S. Karikalan and A. Mohankumar
S. salivarius, S. mitis, L. acidophilus, L. salivarius, L. casei, Staphylococcus spp, Eubacterium spp, Neisse- ria spp, Actinomyces spp, Peptostreptococcus spp and Micrococcus spp, etc. Among them Streptococci was only form the biggest association in the oral cavity (Bhatia and Ichhpujani, 2003).
Cavities are the most common, chronic disease of early childhood. Oral diseases are infectious, progres- sive, often painful and expensive to treat. Poor oral health has been linked to diabetes, heart disease and other long-term problems in adults (CSN-SS, 2012). The primary goal of perinatal oral health care, with regard to caries transmission, is to lower the numbers of cari- ogenic bacteria in an expectant mothers mouth so that Mutans streptococci [MS] colonization of the infant can be delayed as long as possible (AAP, 2013).
Oral health is a significant component of physical con- dition and well-being. Oral diseases, which range from dental carries (cavities) to more prevalent infections, are problematic for millions of Americans and lead to serious consequences, including complications of major chronic conditions, debilitatins pain, absenteeism from work and school, nutrition issues, loss of teeth, impacts on children’s growth and social development, adverse pregnancy outcomes, inefficient use of emergency department services, and even death (INMRCI, 2011). Fortunately, dental disease and poor oral health can be easily prevented with usual access to dental care and efficient patient education. Specialized prophylaxis and fluoride or sealant purpose are proven intrusions that stop cavities and gum disease. Lacking treatment, gum disease may in the end destroy bone, connective tissue, and teeth, necessitate surgery (CDCP, 2011).
Few investigators have reported that it is perhaps non-Mutans streptococci organisms [nonMSO] and Actinomyces that add in the beginning stages of acidifi- cation, demineralization of enamel which results in aid- ing the MS and other oral bacteria to go in for capital- ize the environment (Russell, 2009). Good oral health is necessary for eating and talking, and affects physical form and self-esteem (DHSV, 2003). Recent literature has shown that tongue cleanout show the ways to healthy oral environment (Alms et al., 2005).
A variety of compounds of controlling dental caries have been surveyed, such as antibiotics, enzymes, chlo- rhexidine, organic and inorganic fluoride. Enzymes such as dextranase reduced the amount of actively forming plaque but did not uniformly disperse the plaque already present. Little attention has paid to the elimination of the Streptococci by lytic enzyme and this type of enzymes may not give highly effect the oral and canal flora (Par- sons, 1974).Hence, this present study has been carried out to assess the antibacterial activity of metalloenzyme on Mutans streptococci.
MATERIAL AND METHODS
West coast is located at the Kerala state region of Ara- bian Sea of India (Figure I). Samples were collected and transferred to the laboratory in sterilized translucent container containing Arabian Sea water. Seaweed sam- ples were identified at CMFRI, Mandapam, and Tamil- nadu. It was washed thoroughly with marine water and removed the epiphytes by swab method. Isolated the bio- film and serially diluted; only pure colonies were taken for further process of bacterial identification.Swab the epiphytic bacteria on seaweed. Bacteria were taken into the test tubes containing Zobell marine broth used as a transported media. All the test tubes were stored at 37ºC for 24 to 48 h for incubation period. MYP Agar is the suitable media to Bacillus spp. Polymyxin-B and Egg- Yolk Emulsion were additive chemicals for killing other gram positive and gram negative species on the media. Yellow color appearance of colonies and slightly agar surroundings changed to determined colonies of Bacil- lus spp. which were identified by the Morphological test and Biochemical characterization (Bergey’s Manual of Determinative Bacteriology).
PRODUCTION OF METALLOENZYME
Production and purification of metalloenzyme was done by the methods of Chakraborty and Paul (2008). The production medium was inoculated at 1.0 % [V/V] with 24 h old [OD = 0.6] culture of Bacillus spp and incubated at [15 ± 1] oC in a shaker at 80 rpm / min for 48 h. The growth cultures were then centrifuged at 4 oC [10,000 × g, 15 min]. The supernatant was used for further purification assay. Metalloprotease enzyme was purified from crude extraction of Bacillus spp by Ammonium sulphate precipitation, Dialysis method and DEAE Cellulose column chromatography.
A specific weight of ammonium sulphate crystals was added to the crude enzyme gradually in an ice bath with continuous stirling for 30 min to get saturation percent- age of 40 - 80 % and centrifuged at 10000 rpm at 4 ºC for 20 min. The precipitate was taken and dissolved in minimal amount of 0.1 M phosphate buffer and it was dialyzed against the same buffer for 24 h.
After the purification of dialyzed sample, it was sub- jected to purification by DEAE Cellulose column chro- matography. 4 g of DEAE Cellulose was suspended in 25 mM buffer and kept overnight for equilibration. The column was carefully packed and equilibrated with the buffer at a constant flow rate of 30 ml / h. 3 ml of the dialyzed enzyme extract was diluted to 15 ml and loaded onto the column. The flow through was collected as a single fraction and tested for protease activity. The column was washed with approximately 100 ml of the
