Patent Application: US-9066106-A

Abstract:
the present invention discloses antimicrobial compositions of modified coconut oil and palm kernel oil derived from catalytic activity of 1 , 3 positional specific lipases . said modified oil compositions comprises of free fatty acids , monoglycerides , diglycerides and triglycerides which inhibits the growth of gram positive bacteria i . e . staphylococcus aurous aureus , listeria monocytogenes , sterptococcus pyogene , gram negative bacteria i . e . vibrio cholerae , escherichia coli and yeast i . e . candida albicans .

Description:
in accordance with the object of the invention , efforts have been done in this work to get profiles of the modified coconut oil that contain medium chain fatty acids ( special emphasis on c 8 - c 12 ) and its respective monoglycerides which have antimicrobial activities towards bacteria , yeast , fungi and viruses . coconut oil was selected because by natures it was rich source of medium chain fatty acids . it contains 90 % saturated fatty acids , and of these , 45 - 48 % is lauric acid and 30 - 36 % are other short - and medium chain fatty acids . preliminary work showed that coconut oil in the present form don &# 39 ; t have antimicrobial activities ( contains 7 % diacylglycerol and remaining is the triglycerides ). analysis by hptlc showed that fatty acids and monoglycerides are detected in the oil compositions . the invention also includes methods using 1 , 3 positional specific lipase to obtain not only oils that have antimicrobial properties but also effective and powerful antimicrobial agents . according to the present invention 1 , 3 positional specific lipases is used to modify the coconut oil under specific reaction conditions to obtain profiles of saturated medium chain fatty acids and their respective monoglycerides that have broad antimicrobial spectrum towards bacteria and yeast . the modified coconut oil mixtures preferably contain high amount free fatty acids ( c 8 - c 12 ) and their corresponding monoglycerides that can be obtained through partial hydrolysis or glycerolysis reaction . the enzyme used in the present invention is an enzyme such as lipase , and preferably immobilized onto a suitable enzyme carrier . the said specific lipase possess 1 , 3 - position i . e . lipozyme tl im ( rhizomucor miehei ). reactions to obtain modify oils were as followed . enzymatic reaction was carried out using 2 . 5 g 1 , 3 - positional specific lipases with 250 g coconut oil and 2 . 5 ml distilled water . the reaction was conducted at 45 ° c . at 250 r . p . m . samples were withdrawn for analysis after 24 h reaction ( modified 1 ) and 120 h reaction ( modified 2 ). samples were then passed through funnel containing sodium sulfate powder to remove water form the sample . the reaction mixture was centrifuged to separate the oil phase . high performance thin layer chromatography technique and gas chromatography technique were carried out to determine the lipid classes and fatty acids compositions of the oil sample respectively . the modified oils were then analyzed for their antimicrobial activities of following test 1 : minimal microcidal concentration ( mmc , & gt ; 90 %) and test 2 : time - kill studies twenty ml of coconut oil was added into a 125 ml flask containing 8 g glycerol and 160 ul of distilled water . the reaction mixtures were incubated at 35 ° c ., 300 r . p . m for a time until the incubation temperature reached 35 ° c . lipozyme tl im at 250 mg was then added into the reaction mixture and incubated for 24 h at 35 ° c . consequently , the oil sample was then subjected at 25 ° c . for another 3 days . a mixture of 30 . 4 g glycerol , 1 . 09 ml of water , 1 g of lipozyme tl im and 100 g of coconut oil was prepared . the reaction mixture was first incubated at 30 ° c . for 6 h with constant stirring at 800 r . p . m . the mixture was then transferred to 5 ° c . for up to 3 days before analyze . the reaction mixture was prepared according to modified 4 . the reaction was carried out at 30 ° c ., 800 r . p . m . for 16 h . before analyze . twenty ml oil from modified 2 was added into a mixture containing 8 g glycerol , and 160 μl sterile distilled water . the reaction mixture was pre incubated at 35 ° c ., 300 r . p . m . the reaction was initiated by adding 250 mg lipozyme tl im into the mixture and reaction was carried for 24 h . consequently the mixture was then incubated at 25 ° c . for 3 days all well were inoculated with 120 μl broth ( bhi , contain 0 . 1 % tween 80 , for gram - positive bacteria , tsb for gram - negative bacteria ; pdb for yeast ). 120 μl of antimicrobial agent was inoculated into first well . from first well , 120 μl of the mixture was transferred into the second well and so on until the 12 th well . inoculum which was adjusted to 10 5 - 10 6 cfu / ml was inoculated into each well . the plates were incubated at 37 ° c . ( 2 days ) for bacteria and 32 ° c . ( 3 days ) for yeast . results were expressed in terms of mmc 90 ( minimal bactericidal concentration , & gt ; 90 % killing ) as shown in table 1 . inocula were developed by inoculation of a loopful cell in 50 ml broth ( bhi for gram - positive bacteria ; tsb for gram - negative bacteria ; pdb for yeast ) in flask and shaked at optimum temperature overnight . this was used to inoculate bhi , tsb or pdb broth that contained 50 % of filtered sterilized treated vco . the initial inoculum was adjusted to 10 4 - 10 6 cfu / ml . at time interval , 1 ml of reaction mixture was withdrawn and serial dilution was carried out in ringer solution . viable colony was enumerated by plating dilutions on plate count agar ( pca ). the plates were incubated at 37 ° c . ( 2 days ) for bacteria and 32 ° c . ( 3 days ) for yeast . a control experiment was done in the presence of 50 % sterile distilled water . the following profiles of modified virgin coconut oils and palm kernel oil have been tested for their antimicrobial activities and found to be active against a substantial group of microorganisms according to the invention , methods to obtain antimicrobial compositions containing i . e . medium chain fatty acids and their corresponding monoester through partial hydrolysis and glyecrolysis of coconut oil are provided . modified oil 1 and modified oil 2 were obtained from hydrolysis reaction of virgin coconut oil at 24 h and 120 h , respectively . test for antimicrobial activities as mmc 90 ( table 1 ) and time - kill studies ( table 2 ) showed that the modified 2 have more powerful antimicrobial activities compare to modified 1 . this is probably because of high amount of fatty acids ( 14 %) present in modified 2 ( profile 2 ). the amount of saturated medium chain fatty acids especially caprylic , capric and lauric ( mg / g ) oil were increased ( profile 2 ). however , it was noticed that modified oil compositions from profile 1 and profile 2 can &# 39 ; t stopped the growth of e . coli and p . acne . obviously , from the time - kill studies c . albicans growth was not 100 % inhibited even after 48 h incubation ( table 2 ). modified 1 and modified 2 were effective toward gram positive bacteria where 100 % inhibitions were noted after 8 h and 2 h incubation , respectively . preliminary results indicated that the amount fatty acids i . e . c 8 , c 10 and c 12 might play important role in inactivation of the growth of gram positive bacteria . modified 3 , 4 , 5 and 6 were prepared differently from modified 1 and 2 . they were obtained through glycerolysis reactions . in all reactions glycerol and lipozyme tl im were added . in comparison , modified 3 , 4 , 5 and 6 were more effective in killing c . albicans than modified 1 and 2 . hundred percent inhibitions were noted after 8 hours incubation . minimal microbial concentration towards c . albicans was obtained at 2 . 44 mg / ml whereas modified 5 need higher concentration e . g . 4 . 88 mg / ml . among the modified oil samples , modified 3 and modified 6 contained powerful antimicrobial activities . in addition it was noted that mmc 90 for gram positive bacteria like s . aureus , l . monocytogenes , s . pyogenes of modified 3 , 4 , 5 and 6 were lower than the modified 1 and 2 . the most interesting thing is that these modified coconut oils have antimicrobial activity towards the gram negative , e . coli although the mmc 90 required is still higher compare to the gram positive bacteria . modified 3 and 6 were also proved to be more potent toward gram positive bacteria compare to modified 4 and 5 . detail analysis of the lipid classes of the modified 3 , 4 , 5 and 6 showed that high amount of fatty acids and monoglycerides content play important role in broaden the antimicrobial spectrum of these modified oils . about 8 % to 14 % monoglycerides were required to control and reduce the growth of e . coli and 100 % inhibition of c . albicans . whereas high amount of fatty acids in modified 3 and 6 make the modified oils more potent towards gram positive bacteria ( profile 3 and profile 6 ). the mmc 90 for all gram positive bacteria for modified 3 and 6 were at 4 . 88 mg / ml which are sixteen times lower than that of modified 4 and 5 . fatty acids together with monoglycerides have a synergistic effect that inhibited 100 % growth of c . albicans . palm kernel oil , which fatty acid composition similar to coconut oil , can also be modified using 1 , 3 specific lipase . the modified palm kernel oil was also found to have antimicrobial property towards s . aureus . total inhibition of s . aureus was noted after 24 h exposure . on the other hand it was noted that the amount of medium chain fatty acid and it corresponding fatty acids in the modified palm kernel oil didn &# 39 ; t significant to stop the growth or kill the e . coli and c . albicans modified oils compositions in the present invention were found to exhibit good shelf stability against oxidation and can be safely used to combat bacteria , yeast and viruses that affect human , as food preservatives , products for personal hygiene and prevention of skin infection . as will be apparent to those skilled in the art in the light of the foregoing disclosure , many alterations and modifications are possible in the practice of this invention without departing from the scope thereof . accordingly , the scope of the invention is to be construed in accordance with the substance defined by the following claims . kabara , j . j . 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