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Isolation and characterization of lactic acid bacteria from cereals and legumes for elucidation of potential functional properties.

Prathibha, D. V. (2011) Isolation and characterization of lactic acid bacteria from cereals and legumes for elucidation of potential functional properties. PhD thesis, University of Mysore.

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Abstract

Lactic acid bacteria were isolated from fermented cereals and legumes. Thirty one cultures were isolated and preliminarily screened for gram positive, catalase negative, non motile, non-haemolytic and vancomycin sensitivity strains and were adapted to GIT (gastrointestinal tract) condition. The cultures which were able to grow at pH 3.0 and 2.5 % bile salt mix concentration were further subjected to biochemical and molecular characterization. Finally a single strain which was able to grow at low pH (2.0) and high bile salt concentration (4.0%) as that of gastrointestinal (GI) conditions was selected and identified as Lactococcus lactis. This was deposited to MTCC with an accession number 5441. Further, studies were conducted for its probiotic properties, antimicrobial activity against food borne pathogens like Staphylococcus aureus, Klebsiella pneumoniae, Listeria monocytogenes, Citrobacter freundii, Salmonella typhi and Vibrio cholerae. Cell hydrophobicity assay indicated maximum adhesion to xylene (44.30 ± 0.37%) and toluene (38.50 ± 0.52 %). The presence of β- galactosidase activity shows the ability of the culture to hydrolyze lactose for easy absorption. The antioxidant activity for intact (55.09 %) and intracellular extract (44.20 %) for scavenging DPPH radicals and lipid peroxidation activity was found high, which indicates the ability of the culture to scavenge free radicals. A number of saturated and short chain fatty acids were identified during the growth study. Strain improvement was studied by UV radiations and chemical mutagenesis to develop mutants for enhanced functional properties (antioxidant and antimicrobial activity). Both UV and EMS mutants of L. lactis were tested for its antioxidant and antimicrobial activity. The EMS mutant (treated for 5 min) gave higher antioxidant and antimicrobial activity. UV treated (10s) mutant also gave higher antioxidant and antimicrobial activity. Both these mutants were good whereas the UV treated one had the highest activity and was taken up for all further studies. L. lactis was supplemented into a cereal blend based product (wheat and | 2 ragi) used in equal proportions. Cell viability and functional properties in cereal blend product were studied over a period of five days. Mineral content was higher for Mg (1.3 folds more), Zn (2.64 times more) and Ca (5 times) as compared to the control. Vitamin B1 and B2 contents were found to be high in the probiotic product. The L. lactis was viable in the product even after five days of storage. Further, the viability and stability were enhanced with supplementation of different adjuvants like ascorbic acid, cysteine hydrochloride, casein hydrolysate and tryptone at various concentrations. These were stored at 30° C and 4° C for a period of six days to determine the appropriate temperature of storage. The culture showed viability up to 6 days (1.5 × 106 cfu/ml) with inhibitory activity against Klebsiella pneumoniae and Citrobacter freundii. On supplementation of adjuvants maximum viability was observed with 100 mg/l of ascorbic acid along with increased antimicrobial and antioxidant activity. Storage temperature of 4 °C was found to be the better temperature of storage as compared to 30 °C. These results indicate that the viability of the probiotic culture could be enhanced in the cereal blend with the supplementation of adjuvants. Work was done to study the shelf life of probiotic cereal product. The product found to be good till 30 days. Quorum sensing signalling molecule as piperazine was identified and characterized produced by L. lactis which helps in keeping good quality of the product for a longer shelf life. This is produced when the bacteria coordinate expression after attaining a particular population density. Piperazine was produced after 6 days. Work was done to study the viability of Lactococcus lactis MTCC by using different storage techniques. The culture was dried using different methods like oven, vacuum and lyophilization. Cell viability in lyophilized sample was found to be the best and was studied for six months. Different cryoprotectants like lactose, sucrose and PEG were used as protective agents during drying process. The culture viability after six months was found to be the best with lactose (3.50×105 ±0.01 cfu/ml) followed by sucrose (3.30×105 ±0.91 cfu/ml) and then PEG | 3 (2.10×105 ±0.02 cfu/ml) as compared to the control (1.90×105 ±0.01 cfu/ml). DPPH scavenging activity was found to be the best with lactose (48.1 %±0.04) till six months. Antimicrobial activity against food borne pathogens like Listeria monocytogenes (11.10 ±0.01mm), Salmonella typhi (10.11 ±0.08mm), Staphylococcus aureus (11.01 ±0.01mm), Klebsiella pneumoniae (11.10 ±0.01mm), Vibrio cholerae (6.20 ±0.09mm) and Citrobacter freundii (10.02 ±0.09 mm) was found good till the end of six months storage. Adaptability to low pH (2.0) and high bile salt concentration (4.0%) was also good till six months. Further the culture viability was enhanced by immobilization techniques like matrix and microencapsulation. In case of immobilized techniques, matrix showed maximum colony viability (3.10 ×1010 cfu/ml ±0.08) followed by microencapsulation (1.05×1010 cfu/ml ±0.05) as compared to the control (2.20 ×109 cfu/ml ±0.09) at 4 ºC. DPPH scavenging activity was also found best in matrix (52.83 % ±0.08) followed by microencapsulated beads (50.05 % ±0.01) and control (49.71 % ±0.01) at 4 ºC. Antimicrobial activity in Klebsiella pneumoniae (13.11 ±0.11mm) and Citrobacter freundii (15.03 ±0.01 mm) was maximum in matrix followed by microencapsulated beads (12.01 mm ±0.05; 14.10mm ±0.05) at 4 ºC when compared to the control. In vivo studies were conducted for lipid peroxidation assay after feeding probiotic cereal blend to the rats. The tissue homogenate of different rat organs showed peroxidase activity in lungs (139.9±0.07), kidney (364.1 ± 0.01), liver (257.4 ± 0.05), serum (97.8 ± 0.7) and heart (114.1± 0.02) as compared to the control tissue homogenate sample. The culture was able to inhibit the pathogenic microflora present in the intestinal region. The cell count of E. coli was determined by plating on MacConkey agar media where the cell count was 3.2 × 107 cfu/ml. These results indicate that the culture is safe, has high antioxidant activity and can be used in food.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Lactic acid bacteria, fermented cereals,legumes, functional properties
Subjects: 500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology
600 Technology > 08 Food technology > 21 Cereals
600 Technology > 08 Food technology > 22 Legumes-Pulses
Divisions: Food Microbiology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 14 Nov 2013 07:26
Last Modified: 14 Nov 2013 07:26
URI: http://ir.cftri.com/id/eprint/11295

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