PH26640A - Process for preparing polysaccharide - Google Patents

Description

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This application is a divisional applioation of

Philippine Application Serial No, 36612, filed on March 9, 1988,

BACKGROUND OF THE INVENTION

S Field of the Invention:

This invention relates to a process for preparing a polysaccharides and, more particularly, to a process for preparing polysaocherides induced from plants dy a

Plant-tissue culture method,

Dapaription of the Background:

Polysaccharides induced from plants ere widely uti-~ lised am visoosity increasing sgents, gelatinisebion agents, foan stabilisers, suspension or emilsion stabi- lizers, capsule forming agents, athesives, rhysiological 13 sotive agents, and the like,

Conventionally, these polysaccharides sre commonly produced from seeds, fruits, stems, trunks, leaves, roots, tubers, or tuberous roots of naturally grom or artifi- olally cultivated plants by tapping, extraction, or the like methods,

Production from the natural sources, however, is : liable to be influénced by climatic conditions which tend $0 case fluctuation in the production amount snd rice.

Becaise of this, various trials have been undertsken in

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20k y 0 recent years to produce these polysaccharides from plants of natural origin by culturing calluses or orgsana of puch plents artificially by meens of plaxb~tissue oulture method, thus eliminating the adverse influence of olimatic conditions.

There are very few reports concerning the pro- duction of polysaccharides of plemt origin by plant~tis- sue oulture method application, It is kmown, however, in relation to research about cell wall development mechemism that small amounts of polysaccharides ore secreted in a 14quid culture broth wherein a callus 4s cultured. Re- ports indicate, for instemoce, that Vinaa rogea I. pro- duces 0,22 gn of polysaccharides per liter of culture broth in 10 deys, Ghyoine mex Mexril produces 0,49 gn of 1% polysaccharides per liter of culture broth in 8 days, amd

Phaseolus vulgexis I. produces 1,6 gn of polysaccharides per liter of culture broth in 21 days.

The conventionally utilized plant-tissue culture : : method, however, has a dravback in iis extremely low rate of production, A strong desire has tims existed for the development of a plent-tissue culture method cepsble of a higher productivity.

In this situation, the present inventors heve con- duoted extensive studies and found that a large emount of a5 polysaccharides was produced by using plants belonging to "3 BAD ORIGINAL

906 fu the germs Polianthes LI. as a plent source, and culturing the callus induced therefrom in a culture medium ocontein- ing one or more plant hormones.

SDOURY OF THE INVENTION

Agcordingly, sn objeot of this invention is to provide a process for preparing a polyseccharide compris- ing oulturing a callus induced from a plant belonging to the gems Polienthes T. in a culture medium oombeining one or more plant hormones and oollecting the polysac~ charide from the culture broth,

A more specific objeot of this invention is to provide the above-mentioned process for preparing a poly~- . saccharide using a plant the strain belonging to DPolisnthes uberops IL. 13 Another object of this invention is to provide a process for preparing a polysaccharide camprising as its structural units arsbindse, galactose, mamose, xylose, and uronde acid.

Other and further objects, features, end advamrt- sages of the invention will appear more fully from the fol- lowing description,

BRIE? DESCRIPTIOR OF THE DRAWINGS

Fig, 1 1s a drawing showing infrered spectrum of acidic heteropolysaccharides according to this invention, -4- BAD ORIGINAL le qo and Mig, 2 is a drawing showing Bg FMR ppeotrum of the sane substance,

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The production of polysaccherides according to this invention comprises using a plent belonging to

Polimmthen Iny culturing a onllus induced from the pland in a culture medium aontaining one or more plant hor- mones, smd collecting the polysaccharides fram the oul=- ture hroth, oo

A typicel example of the plant belonging to

Lo | Poliemihes I. 1s Polishes iubsroma Is Portions of the orgen or tissue of the plent such as the flower, stem, leaves, bulb, roots, or the like are used as the explamt,

Among the most desirable portions is a certain tissue of the flower,

The basal medias employed for inducing the gallus may be those conventionally employed in plent-tismie aul- turing, md include Murasige-Skoog medium, Idnsmoier-

Boog medium, CGemborg medium, Yhite medium, Tuleeke me- dium, Niteoh & Fitsch medium, or the like, -

It 1s imperative that one or more plant hormones be added to these medies. FExesuples of the plat hormones to be employed are suxina such ap 2,44-dichlorophenoxy- -5- - BAD ORIGINAL

: Let gy 0 soetio acid (2,4~D), ofnarhthalenessetio acid (NAA »h - ad indolescetio aoid (IM), indolebutylic acid (18a), oytolinins such es furfurylaminopurine (kinetin), ben- syledenins (BA), and dimethylaminopurine (2iP), end the like. Good results are obtsined by the independent use of 2,4-Dy or the combined use of NAA ma BA, or FAA and kinetin, The goncentrations of hormones required for intuoing the callus are 5 x 107% ~ 1 x 10°7 Mt for 2,4-1, / when it is employed independently, md § x 107% = 1 x 10~7 for NAA, when employed in combination with PA or kinetin, wherein BA end ldnetin are used at a concentration of 1 x04 1x10" x respectively, . :

In addition to the basal medium and the plant : hormones, sugars are added to the culture medium os a carbon source for inducing the callus. | Sugars thet oan be employed for this purpose inglude glucose, galactose, fructose, mmmose, xylose, mioross, rhamnose, fuoose, starch, and the like, Among these, the most typically used sugar is suorose,

Although either ®0lid or liquid culture medium ; oan be employed for inducing the callus, the usally em- ployed medium is the s0lid one,

Te callus tims induced oem be subcultured over more then 10 generations, while meintaining the seme form 2% in the same medium in which 1t wes originally induced;

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Culture media employed for subculturing sre those con- taining Idinsmaler—Skoog medium or Mirani ge-Skoog medium o8 a basal medium; 2,4-D at a concentration of 1 x 10~4 - 1x10" M, or the combination of NAA and BA, both at a concentration of 1 x 104 = 1 x 1077 1 a plant hormones and glucose, gelactose, fructose, mmmose, xylose, smiorose, rheamose, fucose, starch, or the like, most desirably, sucrose, at 1-5% based on the culture medium, se» a carbon source,

Por the production of polysaccharides from the 0allus, the callus is cultured in a #olid medium such a» ’ en ager medium or in a liquid medium. Oulturing in a 1l4quid medivm is generally more desirshle,

As a bamal medium, Murasige~Skoog medium, Idns- naior-Skoog medium, Fmmborg medium, White medium, Tulecke medium, Nitsch & Nitsch medium, or the like, is employed.

Among these, the most desirable media are Murasige-Skoog ’ medium and Idnsmeder-Skoog mediim,

The kind end concentration of the plemt hormones is related to the productivity of the polyseccharides, The kinds of the plant hormones employed sre mixins such as 2,4-D, NM, IM, and IBA) oytoldnins such os kinetin, BA end 24Py end gibberellins such as gibberellin A (oa, ).

Among these, the independent use of 2,4-D or NAA, or the use 2% af NAA end BA or kinetin in combination, 1s desirable for -T- BAD ORIGINAL

Dt / G obtaining better results. The concentration of hormones 18, 5x 204 = 1 x 1077 ¥, prefersbly 5 x 10™> - % x 10~% for the independent use of 2,4~D or NAA, amd for the com- bined use of NA and BA or ldnetin, the ¥Mde conoen~ tration of 1 x10 = 1 x 1077 M, preferably 1 x 107% -

Bx 1075 M, and the concentration of BA or kinetin of § x 102 ~ 1 x 1077 M, preferably 1 x 107 - 5x 10™7 N, are used,

Glucose, galactose, fructose, mermose, xylose, suorose, rhamose, fucose, starch, or the like, is umed es a ocarbon mource, The kind of parton source employed : does not have any great effect on the production of the polysnocharides, and sucrose is most usually used, Al- though there is no simificmmt relationship between the : 15 concentration of the earbon source and the amount of poly- seocharides produced, the generally desirable concentra~ tion of the oarbon source 18 1 ~ 6%, and pepécially pre- ferably 1s 3 - 5%,

There 48 nov specific limitation to the conditions of the oulturing. It is usually desirable, however, to omrry out the culturing by shake method at a temperature of 20 - 30°C for 15 - 30 days.

Polysaccharides are collected “rom the culture broth : tus obtained by subjeoting the culture broth to centri- fugation or filtration to separate the cells therefrom, oil URIGINAL

LG eft end condensing using a rotary evaporstor or the like,

Ethanol is then edded to the condensate to obbedn a procipitate, which is freeze-dried to give crude poly- saccharide.

The polysaccharides thus preprred have the fol= lowing charncteristion: (1) Outward appeerance: White — groy-vhite (2) Suger content: 70 - 8% (including uronic acid of 20 to 30%) (3) Oonstituting sugars erabinose, galactose, mamose, xylose, snd uromio acid . (4) Protein content: 1 ~ 6% (3) Molecular weights 10,000 - 6,000,000

According to the present invention it is possible to prepere polysaccharides derived from plents, which have heretofore only been produced by extracting ar bep~ . ping from naturally grown plants, by meens of the plemt= tissue ctilture method, This eliminates the requirememt for a large field for preparing the polysaccharides and overcomes the greatest drawback of natural source, amd price fluctuation due to changes in climatic conditions,

Other features of the invention will become sppa- rent in the course of the following description of the eXemplery embodiments which ere given for illustration of 23 the invention and are not intended to be limiting thereof,

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RX AMPLES

Brauole 1

Comparison of productivity of polysaccharides by : liquid culture of the tuberosa callus and the callus of other plemts: (a) Callus Induction:

A bud of tuberoce 2 ~ 7 drys before blossoming was taken, and sterilised in 707 ethanol for 1 mimute and in 1% equeous solution of sodinm hypochlorite for 10 minutes, and then washed with sterilized woter. The sterilived explant wae cut into a sulteble size and inoculated imbo a medium for oallus inductidn, A tuber of m allium was sterilized in the seme nemner, its exodernia wos removed, . and ths tuber was out into pieces of a suitable mise, he 13 cut pieces were inoculated into n mediim for callus ine duction. Tbbacco was nlso sterilized in the mame marmer ap applied to tuberose, and inoculeted inte a medivm for - eallus induction. As for soybenns, the seeds were gteri- liged in 70% pthenol for 1 mimite and in a 1¥™=aqueous so- lutiom of sodium hypochlorite for 20 mimites, snd then : washed with sterilized water. The sterilized seeds were aseptically germinated and left to grow for 10 days until the embryonic axes had grown es high es 1 cm, whereupon the ootyledons ond embryonic rxes were cut from the germ— free-poedings. The cotyledons and embryonic axes were

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Des then inoculated into a medium for osllus induction.

Idnsmad er—Skoog medium contedning 0.87 agar was used for the basal callus induction medium, The plant hormones used ware 10> M NAA as amin, and 109 MBA oytokinin, Suerose.in the mount of 37 besed on the anount of the medium was ~dded as the crrbon source.

After having been mijusted to pH 5.7 by 0.1 N KON, the neddum wns sterilized for 15 mimites at 1.2 atmospheres,

The cultivation wes carried out under irradiation of eleo- trie light at v temperature of 2% & 1°0. Upon oultive- {ion for 30 - 60 days calluses were found to have been

SI induced from each explent, . ~~ (db) Subculture of callus:

Ench oallus induced in (a) above was subcultured 4n the same medium es used for the callus induction under the same conditions. The callue wes translated into a fresh medium once every 30 days. (eo) Idquid cultures

Idquid Shake culture wns carried out using the eallus which had been cultured for 10 generations es in (b) above end the liquid medium having the sme composi- tion as the medium used in the suboulture, Mighty (80) ml of medium was used in each 200 ml Erlenmeyer flask.

Into this medium 2 gn (fresh weight) of ordlus was ino- culated, end was shake—cultured et a rotation of 120 rm -l1l - Brau URIGINAL

7 cfu and at 28 4 1°0 for 30 dogs. (4) Preparation of polysaccharides

Cells were removed from the culture broth ob- tained in (0) above by centrifugation or filtration, end the flltrete was condensed by rotary evaporator, To this condensate was gdded 3 folds in volume of ethemol, and the mixture wes left to stand at 5° Ce for 24 hours to obtain a precipitete, which was collected by means of centrifugation, washed with 70% ethaol, end freesze- dried to eliminate the water therefrom,

In the above procedure, extrecelluler polysao— : cherides were prepared from 4 ldnds of calluses. As LL shown in Table 1, the amount of polysaccharide produced by tuberose callus, 1.91 gn/1/30 days, is mich higher than thet produced by other calluses,

Zable 1

Production amount of extracelluler polymachherides by callusen originated from different plembm

Kind of callus Amount °f Re ener dee produced tuberose : 1,19 tobacco 0.66 soy been 0.05 : gerlie 0.39 ai BAD ORIGINAL

DY

Exmple 2 :

Anount produced by different kinds of plant hommoness

Cultivation of tuberose callus with different kinds of plomrb hormones was conducted. The tuberose anllus prepared in Expmple 1 was trensplmted into liquid cule ture media each comprised of Idinemed er—Skoog medivm,

The Idnsmed er-Skoog media contedned 1075 nw Han 4 106

BA, 1077 I, 2,4-D, 107° M NM, 1070 W IAA, md 10~> M IBAY

The cultivation was carried out under the mame conditions as desoribed in Example l(c). The qumtity of medium used was 30 ml per 100 ml Erlemeyer flask. The polysesccha- ) rides were collected in the seme method as described in © Exemple 1 (a).

As shown in Table 2, the production inoressed in the order of 2,4-D, NAA, NAA + BA, IBA, IAA, Among these, 2,4~

D attained en especially high productivity of 3.60 gm/1/%0 days. - 1% - 1

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Zable 2

Kinds of plant hormones ve, production of poly- saocharides hy tuberose oallises formones 000000000 ev130ee) 00 5M (1072 uw) 2.6%

Im (107 u) 1.01

BA (107% u) 1.28 : 2,4-D (107 x) 3.60 : 10 ¥a (0%) 4m (10% w) 1.91 oo ——————————— Seem — SSA et etme

Goncentrations of 2,4-D end NAA 4 BA, and production of polysaccharides:

Tuberose oallus was ocultirdi in varying congentra- : tions of plant hormones. Tuberose callus prepsred accord- ing to the methods described Fxemple 1 (a) and (b) was transplanted into liquid culture media comprise of Iins- med ex~Skoog medium containing plemt hormones with varying concentrations, i.e. 5 x 1074 40 1 x 107% M for 2,4-, 1x20%t01x20%MtMAmi1x10 M0120 TH of BA for the NAA end BA combination, The media were oul~- . tured under the same conditions as desoribed in Exemple 1 (0). Polysaccharides were collected according $o the pro- cedure as desoribed in Example 1 (d). As seen from Table - -

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Colm fo 3s in oultivation ab concentrations of 2,4-D in the range of 5 x 1074 to 1x 10~6 M, the polysaccharides production is high at the concentrations of 5 x 107 - 8 x 1075 M, and is exceptionally high (3.41 gm/1/50 days) at the concentration of 1 x 1073 M. As for the combined use of NAA and BA, as shown in Table 4, polysaccherides production wes highest at 1 x 104 M NAA $1 x 107° M BA; lable 3

Ooncentration of 2,4-D va, production of polysaccharides by tuberose calluses

Co, Ooncentration Amount of polysaocheride produced no

Cor 24d (0) (g3/% dare) 5 x 1074 1.26 1x10 x 1553 13 5x 100M 2.73 1x20 u 341 5x10 u 1:90 1x09 Cos . 2 OHIGINAL

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Iabla 4

Concentrations of NAA and BA vs, moduotion of polysaccharides by tuberose calluses

Concentration Oonoentration of NAA of BA TT 1x08 1x120% 1x10%u 1x10 3.27 1.68 0:38 1x10 u 2.53 1390 0.78 1x10" Tu 2.14 2.11 0551 ta nn AA Aer Pete SO a0 eS

Mount of polysaccharide profuced (en/1 /30 days) oo J PRE Obviously, mmerous modifications and variations of the present invention sre possible in light of the above teachings. It is therefore to be understood that the scope of the eppended claims, the invention may be 1s practiced otherwise then as specifically described herein. -16 - i ; LGINAL

Claims (2)

2G ye OLAIMS;

1. A process for prejaring an acidic heteropoly- saocharide comprising culturing a callus induced from a plemt beloning to the gems Polianthese I, in a culture medium containing one or more plant hormones and colleot- ing the acidic heteropolysecoheride from the culture broth,

2. "The process for preparing a polyseccharide according $0 Claim 1, wherein the plant belonging to the : genus Polisnthes I. is Polienthes tuberoma I. Xe The process for preparing sn acidic heteropoly- : saocharide according to (leim 1, wherein the acidic hetero- CT . polysaccharide comprises as its structural units arabinose, galactose, mannose, xyloss, and uromie acid, KAZUYA OTSUJY YASUKI HONDA KIXUHIKO OKAMOTO nventors -17 - } SINAL

- . nae amend