Patent Application: US-200913144470-A

Abstract:
a method of extracting fibers from decorticated plant bast skin involves pre - treating decorticated plant bast skin of a fiber plant with an aqueous solution containing trisodium citrate having a ph in a range of about 8 - 14 at a temperature of about 90 ° c . or less ; and subsequently treating recovered fibers with a protease at alkaline ph .

Description:
treatment of hemp fiber from decorticated bast skin of full - grown hemp , with protease at different concentrations steps 1 and 2 : pre - treatment of hemp bast skin ( or bark ) prior to protease treatment twelve grams of decorticated hemp bast skin was pre - treated by agitation in 360 ml ( 3 . 3 % consistency ) of an aqueous solution containing 0 . 4 % ( w / v ) of trisodium citrate at 85 ° c . for 1 hr . the solution was discarded . this was followed by agitation of the fiber in 360 ml of an aqueous solution containing 0 . 5 % naoh and 0 . 4 % ( w / v ) of trisodium citrate at 85 ° c . for 4 hr . the solution was discarded and the fiber was rinsed by water thrice . the recovered fiber from step 2 , was divided into 6 equal portions , equivalent to 2 gram of the untreated dry fiber . each portion was suspended in 40 ml ( 5 % consistency ) of 0 . 1 % ( w / v ) of trisodium citrate ( ph 9 . 0 ) and was treated by one of the four concentrations of the protease ( 0 , 0 . 2 , 0 . 4 and 0 . 8 μl / ml ), at 55 ° c . for 3 hr . the protease is subtilisin from bacillus licheniformis ( sigma , 94 mg protein / ml , 12 . 9 units / mg protein ). release of total materials , including the insoluble debris , into each of the solutions was monitored via o . d . measured by uv - vis spectroscopy at 280 nm ( table 2 ). after centrifugation to remove the debris , the o . d . of the clear supernatant was again determined at 280 nm ( table 3 ). aliquots ( 1 ml ) were removed to for o . d . measurement at 1 , 2 and 3 hours . in table 2 , without protease ( 0 μl / ml ), the buffer steadily released materials from hemp fiber , including both debris and soluble substances , represented by the od 280 of the supernatant as 0 . 855 , 1 . 041 and 1 . 269 in 1 , 2 and 3 hr respectively . however , with addition of protease at different concentration of 0 . 05 , 0 . 1 and 0 . 2 μl / ml , there was a consistent increase in the rate of release of materials ( od 280 ) in the supernatants in the same periods . as comparison , with protease at 0 . 2 μl / ml , the od 280 of the supernatant as 1 . 540 , 1 . 842 and 2 . 018 in 1 , 2 and 3 hr respectively . such increase of od 280 of the supernatant cannot be accounted by the insignificant background od 280 ( 0 . 087 ) of protease , which is 0 . 084 at that concentration . it is obvious that protease expedited the release of both debris and soluble materials from fiber . at the higher concentrations of 0 . 4 and 0 . 8 μl / ml , there did not seem to speed up the release significantly , as compared to 0 . 2 μl / ml . after the removal of the debris via centrifugation , the od of the same solutions was re - determined to show only the release of soluble substances detected at 280 nm . in table 3 , without protease ( 0 μl / ml ), the release of soluble materials by buffer was represented by increase of od 280 of the supernatant ( 0 . 443 , 0 . 607 and 0 . 710 ) in 1 , 2 and 3 hr respectively . the addition of protease at the concentrations of 0 . 05 , 0 . 1 and 0 . 2 μl / ml , also resulted in faster rates of release of the soluble materials in the same periods . it therefore indicated that protease has expedited the release of soluble materials from fiber . at the higher concentrations of 0 . 4 and 0 . 8 μl / ml , there did not seem to speed up the release significantly , as compared to 0 . 2 μl / ml . based on tables 2 and 3 , it is evident that protease can expedite the release of both the debris and soluble substance from the treated fiber . significant release can be accomplished in 1 hr at a concentration of protease at 0 . 2 μl / ml . generally o . d . at 280 nm is used to determine the presence of aromatic ring - containing compounds that include substances like lignin or plant protein with aromatic amino acid residues . since the release of the soluble substances was effected by protease . the target substrate in the hemp fiber would be plant proteins . the present protease treatment of the hemp fiber has likely released short soluble peptides and other substances physically or chemically associated . the present protease treatment of decorticated bark at alkaline ph is therefore different from that by the aspergillus enzyme mixture at acidic ph described in various prior art . after the protease step , the supernatant was discarded and the fiber was rinsed by water thrice . the recovered fiber ( equivalent to 2 g of the starting dry bast fiber ) was treated in 40 ml ( 5 % consistency ) of an aqueous solution containing the enzyme pectinase ( novozyme pectinase ( polygalacturonase ) from aspergillus niger ) at 0 . 2 μl / ml in 50 mm sodium citrate ( ph 5 ) at 55 ° c . after 0 . 5 hr , the enzyme solution could be recovered for recycling . the fiber was rinsed twice with water . the fiber from step 4 was bleached in 20 ml ( 5 % consistency ) of a solution of 0 . 35 % h 2 o 2 and 0 . 2 % naoh , 70 ° c . for 1 hour . the bleaching solution was discarded and the fiber was washed with water thrice . comparison of the different fiber samples indicated those processed with protease at concentration of 0 . 1 μl / ml or higher in step 2 , were more separated into finer , softer and brighter fibers than the control sample without protease treatment . treatment of hemp fiber from decorticated bast skin of full - grown hemp , with protease at different temperatures and ph bast fiber was pre - treated as described in steps 1 and 2 of example 1 . then the pre - treated fiber ( equivalent to 1 g of the dry starting bast fiber ) was treated with bacillus licheniformis protease subtilisin ( 0 . 2 μl / ml ) in 20 ml ( 5 % consistency ) of 0 . 1 % ( w / v ) of trisodium citrate ( ph 9 . 4 ), at 55 and 65 ° c . for 3 hr . release of soluble materials , free of the debris , into each of the solutions was monitored via o . d . measured by uv - vis spectroscopy at 280 nm ( table 4 ). after centrifugation to remove the debris , the o . d . of the clear supernatant was again determined at 280 nm ( table 4 ). aliquots ( 1 ml ) were removed for o . d . measurement at 1 , 2 and 3 hours . in table 4 , the supernatants with protease ( 55 ° c . and 65 ° c .) have much higher od than the control which is a buffer without protease . there was little difference in the od between supernatants at 55 ° c . and 65 ° c . the fiber samples ( equivalent to 1 g of dry starting bast fiber ) pretreated by naoh as described in step 2 of example 1 , was processed with bacillus licheniformis protease subtilisin ( 0 . 2 μl / ml ) in 40 ml of 0 . 1 % ( w / v ) of trisodium citrate at different ph ( 8 . 0 , 8 . 5 , 9 . 0 and 9 . 5 ) and 55 ° c . for 3 hr . release of soluble materials , free of the debris , into each of the solutions was monitored via o . d . measured by uv - vis spectroscopy at 280 nm ( table 5 ). after centrifugation to remove the debris , the o . d . of the clear supernatant was again determined at 280 nm ( table 5 ). in table 5 , based on the value of od 280 , it is evident that that the protease subtilisin was efficient at ph 8 . 0 , 8 . 5 , 9 . 0 and 9 . 5 , but slightly more at 9 . 0 than the rest . the use of alkaline ph in the present protease treatment is therefore in big contrast to the use of acidic ph of the aspergillus enzyme mixture described in various prior art . treatment of hemp fiber from decorticated bast skin of young hemp ( 70 days ), with proteases in order to confirm that protease treatment is applicable to other hemp fiber sample , the protocol used in example 1 was repeated for the processing of the young hemp grown for 70 days in the region of peace river , alberta , canada , including steps 1 to 5 . in step 3 involving protease treatment , 2 samples were treated with or without the protease subtilisin at 0 . 2 μl / ml . the od 280 of both the raw and the centrifuged supernatants was determined ( table 6 ). the od 280 of the protease supernatant were consistently higher than the control . it therefore indicated that the protease treatment is effective to release both the debris and the soluble material from the canadian hemp fiber . the full - grown hemp bast fiber was also purified by a shorter procedure , as compared to example 1 , including a much shorter pretreatment in naoh ( from 3 hr to 1 hr ) and shorter treatment in protease subtilisin ( 3 hr to 1 . 5 hr ), without the subsequent pectinase treatment as described as step 4 in example 1 . steps 1 and 2 : pre - treatment of hemp bast skin ( or bark ) prior to the protease treatment decorticated hemp bast skin was pre - treated by agitation in an aqueous solution ( 3 . 3 % consistency ) of containing 0 . 4 % ( w / v ) of trisodium citrate at 85 ° c . for 30 min . the solution was discarded and the fiber was rinsed by water thrice . the solution was discarded . this was followed by agitation at 3 . 3 % consistency in an aqueous solution containing 0 . 5 % naoh and 0 . 4 % ( w / v ) of trisodium citrate at 85 ° c . for 1 hr . the solution was discarded . the fiber was sprayed with a waterjet to facilitate the removal of a good amount of plant debris loosely attached to the fiber . the pre - treated hemp fiber from step 2 was suspended at 5 % consistency in a solution of 0 . 1 % ( w / v ) of trisodium citrate ( ph 9 . 0 ) with or without protease subtilisin at 0 . 2 μl / ml at 55 ° c . for 1 . 5 hr . the solution was discarded and the fiber was washed by water twice . without the pectinase treatment described in example 1 , the washed fiber was bleached . the hemp fiber from step 3 of protease treatment was bleached in 20 ml ( 5 % consistency ) of a solution of 0 . 35 % h 2 o 2 and 0 . 2 % naoh , 70 ° c . for 1 hour . the bleaching solution was discarded and the fiber was washed with water thrice . this yielded bright , fine and soft fibers comparable to the sample processed with the long protocol described in example 1 . as the pre - treatment with trisodium citrate / sodium hydroxide proceeding at ph 9 - 14 and the subsequent protease treatment proceeding at ph 9 , all steps in the present purification of fiber have been conducted in alkaline ph . this has avoided any long exposure of fiber in acidic condition that may damage its integrity . extraction of hemp fiber from decorticated bast skin of the young hemp , without the use of pectinase the young hemp bast fiber was also purified by a shorter procedure , as compared to example 1 , including a much shorter pretreatment in naoh ( 3 hr to 2 hr ) at lower temperature ( 70 ° c . vs . 85 ° c . ), and shorter treatment in protease subtilisin ( 3 hr to 1 . 5 hr ), without the subsequent pectinase treatment as described as step 4 in example 1 . steps 1 and 2 : pre - treatment of hemp bast skin ( or bark ) prior to the protease treatment decorticated hemp bast skin was pre - treated by agitation in an aqueous solution ( 3 . 3 % consistency ) of containing 0 . 4 % ( w / v ) of trisodium citrate at 70 ° c . for 30 min . the solution was discarded and the fiber was rinsed by water thrice . the solution was discarded . this was followed by agitation at 3 . 3 % consistency in an aqueous solution containing 0 . 5 % naoh and 0 . 4 % ( w / v ) of trisodium citrate at 70 ° c . for 2 hr . the solution was discarded . the fiber was sprayed with a waterjet to facilitate the removal of any plant debris loosely attached to the fiber . the pre - treated hemp fiber from step 2 was suspended at 5 % consistency in a solution of 0 . 1 % ( w / v ) of trisodium citrate ( ph 9 . 0 ) with or without protease subtilisin at 0 . 2 μl / ml at 55 ° c . for 1 . 5 hr . the solution was discarded and the fiber was washed by water twice . without the pectinase treatment described in example 1 , the washed fiber was bleached . the hemp fiber from step 3 of protease treatment was bleached in 20 ml ( 5 % consistency ) of a solution of 0 . 35 % h 2 o 2 and 0 . 2 % naoh , 70 ° c . for 1 hour . the bleaching solution was discarded and the fiber was washed with water thrice . this yielded bright , fine and soft fibers . like example 4 , all steps including the pre - treatment with trisodium citrate / sodium hydroxide proceeding at ph 9 - 14 and the subsequent protease treatment proceeding at ph 9 , have been conducted in alkaline ph . this has avoided the long exposure of fiber in acidic condition that may damage its integrity . treatment of flax fiber from decorticated bast skin of flax , with protease flax fiber was purified by a shorter procedure , as compared to example 1 , including a 1 - step pretreatment without naoh , without subsequent pectinase treatment . step 1 : pre - treatment of flax bast skin ( or bark ) prior to the protease treatment decorticated flax bast skin was pre - treated by agitation in an aqueous solution ( 5 % consistency ) of containing 0 . 4 % ( w / v ) of trisodium citrate at 85 ° c . for 1 hr . the solution was discarded and the fiber was rinsed by water thrice . without naoh pre - treatment described in step 1 of example 1 , the fiber was treated with the protease subtilisin as described in step 2 below . the pre - treated flax fiber from step 1 was suspended at 5 % consistency in a solution of 0 . 1 % ( w / v ) of trisodium citrate ( ph 9 . 0 ) with or without protease subtilisin at 0 . 2 μl / ml at 55 ° c . for 3 hr . the release of total materials , including the debris , into each of the solutions was monitored via o . d . measured at 280 nm ( table 7 ). aliquots ( 1 ml ) were removed to for the o . d measurement of the raw supernatant and the clear centrifuged supernatant at 1 , 2 and 3 hours . it was evident that the protease has accelerated the release of debris and other soluble materials from the flax fiber . the flax fiber from step 2 of protease treatment was washed by water twice . without the pectinase treatment described in example 1 , the fiber was bleached in 20 ml ( 5 % consistency ) of a solution of 0 . 35 % h 2 o 2 and 0 . 2 % naoh , 70 ° c . for 1 hour . the bleaching solution was discarded and the fiber was washed with water thrice . comparison of the fiber samples indicated those processed with protease was more separated into finer fibers and softer than the control sample without protease treatment . both pre - treatment and protease treatment in the present purification of fiber have been conducted in alkaline ph . this has avoided any long exposure of fiber in acidic condition that may damage its integrity . extraction of hemp fiber from retted bast skin of hemp , without the use of pectinase retted hemp bast fiber was also purified by a shorter procedure , as compared to example 1 , including a much shorter pretreatment in naoh ( 3 hr to 2 . 5 hr ) at 85 ° c ., and shorter treatment in protease subtilisin ( 3 hr to 2 hr ) at lower concentrations , without the subsequent pectinase treatment as described as step 4 in example 1 . steps 1 and 2 : pre - treatment of retted hemp bast skin ( or bark ) prior to the protease treatment retted and decorticated hemp bast skin was pre - treated by agitation in an aqueous solution ( 3 . 3 % consistency ) of containing 0 . 4 % ( w / v ) of trisodium citrate at 85 ° c . for 30 min . the solution was discarded and the fiber was rinsed by water thrice . the solution was discarded . this was followed by agitation at 3 . 3 % consistency in an aqueous solution containing 0 . 5 % naoh and 0 . 4 % ( w / v ) of trisodium citrate at 85 ° c . for 2 . 5 hr . the solution was discarded and the fiber was rinsed by water thrice . the pre - treated hemp fiber from step 2 was suspended at 5 % consistency in a solution of 0 . 1 % ( w / v ) of trisodium citrate ( ph 9 . 0 ) with protease subtilisin at 0 , 0 . 01 , 0 . 05 , 0 . 1 and 0 . 2 μl / ml at 55 ° c . for 2 hr . release of soluble materials into the solutions of each run was monitored via uv - vis spectroscopy at 280 nm . aliquots ( 1 ml ) were removed for o . d . measurement at 0 , 0 . 5 , 1 , 1 . 5 and 2 hr . after centrifugation to remove debris , the o . d . of the clear supernatant was determined at 280 nm via uv - vis spectroscopy ( table 8 ). after 2 hr , the solution was discarded and the fiber was washed by water twice . without the pectinase treatment described in example 1 , the washed fiber was bleached . the hemp fiber from step 3 of protease treatment was bleached in 20 ml ( 5 % consistency ) of a solution of 0 . 35 % h 2 o 2 and 0 . 2 % naoh , 70 ° c . for 1 hour . the bleaching solution was discarded and the fiber was washed with water thrice . fiber samples which were previously treated with the protease at concentration of 0 . 01 to 0 . 2 μl / ml in step 3 , yielded bright and soft fine fibers . example 4 taken with example 1 shows that the process involving protease alone results in fibers of better quality than the pectinase process of the prior art ( sung 2007 ). in example 1 , the protocol for testing protease has five steps : steps 1 & amp ; 2 of pretreatment , step 3 of protease , step 4 of pectinase and step 5 of bleaching . in example 1 , there is also a parallel control run without step 3 of protease , which is equivalent to the “ pectinase process ” of sung et al ( sung 2007 ). the control run is of four steps : steps 1 & amp ; 2 of pretreatment , step 3 of pectinase and step 4 of bleaching . in table 2 , the control run is represented by the run with concentration of protease at 0 μl / ml . as indicated in example 1 , comparison of the different fiber samples indicated those processed with protease at concentration of 0 . 1 μl / ml or higher in step 2 , were more separated into finer , softer and brighter fibers than the control sample without protease treatment . therefore example 1 teaches that with both protease and pectinase treatment , the fiber is better than with pectinase treatment alone . further , example 4 describes a protocol with four steps , i . e . to eliminate the pectinase step . therefore there are four steps : steps 1 & amp ; 2 of pretreatment , step 3 of protease and step 4 of bleaching . in this protocol , there is only protease treatment without pectinase treatment . as described in example 4 , this process ( i . e . protease alone ) yielded bright , fine and soft fibers comparable to the sample processed with the long protocol ( i . e . protease plus pectinase ) described in example 1 . therefore , example 4 teaches that the protease alone process is comparable to the protease / pectinase process . since example 1 demonstrates that the long protocol with both protease and pectinase is better than pectinase alone , and example 4 demonstrates that the protease alone process is comparable to the protease / pectinase process , it is evident that the protease alone process provides improved results over pectinase alone . therefore the instant protease process is better than the pectinase process of the prior art . adamsen a p s , akin d e , rigsby l l ( 2002 ) textile res . j . 72 : 789 - 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267 . zhang j ( 2006 ) doctoral thesis dissertation entitled “ biochemical study and technical applications of fungal pectinase ”. digital comprehensive summaries of uppsala dissertations from the faculty of science and technology 137 . other advantages that are inherent to the structure are obvious to one skilled in the art . the embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed . variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims .