The colonial diameter of Lactococcus lactis spp. lactis isolates on MRS agar was promoted by providing negative atmospheric pressure and /or enrichment of negative atmosphere by CO2 gas in vacuum desiccator. In this study, application of negative atmospheric pressure or enrichment of negative atmosphere by CO2 gas at 722 mmHg was observed for maximum (2.5 mm) colony size of L. lactis spp. lactis on agar surface. No significant difference was observed in the colonial diameter in the presence of negative atmospheric pressure or enrichment of negative atmosphere by CO2 gas at 722 to 608 mmHg pressure.
Key Words: Lactococcus. lactis spp. lactis; O2 concentration; CO2 concentration; negative atmospheric pressure; colonial diameter
Lactococcus lactis is one of the beneficial and food grade microflora of dairy and food industry. Its four sub-species, L. lactis spp. cremoris, L. lactis spp. hordniae, L. lactis spp. lactis (including biovar diacetylactis) and L. lactis spp. tructae have since been recognized (Pérez et al., 2011). Amongst, L. lactis spp. lactis and spp. cremoris are widely important in dairy and food industry in manufacturing several kinds of chesses such as Cheddar, Monterey, Jack, Gouda, Edam, Muenster, Feta, Blue, Gorgonzola, Roquefort, Brie, Camembert, Havarti, Colby, Swiss, and Mozzarella, sour milk, butter milk, kefir, fermented sausages, cucumber pickles, sauerkraut and vegetable pickles (Salminen and Von Wright 1993; Vanniel and Hahn-Hagerdal 1999; Boonmee et al., 2003). L. lactis is defined as Gram +ve, catalase –ve, homo-fermentative microaerophilic cocci (Samarzija et al., 2001). Being micro-aerophilic, L. lactis requires less O2 to grow well or develop colony on agar surface because oxygen has negative effect on the growth and survival of L. lactis (Duwat et al., 2001). Although having O2 tolerance capability, L. lactis is able to develop colony on agar surface of the diameter of ≤1mm. As a result, substantial interests exist in searching of some approaches which could increase the diameter of colony on agar surface of >1mm diameter. Moreover, during the literature scan, no report was observed related to the effect of different concentrations of O2 and CO2 or negative atmospheric pressure on the colony size of L. lactis. Therefore, minimizing the O2 concentration by generating negative atmospheric pressure and /or enrichment of negative atmosphere with CO2 gas could be one of the approaches for enhancing colony diameter of L. lactis on agar surface. In the present study, an attempt has been made to investigate the effect of different O2 concentration by negative atmospheric pressure and /or enrichment of negative atmosphere with different levels of CO2 gas on the diameter of colony of L. lactis spp. lactis isolated from different dairy and non-dairy sources.
MATERIALS AND METHODS
A variety of samples from dairy and non-dairy sources were collected from different regions of district Varanasi (UP), India. Among dairy samples, milk samples of cow (35), buffalo (18), goat (08) along with cheddar cheese (08) and in non-dairy samples, samples of vegetables (16) cattle hair (08), cattle dung (12), cattle saliva (07), cattle fodder (06), cattle urine (05) and poultry faeces (08) were collected from local herd. Cheddar cheese samples were procured from experimental dairy of National Dairy Research Institute, Karnal, India.
Bacterial strains and growth conditions
Lactococcus lactis spp. lactis NCDC 094, Lactobacillus acidophilus NCDC 015 and Lactobacillus brevis NCDC 01 were procured as reference strains from National Collection of Dairy Cultures (NCDC), National Dairy Research Institute (NDRI), Karnal (India) and L. lactis spp. cremoris ATCC 19297 was purchased from Himedia Pvt. Ltd. India. Lactococcus and Lactobacillus strains were grown in MRS broth (Himedia Pvt. Ltd. India) at 30oC for 24 h. All strains were maintained as glycerol stocks in their respective broth at -20oC.
Phenotypic identification of L. lactis spp. lactis was carried out as described by Khemariya et al., (2012a). Gram-positive cocci, homo-fermentative, catalase-negative and acid-producing LAB were isolated and identified phenotypically at genus level by litmus milk reduction test and growth at 10, 40 and 45°C and at subspecies level by arginine hydrolysis test and growth at pH 9.2 and 4 % (w/v) NaCl. Molecular identification at subspecies level was carried out by PCR amplification of glutamate decarboxylase gene (gad B) followed by restriction digestion by Ase I, 16S rRNA sequencing and PCR-RFLP assay by htrA-Taq I and yueF-Alu I (Khemariya et al., 2012b).
Effect of different levels of O 2 and CO2 gases on colony formation
A total of 10 isolates of L. lactis spp. lactis were selected for the observation of the effect of different levels of O2 and CO2 on colony formation. Strains were grown at 30±0.5°C in MRS medium (pH 6.8) containing 2% (w/v) glucose, 1% peptone, 0.5% yeast extract, 0.5% meat extract, 0.5% sodium acetate, 0.2% K2HPO4, 0.2% tri-ammonium citrate, 0.005% MnSO4, 0.001% MgSO4, 0.1% tween -80 and 2% agar. After autoclaving, 20 ml aliquots of medium were poured into 90 mm plates. An aliquot of 5 µl culture broth in MRS broth (optical density at 600 nm of 1.0) was used for streaking the test strains on the agar surface. Triplicate plates for each strain were streaked and incubated at 30±0.5°C for 48 h under various levels of O2 gas i.e. 760, 722, 684, 646 and 608 mmHg generated in vacuum desiccator by vacuum pump. Similarly, another set of triplicate plates of each strain was streaked and incubated at 30±0.5°C for 48 h under various levels of CO2 gas (760, 722, 684, 646 and 608 mmHg) generated in vacuum desiccator by vacuum pump. The generation of CO2 gas was performed by the fermentation of sucrose solution (2% w/v) using Saccharomyces cerevisae and availability of CO2 gas to L. lactis spp . lactis was made possible by replacing the negative atmospheric pressure generated in vacuum dessicator by vacuum pump. The colony development was evaluated by diameter of colony using mm scale.