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

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
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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
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(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.