Patent Application: US-30594606-A

Abstract:
provided are methods of producing , via induction , plant products such as bromelain from plants of the genus ananas established in vitro . a process for extracting bromelain from tissues of the pineapple plant that have been established and induced in vitro is provided . the plants have a higher proteolytic activity — and in high concentrations — as compared with the protein generated naturally in plants and as currently extracted using conventional methodologies .

Description:
the main object of this invention is a process for acquiring bromelain with significant proteolytic activity and in greater concentration in the vegetable tissue of the pineapple plant obtained and induced in vitro , as compared to the protein generated naturally in the plant in the field and that is extracted using conventional methodologies . this acquisition process for bromelain using protein inducer substances in pineapple plants ( ananas comosus ), includes the following stages : ananas comosus plants in the field were selected , which should be between 1 and 2 years of age , 50 cm tall , with a leaf diameter of 90 cm , and that have at least 2 shoots coming from the root , where said shoot is between 15 and 20 cm in height , with a diameter of 4 to 6 cm , weighing between 200 and 500 g , and 5 months old ; said characteristics make it easier to select a young shoot , which will facilitate the in vitro establishment . the shoots selected in the previous stage are removed manually , and once in the laboratory , the leaves and rosette are removed from the stem to uncover the apical meristem , which is immediately submerged in a 20 % sodium hypochlorite solution for 20 minutes . afterwards they are taken to a laminar flow hood to perform continuous rinsing with sterile , room temperature water , in order to remove sodium hypochlorite residues . the apical meristems that have not be damaged by the sodium hypochlorite are then selected to obtain explants measuring 6 × 6 × 4 mm , for which cuts are made in the perimeter for the purpose of removing external tissue oxidized by the effects of the sodium hypochlorite . said explants are planted in a modified murashige & amp ; skoog ( 1962 ) culture medium , considered for this stage “ establishment ss ”. 1 mg / l of bap ( 6 - benzylaminopurine ), 30 g / l of sacarose , 2 g / l of gel - rite and with a ph of 5 . 7 ± 0 . 01 is added . finally , the explants planted are incubated in a growth chamber at 27 ± 2 ° c ., a photo period of 16 hrs / light and 8 hrs / dark for 30 days , where a sprout is obtained measuring 1 - 2 cm in height with at least four leaves , but said sprouts still does not have axillary sprouts . a central lengthwise cut is applied to the sprouts from the previous stage ( maintaining aseptic conditions ), in such a way that two parts are obtained , which are planted in vitro in a modified murashige & amp ; skoog ( 1962 ) culture medium , considered for this stage “ propagation ss ”. 4 - 8 mg / l of bap ( 6 - benzylaminopurine ), 30 g / l of sacarose , 2 g / l of gel - rite and with a ph of 5 . 7 ± 0 . 01 is added . these sprouts are incubated in a growth chamber at 27 ± 2 ° c ., a photo period of 16 hrs / light and 8 hrs / dark for 30 days . during this period each sprout has a production of 4 - 12 side sprouts which are between 0 . 5 - 0 . 7 cm in height . this stage is repeated at least once , according to the number of sprouts desired , accordingly the sprouts obtained in this stage are grown under the same propagation conditions as were used for their progenitors . once the amount of sprouts desired is obtained , the sprouts pass to the next stage which is the induction of bromelain . in this stage a selection is made of the plantlets obtained in the previous stage which are between 1 and 2 cm in height , with an average of 3 - 5 leaves , and with a weight of 120 - 250 mg , which are planted in vitro in a modified murashige & amp ; skoog ( 1962 ) culture medium , considered for this stage “ induction ss ”. 30 g / l of sacarose , 2 g / l of gel - rite is added and sacarose ( 30 - 90 g / l ) as an osmotic stress inductor . the sprouts are placed in a growth chamber at 27 ± 2 ° c ., a photo period of 16 hrs / light and 8 hrs / dark for 7 - 45 days , which is where the sprouts achieve a size of 2 - 5 cm , a formation of 6 - 10 leaves and a complete root system . in the course of this stage it is advisable to evaluate the concentration of the total bromelain ( μg protein / g tissue ) using the bradford method , the specific enzyme activity ( u / mg protein ) using the dapeau method ( 1976 ), at 7 , 15 , 30 , and 45 days in order to discover the effect of the induction period in relation to the production of bromelain . the bromelain can be extracted when an enzymatic activity of ( 5 - 6 u / mg of protein ) is obtained . the plants are washed in running water to remove the remains of the culture . afterwards , they are submitted to a first extraction , passed through a filter press to obtain a first juice that contains between 60 and 80 % bromelain , which is stored at ( 0 - 5 ° c .). the resulting pulp , which obviously contains bromelain , is dissolved in an extraction buffer , that contains cold ( 0 - 5 ° c .) potassium phosphate ( k 2 po 4 ) to avoid denaturalization of the enzyme , at a concentration of between 0 . 03 and 0 . 2 m , at a ph of between 5 . 3 and 7 . 2 . this is allowed to repose for 20 to 30 minutes , in order to afterwards pass the mixture through a filter press and obtain a second extraction juice ; then the first juice is mixed with the second juice at between 2000 to 5000 rpm for 1 - 2 minutes , and after centrifuging at 5000 - 10 , 000 rpm for 15 - 20 minutes , at 0 - 4 ° c . to remove precipitate and recover the supernatant which is preserved at between − 80 and 2 ° c . and finally it is lyophilized for which said extract should contain at least 10 - 15 % of solids . one gram of raw extract , obtained by the aforementioned process , is made up of : 0 . 42 - 3 . 7 mg of phenol compounds , 0 . 9 - 1 . 038 mg of chlorophyll , and 0 . 053 - 0 . 106 μg of total protein . therefore , one gram of raw extract has an enzymatic activity of 4 . 5 - 6 . 5 u / mg of protein , ( this enzymatic activity has not been found in conventional extracts ) and a peroxidase activity of 0 . 97 - 2 . 3 u / mg of protein . the raw extract obtained by using this invention may be used in biotechnical and pharmaceutical applications due to its high enzymatic activity . due to the confusion that has originated in the preparation of the murashige & amp ; skoog ( 1962 ) culture medium which is modified from the conventional medium , it is important to point out that the preparation method used starts with a stock solution ( chart 1 ). starting with this medium the establishment ss , propagation ss , and the induction ss mediums are prepared . therefore , the invention also includes two innovative culture mediums , the propagation and the induction mediums . the modification of the murashige and skoog ( 1962 ) culture medium consists in that the nitrate solution is not prepared to keep it in stock , but it is prepared when it will be applied in the preparation of the medium , as indicated , in the following medium preparation process from stock solutions . 1 . in a precipitation flask place 850 ml of distilled h 2 o , add the volume indicated of the concentrated solutions : 2 . weigh , add , and stir until the following reagents are dissolved : 3 . adjust the ph of the medium to 5 . 7 with sodium hydroxide ( naoh ) at in or hydrochloric acid ( hcl ) at 1 n and dilute to 1 liter . 4 . add the gelling agent ( gel - rite ) 2 g / l and heat until it dissolves . 5 . empty 30 ml of the conventional ss in 460 ml flasks and add growth regulators , sterilize at 121 ° c ., at a pressure of 1 . 2 kg / cm 2 for 15 minutes . at this point the growth regulator in the amounts of 1 mg / l of bap and ss is called establishment or if 4 - 8 mg / l of bap is added , it is called propagation ss . 6 . if induction ss is to be prepared , the solution up to point number 4 is made and the inducing substances are added , i . e ., sodium chloride ( nacl ) at 0 . 1 - 4 . 5 g / l , sacarose at 40 - 90 g / l and salicylic acid at 0 . 001 - 2 . 0 mm in combination or separately , once the inducer is added , 30 ml is emptied into 460 ml flasks and it is sterilized at 121 ° c ., at a pressure of 1 . 2 kg / cm 2 for 15 minutes , except when the medium contains salicylic acid . in this case , the medium is sterilized without the inducer and the inducer is added to the medium once it is sterilized by filtration and under aseptic conditions . the objective of this stage was to induce the production of bromelain in pineapple plants through induction substances ( sacarose , nacl , and salicylic acid ) with a high enzymatic activity . in order to perform the evaluation of the inducers , a factor design was carried out of 3 × 3 × 3 with three inducers at three different concentrations and three monitoring times ( chart 4 ), for each treatment the evaluations are carried out using the inducers separately or in combination giving a total of 64 treatments . the parameters evaluated are the concentration of the total protein ( μg protein / g tissue ) using the bradford method , the specific enzyme activity ( u / mg protein ) using the dapeau method ( 1976 ), at 7 , 15 , 30 , and 45 days in order to discover the effect of the induction period . the induction was begun with the selection of the sprouts previously established in vitro with a size of 2 cm and an average of 3 leaves and weighing 120 mg . these plants were planted in a induction ss without growth regulator and adding sodium chloride nacl at 0 . 1 - 4 . 5 g / l as a hydric stress inductor , sacarose at 30 - 90 g / l . as an osmotic stress inducer and salicylic acid at 0 . 001 - 2 . 0 mm as an inducer of resistance to pathogens , said inducer substances are added in combination ( two or more inducers ) or separately ( one single inducer ) following the treatment ( chart 5 ). z11 = modified ss + sa ( 0 . 01 mm ) + nacl ( 0 . 5 g / l ) z12 = modified ss + sa ( 0 . 01 mm ) + nacl ( 1 . 5 g / l ) z13 = modified ss + sa ( 0 . 01 mm ) + nacl ( 2 . 5 g / l ) z14 = modified ss + sa ( 0 . 1 mm ) + nacl ( 0 . 5 g / l ) z15 = modified ss + sa ( 0 . 1 mm ) + nacl ( 1 . 5 g / l ) z16 = modified ss + sa ( 0 . 1 mm ) + nacl ( 2 . 5 g / l ) z17 = modified ss + sa ( 1 . 0 mm ) + nacl ( 0 . 5 g / l ) z18 = modified ss + sa ( 1 . 0 mm ) + nacl ( 1 . 5 g / l ) z19 = modified ss + sa ( 1 . 0 mm ) + nacl ( 2 . 5 g / l ) z20 = modified ss + sa ( 0 . 01 mm ) + sac ( 30 g / l ) z21 = modified ss + sa ( 0 . 01 mm ) + sac ( 60 g / l ) z22 = modified ss + sa ( 0 . 01 mm ) 4 - sac ( 90 g / l ) z23 = modified ss + sa ( 0 . 1 mm ) + sac ( 30 g / l ) z24 = modified ss + sa ( 0 . 1 mm ) + sac ( 60 g / l ) z25 = modified ss + sa ( 0 . 1 mm ) + sac ( 90 g / l ) z26 = modified ss + sa ( 1 . 0 mm ) + sac ( 30 g / l ) z27 = modified ss + sa ( 1 . 0 mm ) + sac ( 60 g / l ) z28 = modified ss + sa ( 1 . 0 mm ) + sac ( 90 g / l ) z38 = modified ss + sa ( 0 . 01 mm ) + nacl ( 0 . 5 g / l ) + sac ( 30 g / l ) z39 = modified ss + sa ( 0 . 01 mm ) + nacl ( 0 . 5 g / l ) + sac ( 60 g / l ) z40 = modified ss + sa ( 0 . 01 mm ) + nacl ( 0 . 5 g / l ) + sac ( 90 g / l ) z41 = modified ss sa ( 0 . 01 mm ) + nacl ( 1 . 5 g / l ) + sac ( 30 g / l ) z42 = modified ss + sa ( 0 . 01 mm ) + nacl ( 1 . 5 g / l ) + sac ( 60 g / l ) z43 = modified ss + sa ( 0 . 01 mm ) + nacl ( 1 . 5 g / l ) + sac ( 90 g / l ) z44 = modified ss + sa ( 0 . 01 mm ) + nacl ( 2 . 5 g / l ) + sac ( 30 g / l ) z45 = modified ss + sa ( 0 . 01 mm ) + nacl ( 2 . 5 g / l ) + sac ( 60 g / l ) z46 = modified ss + sa ( 0 . 01 mm ) + nacl ( 2 . 5 g / l ) + sac ( 90 g / l ) z47 = modified ss + sa ( 0 . 1 mm ) + nacl ( 0 . 5 g / l ) + sac ( 30 g / l ) z48 = modified ss + sa ( 0 . 1 mm ) + nacl ( 0 . 5 g / l ) + sac ( 60 g / l ) z49 = modified ss + sa ( 0 . 1 mm ) + nacl ( 0 . 5 g / l ) + sac ( 90 g / l ) z50 = modified ss + sa ( 0 . 1 mm ) + nacl ( 1 . 5 g / l ) + sac ( 30 g / l ) z51 = modified ss + sa ( 0 . 1 mm ) + nacl ( 1 . 5 g / l ) + sac ( 60 g / l ) z52 = modified ss + sa ( 0 . 1 mm ) + nacl ( 1 . 5 g / l ) + sac ( 90 g / l ) z53 = modified ss + sa ( 0 . 1 mm ) + nacl ( 2 . 5 g / l ) + sac ( 30 g / l ) z54 = modified ss + sa ( 0 . 1 mm ) + nacl ( 2 . 5 g / l ) + sac ( 60 g / l ) z55 = modified ss + sa ( 0 . 1 mm ) + nacl ( 2 . 5 g / l ) + sac ( 90 g / l ) z56 = modified ss + sa ( 1 . 0 mm ) + nacl ( 0 . 5 g / l ) + sac ( 30 g / l ) z57 = modified ss + sa ( 1 . 0 mm ) + nacl ( 0 . 5 g / l ) + sac ( 60 g / l ) z58 = modified ss + sa ( 1 . 0 mm ) + nacl ( 0 . 5 g / l ) + sac ( 90 g / l ) z59 = modified ss + sa ( 1 . 0 mm ) + nacl ( 1 . 5 g / l ) + sac ( 30 g / l ) z60 = modified ss + sa ( 1 . 0 mm ) + nacl ( 1 . 5 g / l ) + sac ( 60 g / l ) z61 = modified ss + sa ( 1 . 0 mm ) + nacl ( 1 . 5 g / l ) + sac ( 90 g / l ) z62 = modified ss + sa ( 1 . 0 mm ) + nacl ( 2 . 5 g / l ) + sac ( 30 g / l ) z63 = modified ss + sa ( 1 . 0 mm ) + nacl ( 2 . 5 g / l ) + sac ( 60 g / l ) z64 = modified ss + sa ( 1 . 0 mm ) + nacl ( 2 . 5 g / l ) + sac ( 90 g / l ) * treatment without the application of inducer substances , used as a test control to verify the effect of the inducers on the production of bromelain . once the inducers are added to the modified ss , it is sterilized at 121 ° c ., at a pressure of 1 . 2 kg / cm 2 for 20 minutes . in the treatments that contain salicylic acid , the modified ss medium is first sterilized and later the acid is added , which was sterilized through filtration , under aseptic conditions in a laminar flow hood . here it is advisable to add the acid to the medium before it is cooled to achieve the homogenization of the inducer with the medium before it is solidified . the sprouts are planted under aseptic conditions in 460 ml flasks that contain 30 ml of the induction ss , said flasks are closed and sealed with clear tape in order to avoid any filtering of air and with the air , contamination of same . the treatments are moved to a growth room under controlled conditions at 27 ± 2 ° c . and a photoperiod of 16 hr / light and 8 hr / darkness for a period of 7 , 15 , 30 and 45 days . once the first 7 days of induction have passed , enzymatic extraction is carried out in order to obtain raw extract . three 30 μg / ml samples are taken for each treatment to determine in triplicate the specific activity . during the evaluation of the inducers it can be concluded that all the inducers applied present statistical differences , e . g ., all the treatments to which an inducer substance was applied showed a positive effect in producing bromelain in greater enzymatic activity than the treatments without inducers ( controls ). chart 6 shows the differences in the treatment means that resulted to be statistically significant , e . g ., those that presented a maximum of specific activities during 15 , 30 and 45 days . the behavior of the inducers with regard to time is the following . at 7 days there is a significant presence of the enzyme . at 15 days the production of bromelain increases . at 30 days it decreases and at 45 days it increases . when the plant discovers that it is being threatened it produces defense proteins , in this case , bromelain which gives rise to the assumption that it is a rapid response protein due to presenting enzymatic activity at 7 days after induction and presents its maximum specific activity at 15 days . after 30 days , the plants adapted to their conditions and in turn produced bromelain in lower quantities . at 45 days after induction , the plants were submitted to stress due to a shortage of nutrients in the medium and they began to produce bromelain again , but in lower quantities than the amounts at the beginning . the plantlets grown in vitro in different culture mediums indicated in this invention were taken and rinsed in running water to remove the induction ss medium . 330 g of plants were weighed , placed in an extractor to be ground and 200 ml of juice was obtained ( first extraction ) that was stored at a temperature of 4 ° c . 670 ml of extraction buffer ( monobasic potassium phosphate and dibasic potassium phosphate ) is added to the pulp obtained from the extraction at a ph of 6 . 1 and a concentration of 0 . 05m ; this is allowed to repose for 20 minutes in order to afterwards collect the enzyme and pass it through an extractor and thus obtain the second juice . the following step is to mix and homogenize the juice from the first extraction with the second extraction for 1 minute at 2000 rpm and to then centrifuge at 10000 rpm for 20 min . at 4 ° c ., to afterwards recover the supernatant with a volume of approximately 800 ml which is frozen at − 80 ° c . and lyophilized ( 10 % of solids ) for its preservation . the extraction procedure is applied for all the treatments of which the amount of protein present is determined ( bradford ) expressing the protein per ml of extract in mg . a standard curve is used for the protein tests . the standard used was bovine serum albumin at 0 . 20 - 1 . 0 mg / ml . based on the results , the following specific activities were obtained compared with imported commercial bromelain and bromelain induced from in vitro plants . upon performing various induction tests for the production of bromelain , greater specific activity is obtained using sacarose as an inducer in a range of 30 - 90 g / l ; in this case the addition of sacarose to the medium caused osmotic stress in the plant which used the production of bromelain as a natural response . with the use of the inducers in the in vitro propagation of pineapple plants a greater enzymatic activity is generated ranging from 200 up to 600 % in comparison with the bromelain produced naturally in conventionally grown pineapple plants . the bromelain extracted through this process do not require purification methods to increase its specific proteolytic activity which is now greater than the proteolytic activity of bromelain obtained by current extraction methods that include a purification stage . not submitting the bromelain to a purification process makes its production much more profitable and minimizes the losses of raw material which occur during the purification process . 1 . the method for obtaining bromelain from pineapple plants is characterized in that it may include the following steps : a . selection of the vegetative material . select ananas comosus plants in the field , which should be between 1 and 2 years of age , 50 cm tall , with a leaf diameter of 90 cm , and that have at least 2 shoots originating from the root where said shoot should be 15 to 20 cm tall with a diameter of 4 - 6 cm , and a weight of 200 - 500 g and 5 months of age . b . establishing the culture . remove the shoot manually , and once in the laboratory , remove its leaves and rosette from the stem to uncover the apical meristem ; perform disinfection of the central meristem by submerging it in a 20 % sodium hypochlorite solution for 20 minutes . afterwards they are taken to a laminar flow hood to perform continuous washings with sterile room temperature water , in order to remove sodium hypochlorite residues ; select those apical meristems that have not be damaged by the sodium hypochlorite to obtain explants measuring 6 × 6 × 4 mm , for which cuts are made in the perimeter for the purpose of removing external tissue oxidized by the effects of the sodium hypochlorite ; plant said explants in a modified murashige & amp ; skoog ( 1962 ) culture medium , considered for this stage “ establishment ss ”. 1 mg / l of bap ( 6 - benzylaminopurine ), 30 g / l of sacarose , 2 g / l of gel - rite and with a ph of 5 . 7 ± 0 . 01 is added ; incubate the explants in a growth chamber at 27 ± 2 ° c ., a photo period of 16 hr / light and 8 hr / dark for 30 days , where a sprout is obtained measuring 1 - 2 cm in height with at least four leaves , but said sprouts should still not have axillary sprouts ; c . multiplication of propagates make a central lengthwise cut on the previously mentioned sprouts so as to obtain two parts ; plant the parts obtained from the cut in a modified murashige & amp ; skoog ( 1962 ) culture medium , considered for this stage “ propagation ss ”. 4 mg / l of bap ( 6 - benzylaminopurine ), 30 g / l of sacarose , 2 g / l of gel - rite and adjusted to a ph of 5 . 7 ± 0 . 01 is added ; incubate the sprouts in a growth chamber at 27 ± 2 ° c ., a photo period of 16 hrs / light and 8 hrs / dark for 30 days . during this period each sprout has a production of 4 - 12 side sprouts which are between 0 . 5 - 0 . 7 cm in height ; plant the sprouts again ( maintaining aseptic conditions ) according to the number of sprouts desired , accordingly , the sprouts obtained in this stage are grown under the same propagation conditions as were used for their progenitors ; d . induction of bromelain . select plantlets obtained in the previous stage , which are between 1 and 2 cm in size with an average of 3 - 5 leaves and a weight of 120 - 250 mg ; plant the plantlets selected in a modified murashige & amp ; skoog ( 1962 ) culture medium , considered for this stage “ induction ss ”. 30 g / l of sacarose , 2 g / l of gel - rite and sacarose ( 30 - 90 g / l ) as an osmotic stress inducer is added ; incubate the sprouts in a growth chamber at 27 ± 2 ° c ., a photo period of 16 hrs / light and 8 hrs / dark for 7 - 1 - 45 days , which is where the sprouts achieve a size of 2 - 5 cm , a formation of 6 - 10 leaves and a complete root system ; and 2 . extraction of bromelain . extract the bromelain when an enzymatic activity of ( 5 - 6 u / mg of protein ) is obtained ; submit the plantlets to a first extraction , passing them through a filter press to obtain a first juice that contains between 60 and 80 % bromelain , which is stored at ( 0 - 5 ° c . ); suspend the resulting pulp that obviously still contains bromelain in an extraction buffer that contains cold ( 0 - 5 ° c .) potassium phosphate ( k 2 po 4 ) to avoid denaturalization of the enzyme , at a concentration of 0 . 03 to 0 . 2 m , with a ph of between 5 . 3 and 7 . 2 , allowing it to repose for 20 to 30 minutes , and afterwards pass the mixture through the filter press to obtain a second juice from this extraction ; mix and homogenize the first juice with the second juice at 2000 - 5000 rpm for 1 - 2 minutes , and afterwards centrifuge it at 5000 - 10 , 000 rpm for 15 - 20 minutes at 0 - 4 ° c . to remove precipitate and recover the supernatant which is preserved at − 80 to 0 ° c . ; lyophilize the extract which should contain at least 10 - 15 % of solids . 1 . chao l . p . and i . e . liener . 1967 . biochem . biophys . res . commun . 27 : 100 . 2 . chavez p . m ., marquez p . m ., hernandez , rodriguez a . g . and b . santos . 1998 . “ inventor &# 39 ; s certificate ”. office of cuban industrial property . 3 . feinstein g . and j . r . whitaker . 1964 . biochemistry 3 : 1 - 050 . 4 . felton g . e . 1980 . fibrinolytic and antithrombotic action of bromelain may eliminate thrombosis in heart patients . med hypotheses ; 6 : 1123 - 1133 . 5 . fernandez g . m . 200 - 1 . bromelain : the useful side of eating pineapples . cellular biology unit . university of chile . 6 . heinicke r . 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