reducing sugar released, as described in Chapter 2, Section 2.2.4.1. Enzyme activity was measured by quantifying the amount of reducing sugar released, as described in Chapter 2, Section 2.2.5.
Results and Discussion
- Purification of dextransucrase from Weissella cibaria RBA12
- Purification of dextransucrase by size exclusion chromatography
- Identification and purity analysis of dextransucrase from 25% (v/v) PEG 400 and column purified fractions by Silver and PAS staining
- Optimization of reaction conditions for dextransucrase activity
- Effect of temperature pH and ionic strength on dextransucrase activity The purified dextransucrase (specific activity 20 U/mg) was used to study the effect of
- Effect of sucrose concentration on dextransucrase activity
- Effect of metal ions on dextransucrase activity
- Effect of denaturing agents on dextransucrase activity
- Thermal and pH stability of dextransucrase
The optimum temperature of dextransucrase from Weissella cibaria JAG8 (Rao and Goyal 2013) and Weissella confusa Cab3 (Shukla et al. 2014) was 35°C. The pH profile of dextransucrase from Weissella cibaria RBA12 showed maximum enzyme activity at pH 5.4 (Fig. 3.3.5 B). Similar results on the effect of urea were reported for dextransucrase from Weissella cibaria JAG8 (Rao and Goyal 2013).
- Effect of additives on stability of dextransucrase
- Conclusions
The thermal and pH stability results were similar to the earlier results of dectransucrase from L. The residual activity of dectransucrase from Weissella cibaria RBA12 with glutaraldehyde at 30°C after 24 hours was only 9% (Table 3.3.4). Characterization of the multiple forms and major component of dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Stabilization of enzymes with polyvinyl saccharides I: physical stabilization of horseradish peroxidase.
Structural and physicochemical characterization of dextran produced from Weissella cibaria RBA12
Introduction
The genus Weissella was proposed in 1993 after the reclassification of some Leuconostoc-like bacteria (Collins, et al., 1993). The higher percentage of branching in dextran confers different chemical (resistance to enzyme hydrolysis) and physical properties (water solubility, viscosity and diffusion) (Vettori et al., 2012). MNPs (2:1) showed superior biocompatibility results over dextran coated: MNPs (1:1) and uncoated MNPs (Tingirikari et al., 2016).
Materials and Methods .1 Chemicals and reagents
- Microorganism and culturing condition
- Production of dextran from Weissella cibaria RBA12
- Purification of dextran from Weissella cibaria RBA12 .1 Purification of dextran by ethanol precipitation
- Purification of dextran by gel filtration
- Structural characterization of dextran from Weissella cibaria RBA12 .1 Monosaccharide composition analysis of dextran
- Fourier Transform Infrared Spectroscopic analysis of dextran
- Molecular weight determination of dextran
- Differential scanning calorimeter (DSC) analysis of dextran
- Physicochemical properties of dextran from Weissella cibaria RBA12 .1 Scanning Electron Microscopic analysis of dextran
Solubility of dextran from Weissella cibaria RBA12 in water was determined by the method established by Chang and Cho, (1997). The emulsifying activity of dextran from Weissella cibaria RBA12 was tested by the method described by Bramhachari et al., (2007). The flocculating activity of dextran from Weissella cibaria RBA12 was determined by following the method of Lim et al., (2007) using activated carbon charcoal.
Result and Discussion
- Purification of dextran from Weissella cibaria RBA12
- Structural characterization of dextran from Weissella cibaria RBA12 .1 Monosaccharide composition analysis of dextran
4.3.2.3 1H and 13C Nuclear Magnetic Resonance (NMR) spectral analysis of dextran The 1H NMR and 13C NMR spectra of purified dextran-RBA12 are shown in Fig. Therefore, molecular weights of the standards used were expressed as Log molecular weight (Mw). to obtain a linear equation for the determination of molecular weight of dextran samples. This is the first report of dextran with different molecular weight produced at different times of fermentation.
Retention Time (min)
Physicochemical properties of dextran from Weissella cibaria RBA12 .1 Scanning Electron Microscopic analysis of dextran
The images of dextran of Weissella cibaria RBA12 from an aqueous solution of 0.1 mg/ml were obtained by AFM (Fig. 4.3.11 A & B). The emulsion stability of dextran from Weissella cibaria RBA12 was compared with guar gum, a natural polysaccharide, and sodium alginate, a synthetic hydrocolloid, both of which are commercially used emulsifiers. The flocculation activity of dextran from Weissella cibaria RBA12, ranging from 0.05 to 0.8 mg/ml in a 5 mg/ml dispersion of activated carbon containing 6.8 mM CaCl2 solution, was compared with guar gum and is shown in Fig .
Conclusion
Structural analysis and characterization of dextran produced by wild-type and mutant strains of Leuconostoc mesenteroides. Characterization of superparamagnetic nanoparticles coated with a biocompatible polymer produced by dextran sucrase from Weissella cibaria JAG8. 2012) Structural characterization of a novel low branching dextran produced by Leuconostoc mesenteroides FT045B dextransucrase.
Production and scale up of dextran from Weissella cibaria RBA12 in bioreactor
Introduction
In this study, the optimal conditions for dextran production by Weissella cibaria RBA12 were standardized. Dextran production was then scaled up from a 100 mL shake flask to a 2.5 L bioreactor using optimized conditions. Batch fermentation of Weissella cibaria RBA12 was carried out to obtain a higher yield of dextran.
Materials and Methods
- Microorganism and culturing condition
- Production and estimation of dextran
- Optimization of culture conditions for the production of dextran .1 Effect of temperature and aeration on dextran production
- Scale up of dextran production to bioreactor level .1 Bioreactor
- Fed-batch fermentation for dextran production from Weissella cibaria The production of dextran from Weissella cibaria RBA12 was carried out by
Dectran production from Weissella cibaria RBA12 was scaled up from 100 mL shake flask culture to 2.5 L working volume of optimized medium in a 5L bioreactor (New Brunswick, model BioFlo115). The biomass yield was calculated in relation to the amount of sucrose used for cell growth and dextran production i.e. The dextran yield was calculated in relation to the amount of sucrose used for cell growth and dextran production, i.e.
Results Discussion
- Effect of temperature and orbital shaking speed on dextran production The optimum temperature for dextran production from Weissella cibaria
- Effect of sucrose concentration on dextran production
- Effect of other medium nutrients on dextran production
- Determination of bacterial growth
- Batch fermentation for dextran production from Weissella cibaria RBA12 Production of dextran from Weissella cibaria RBA12 by batch fermentation
- Kinetics of dextran production from Weissella cibaria RBA12 under batch fermentation
The optimum sucrose concentration for dextran production by Weissella cibaria RBA12 was 2% (w/v) producing 8.9 mg/ml dextran concentration reaching 89%. Similar results from Weissella confusa Cab3 in dextran production from K2HPO4 were reported (Shukla and Goyal, 2011). Kinetic parameters for dextran production by Weissella cibaria RBA12 were determined for batch fermentation in a bioreactor at 20°C and pH 6.9.
Parameters Calculated value
