strains of microorganisms separated from kefir grains include acetobacter cerevisiae siid1719 - 2b , gluconobacter oxydans siid1719 - 3b , lactobacillus sp . siid1719 - 6b , which is a new species of the lactobacillaceae family , pichiamembrani faciens siid1719 - 1y , saccharomyces cerevisiae siid1719 - 4y and picchia anomala siid1719 - 5y . in order to find out whether microorganisms separated from kefir grains and a microorganism culture obtained in a single culture of lactobaccilus sp . siid1719 - 6b , which is a new species of the strain lactobacillaceae family , have antioxidant properties , we conducted the following evaluation tests . as a result , we found that these microorganisms and the lactobacillus sp . siid1719 - 6b itself have antioxidant properties . moreover , active principles of the culture that is removed of bacteria in a centrifugal separator and sterilized using a sterilizing filter were also found to have antioxidant properties . according to the present invention , anything that shows the same properties in symbiosis with microorganisms whose safety with lactobacillus sp . siid1719 - 6b is confirmed can be used . the lactobacillus sp . siid1719 - 6b , which is a new species of the lactobacillaceae family , is deposited at the international patent organism depository of the national institute of advanced industrial science and technology . as a typical example , a strain of lactobacillus sp . siid1719 - 6b , which is a new species of the family lactobacillaceae , was cultured and its antioxidant properties were evaluated as described in the following paragraphs . a strain of lactobacillus sp . siid1719 - 6b , which is a new species of the family lactobacillaceae , is cultured in a medium m . r . s . broth ( oxoid england , uk ). the composition of the medium m . r . s . is shown in table 1 . 2 × 10 6 cells / ml of bacteria are cultured statically at a culture temperature of 30 ° c . and sampled every 24 hours to measure the number of bacteria and ph . the growth changes in the number of bacteria are shown in fig1 , and the changes of ph are shown in fig2 . there is no limitation as to the medium composition , substrate and temperature of the culture as long as the conditions allow the bacteria to grow well . the inventors of the present application tested the antioxidant properties of the above culture . first , 190 μl of 0 . 1 mm dpph ( dissolved in 100 % ethanol ) is added to each of 96 wells on a microplate . next , samples of the culture ( 0 , 24 , 48 , 72 , 96 and 120 hours ) and 10 μl of the mrs medium ( blank ) are added to each well to start radical - scavenging reactions . the light absorption immediately after ( 0 minute ) and 30 minutes after the addition of the samples were measured with a microplate reader . the radical - scavenging rate was calculated from the light absorption of the blanks and samples . the results are shown in fig3 . as the culture time becomes longer , the microorganisms grow and their ph changes . as the microorganisms grow , the antioxidant properties also become more evident . the inventors have identified lactobaccilus sp . siid1719 - 6b to be of a new species . our method of identification and the results of the microorganisms test are described in the following paragraphs . as a first - stage bacteria test , we observed the cell shape , gram stainability , the existence of spores and the motility of flagella using an optical microscope ( u - lh1000 , olympus , japan ). we also observed the shapes of colonies on an mrs broth ( oxoid , england , uk )+ agar . we tested for catalase reactions , oxidase reactions , acid and gas generation from glucose and oxidation and fermentation of glucose . the results of the mycological properties test are shown in table 2 , and a photo of a gram stained culture is shown in fig4 . the inventors used the prepman method ( applied biosystems , ca , usa ) for extraction of genome dnas . using the extracted genome dnas as templates , we amplified the region of approximately 1500 to 1600 bp of all of the base sequence of 16s ribosomal rna genes ( 16s rdna ) by pcr . after that , we sequenced the amplified 16s rdnas and obtained a base sequence . for purification of pcr products and cycle sequence , we used the microseq full 16s rdna bacterial sequencing kit ( applied biosystems , ca , usa ) ( microseq is a registered trademark ). for the thermal cycler , we used the geneamp pcr system 9600 ( applied biosystems , ca , usa ), and for the dna sequencer , we used the abi prism 3100 dna sequencer ( applied biosystems , ca , usa ). for the basic operations from the extraction of genome dnas to cycle sequence , we followed the appliedbiosystems &# 39 ; protocol ( p / n4308132 rev . a ). the base sequence of 16s rdna of siid 1719 - 6 b is shown in table 4 . using blast , we conducted a homology search of the above base sequence by comparing it against the dna base sequence database , genbank . from the base sequence of the 16s rdna that has been obtained , we conducted a homology search for species that are considered to be close relatives of the samples and identified the top five strains . the results of the homology search are shown in table 5 . based on the information obtained so far , we constructed a molecular pedigree using the 16s rdna base sequence and the 16s rdna base sequence of strains that are considered to be their close relatives . we downloaded the base sequence alignment and the 16s rdnas used in the construction of the molecular pedigree from the genbank / ddbj / embl on the basis of the strain - derivative alignment . for computation of the molecular pedigree , we used the neighbor - joining method , in which 1 , 000 bootstraps were generated to test the validity of the topology . for analysis , we used dnasis pro ( hitachi software engineering , yokohama ). the analysis results of the molecular pedigree are shown in fig5 . the inventors extracted and purified dnas from cultured bacteria , and obtained dnas for measuring the g + c content . we used high performance liquid chromatography ( hplc ). we added 20 liters of nuclease p 1 solution ( 0 . 1 mg / ml of nuclease p 1 ( dna - gc kit , made by yamasa corporation , sold by seikagaku corporation ), 40 mm of sodium acetate and 2 mm of zinc sulfate [ ph 5 . 3 ]) to the same amount of dnas ( 350 g / ml ) that are dissolved in sterilized water and thermally denatured , and treated the solution for one hour at 50 ° c . to dissolve it into nucleotides and used it as a sample for hplc measurement . we performed measurement using an hplc device ( lc - 10 , shimadzu , kyoto ) under the following conditions . moving phase : 10 mm phosphate buffer ( 10 mm potassium phosphate , 10 mm monobasic potassium phosphate [ ph 7 . 0 ]). column : develosil rpaqueous , φ4 . 6 mm × 250 mm ( nomura chemical , co ., ltd ., aichi ). the inventors identified each nucleotide from the retention times of the standard nucleotide compound ( containing equal moles of dcmp , damp , dgmp and dtmp ) contained in the dna - gc kit ( made by yamasa corporation , sold by seikagaku corporation ) and the sample . at the same time , we calculated the dna base composition of the sample expressed as a gc content from the ratio of the peak areas of the standard nucleotide compound and the sample using the following equation . nx : value of the peak area of dcmp , damp , dgmp and dtmp of the sample . ns : value of the peak area of dcmp , damp , dgmp and dtmp contained in the standard nucleotide compound . the results of the g + c content measurement are shown in table 6 . according to the analysis of the 16s rdna base sequence , the siid1719 - 6b &# 39 ; s similarity to its closest relative strain lactobacillus mali is 95 . 3 %. while the g + c content of lactobacillus mali is 32 . 5 - 33 . 0 %, that of siid1719 - 6b is 40 . 5 %. in taxonomy , there is a guideline for two species to be considered the same species . according to this guideline , in order for any two species to be considered the same species , the result of their dna - dna hybrid formation test must be 70 % or higher . that is the definition of “ being the same species ”. in order for the result of a dna - dna hybrid formation test to be 70 % or higher , the result of a 16s rdna base sequence analysis must exceed 97 % and the difference in the g + c content between the two species compared must be smaller than 3 %. our test results do not meet these criteria . accordingly , it has been found that siid1719 - 6b is a new species .
a single or mixed culture performed under conditions in which a new species of the lactobacillaceae family having antioxidant properties and separated from kefir grains can be cultured . an antioxidant having the obtained culture or bacteria as active principles is formulated and used as a raw material for or added to drinks , foods , cosmetics or animal feeds .