IW‘ I w I ) I I 1 | W” I WI 137 088 TH . A QUANTITATIVE STUDY OF WATER SOLUBLE AND ALCOHOL SOLUBLE CARBOHYDRATES IN BRDME GRASS {BROMUS INERMIS) 3N R‘ELATLON TO VARIOUS DRYING METHODS FOR ”5 PRESERVATMN flash for the Degree of M. 5. MICHIGAN STATE COLLEGE Marta Tyse 1948 This is to certify that the thesis entitled A @an 757475 ye $7on 9L Ida/er Ja/aA/p 4/74 Aka/ml grab”? @:Aa£7 «Ire/N ”7 8rd»)? vafidn I'll Var/all J 9727:: (W Dry/n7 ”If! «(.1 f0 rpm/Sgt!“ bgPI’R-rerm f/ a); M% has been accepted towards fulfillment of the requirements for 0 M M Major professor Date 2011/ /L‘IA/’€ggf\ M-796 n n" "m m run”? v '1 r r n ‘) 3 TT" '3 t?‘3 ' A R~,AA~...LIi AAA-ml mrJo i H IiJLELJ'L 31.] .7 JLJI‘C: LCII ‘\ T7“ v‘ )\ VV‘r‘u -’ fifif‘ ..v 1-5 I - —--1 H \ ,‘fl ) I a “7"!“ an ‘ .l" 1-H" T“ SQ It- A I 1 \ ILA»; ._ J «.1; .1!) 1 2‘ LL. “1 J ukklj') ‘\ UL J I .31 n. L) .L - .LLJ m r k ." "tTTC' . ‘f‘T""?‘r' ‘. "“1 ’1 f" f! 'fi \ 3 (It!) T‘=".“|’:‘ f‘l-T 4x 'I‘. "Lb- -) D..L.LlL,‘J , 1”.ij (CD. I '.O Jimburn \'a:!TA1’I\ ' 73"? “d -L- f 1' 31"» -JJ 8 e T '7‘“ m ‘r(‘ .L‘L -AilJa—n) "“ ‘ r . 4“" I ’0 \ 4‘" ' w .7 ’ ‘ 1 ‘fi 'V‘ ,- 1" L‘. 1'7 : x,’l ,".‘ ‘- . r- s '\ 0-10-flltbex1tb -1,iJ \)C LC/C/J- C- .L'L‘..l”.8.b‘3 x)‘J1—L-L..es (,1. J.C:L| fill; n" “ fl ' 1 " “a "i .“ . ‘ 1 '- . " ‘\ " " fi‘ . p 1“ . ‘ QQqu Loire e or Agricundnre cal “‘sllel ”Glance O .1- -. . ‘0. :1“ C 1n rartrgl -a lillmen f requlrements OF I‘ 090 »r~ lCQL ) 11 ~mT 4,-5‘-’ C" awm- u... ' 7:53“ / ~ / ‘7 ’7“ ‘f C? 4' TP1YC'rrI Vr“ IV“ "'1‘“ v,“ AC1. . '0! .JAJ u. «L J. I want to express my sincere thanks to Professor C. D. 7all, for his help and criticism of the work leading up to this thesis, and of the thesis itself. I also Association it possible give my great appreciation to the.American of University‘fionen, whose Fellowship made for me to study at an American College. --Yarta Tyse *********** **$****** ***$*** ***** *** 4: TAQTE Cy CCNT'E"':"S Ivm 7""?! h‘T .L 10:); 'LCLI _-ooo00000000000000.0000. U om .~~~ -LIQ-O.Ll\/A“ALQOooooooooooooooooooooooo 1“,? P“ " ‘T'1 LJI-h JAL .11“ A 1) 2) 3) 4) -314 P-LCCHDVTAL............. Sampling of materials...... Sater extraction........... Ethanol extraction......... Carbohydrate determination. 3:3 Us"ICU 4..OOOCOOOOOOOOOCOOOOOOOOO ESTI’T‘leCllf ?ISLICT: *3 CC‘TCL.-S.L CISoooooooooooo 3 ~ ")Urv LC; J-ooooooooooooooooooooooo ll 12 13 IETRCDUCTICN For several years research workers at the Department of Agricul- tural Chemistry at Hichigan State College have studied different methods of determining carbohydrates in fresh plant material (1). They found that for many of the common creps the amount of hydro- lyzable carbohydrates was the same whether water or 80% ethanol (which is the commonly used extraction solvent) was used for the carbo- hydrate extraction. Young brome grass, however, was one of the except- ions. The amount and type of carbohydrates - reducing and nonreducing - obtained from this tissue appeared to be greatly influenced, both by the solvent used and by the hydrolytic agent. The highest total sugar values were obtained when water was used as the extraction medium and hydrochloric acid as the hydrolytic agent. The water extract hydrolyzed with invertase or the alcoholic extract hydrolyzed with invertase both gave considerably lower values. The variations obtained with brome grass showed a complexity likely to be encountered in the determination of carbohydrates in crop plants. These authors emphasize the need for sufficient preliminary work with each different crop to insure that the values obtained with a given analytical procedure actually represent the sugar content of the tissue. In the work for this thesis a comparative study has been made of the amounts of carbohydrates obtained by water extraction and BOfl ethanol extraction of brome grass. The extracts were obtained both from fresh material and from dried samples of the grass. HISTORICAL According to Miller (2) the principle carbohydrates in green plants can be divided into two groups: monosaccharides l)sugars disaccharides trisaccharides and celluloses starch amyloses dextrins or levulosans hexosans starches 2)non sugars-{polysaccharides mannosans galactosans entosans araban xylan The levulosans or fructosans which are polymers of fructose occur commonly in monocotyledons (5). They may be found as the inulin type which is insoluble in water and where the fructofuranose units are linked together in glycosidic linkages through their carbon atoms 1 and 2 (4); or they may occur as in the water soluble fructosan group where the glycosidic linkage is between the carbon atoms 2 and 6 (4). In many of the common grains and grass species there has been shown the presence of the fructosan group - brome grass being one in which rather large amounts have been found. The naturally occuring fractosans have been studied very little in comparison to studies made on other carbohydrates. Inulin was dis- covered in 1805, and about 1870 - 1900 a number of other fructose polymers were discovered in the monocotyledons (3). They were very water soluble, and thus could be distinguished from the inulin group which was only slightly water soluble. For the water soluble carbohydrates the term fructosans has been employed, or levolusans. As a rule inulin is not included when using the term fructosans. The early Workers reported fructosans present in barley, rye, wheat and many other plants (3). They were found in a number of grass rhizomes,. in stems of rye, in unripe grains of barley, wheat and rye, and in leaves of Yucca filamentosa (5); the occurrence, therefore, was not limited to special parts of the plants. After the early years of discovery, the significance of these l-rotatory fructosans was over- looked, and the interest did not arise again until twenty years later. The early Workers had been German and Swedish; the modern work has been done mainly in France and German‘, but also in America, England and other countries. Of recent interest are the studies of Colin (3) and his collabora- tors and of other workers (5) who studied the distribution of fructo- sans in cereals, grasses and other plants, and discussed their signifi- cance in the general carbohydrate metabolism of the plants, and the inter relationships of the several fructosans. Archbold (8) and Barnell (7) have investigated the distribution and seasonal fluctuation in wheat and barley respectively, whereas Russel (8) has studied the effect of various mineral deficiencies on the fructosan metabolism in barley. Because fructosans have been found distributed throughout the plant, the earlier concept that they served mainly as a reserve carbohydrate has been changed. No certain evidence has been available as to the fate of the frucosans, whether they are re-utilised in respiration, or used in polysaccharide or even protein synthesis. at the present time it can be said that fructosans may be in part the precursor of starch, and that they are secondary products of sugar changes. The exact constitution of the fructosans is a matter for fur- ther research. It seems that the majority of isolated compounds differ only slightly from each other. Fructosans may also result from the activity of bacteria and from mold spores when these latter act upon sucrose (9). Hestrin and Avineri-Shapiro have even suggested a mechanism for levulosan produc- tion from sucrose and raffinose by enzymes from the autolyzate from A. 1evanicum.(lO). In some cases the product of bacterial activity seems to be identical with some of the fructosans found in plants. As already mentioned the fructosans have been found in brome grass. deCugnac (11) in 1951 reported that Bromus sterile contained fructosans. In 1941 workers at Iowa State College (12) reported that they had found Z-Bfi in Bromus Inermis. These findings are in agreement with the results obtained at Nichigan State College (1) where the workers stated that apparently the variations in the results they obtained were due to the fact that brome grass contained sub- stances which were more soluble in water than in 80$ ethanol and yielded reducing sugars more readily when hydrolysed with acid than with invertase. These compounds are probably mainly fructosans. -4- 711' q .3 1 TT"T"Tm P L 131,} .C‘l“? 1"") 7’1 J4“- JJJA ‘bI. JJJ.‘ LIX ‘ a .- LC m‘LDLI -:L._.J -- »_~_.’_ The experimental work was planned as a quantitative study of the water soluble and water insoluble carbohydrates. The work was divided into four parts: ) sampling of material, ) preservation of material, ) ) extraction, determination of the carbohydrates. PPCNNii—J Sampling of materials Samples were taken five times during the spring and summer of 1948; Tay 17th, June lst, June 17th, July lst, and August 15th. The samples :1 (D ’1 CD 93 I.) .._l (+- p 'F‘" (D :3 93 rt (1' 2 O O O 1-4 O O '5." I...) a :5 (1- D“ (D 93 to d‘ (D ’1 :3 O O :3 m E? 1..“ E D" (D :5 Cf- {3‘ 0 sun was shining—~altnough the 17th of hay was a partly cloudy day. The field in which the samples were taken belonged to the college and was not used for pasture. There were, therefore, no animals grazing and thus the grass was allowed to grow undisturbed. The samples were out each time with a pair of scissors and were clipped about 10 cm. from the ground. The material was scattered over an area of about 250 square meters. The first two and the last samplingS'were taken from the same section, whereas the third and fourth samplings were taken on a similar lot about 50 meters away from the first one, but in the same field. The reason for changing places, was that the grass had been cut in the section where the first and second samplings had been taken. The fifth samples contained grass of second growth. Before subjected to any treatment, the dead grass and diseased straws and the weeds were removed in order to get the samples as even as possible. A description of the samples is given in Table I. 1‘1!le I o 37""L3IPTICTT CF ° ”'PIFS Sample 1*0. Sampling Date Length of Cut Grass f'oisture Content DJ 00 6-17-48 40-60 cm. 15-30 cm. Rust spots C ommon . Part of grass in bloom. Rust common. Crass in bloom Rust common Part of grass in bloom; part through blooming. dust common, Second crop. Growth very uneven due to drought. Rust common . 6‘ . 5‘27, 67.87fi The grass was first divided into five parts of apprcx'mately the same size, respectively A, B, C, D, E, and were subjected to treatments as described in the following paragraphs. Part A. The grass was cut as finely as possible with a pair of scissors and four 50 g. samples were weighed out, two for aqueous extraction and two for alcoholic extraction. At the same time four 10 g. samples were weighed out for moisture determinations. All the moisture determinations during the experiment were made with a Yrabender Semi—automatic Toisture Tester. The combined re- sults are she?“ in Table II. TABIE II. arc-73w“: ? 4.. ULLJ CCNTEFT IN BRCVE CdASS (Bromus inermis) _ . Fresh Sample Sample Sample Sample Sampling Sample dried at dried at dried dried Data 100° c. 450 0. inside outside Woisture percentage 5-17—48 75.40 6.88 6.38 11.13 14.13 6-1-48 75.70 10.20 5.52 16.63 17.10 6-17-48 £5.99 4.16 6.13 15.26 15.10 7-1-46 60.93 4.75 8.62 9.70 9.60 6-15-42 67.67 6.55 5.25 10.70 10.83 Four samples were used when the material was fresh, and three W‘ " 1 d b e ' 1r " t d t xp rimental drVina and when it 1a eon prev1ous 5 suogec e o e Le ‘ v v .rinding. The loss of moisture was considered complete when three fJ succeeding readings taken fifteen minutes apart gave the same values in the tester. The two samples for water extraction were put in separate 600 ml. beakers containing 300 ml. boiling water and lg. of CaCO5 - for neu- tralization of the extract - and boiled for fifteen minutes. There- after most of the water was decanted and the grass and remaining water poured into a flaring Blender fitted with a screw lid. The sam- ple was then blended for nine minutes as recommended by waldron etal. (1). The blended material was poured onto a No. 65 Buechner funnel containing a layer of paper pulp, about one cm. thick. The material was washed by suction with five hundred m1. of warm water. The fil- trate was transferred to a one liter volumetric flask and cooled. Five m1. of saturated neutral lead-acetate were added and the extract was brought up to volume with distilled water. Excess lead was removed with anhydrous potassium oxalate, the solution was filter- ed and Toluene added. The samples for alcoholic extraction were added to 300 ml.-of boiling ethanol, containing 1 g. of CaCOS and enough water - the water contained in the sample being taken into consideration - to I make the final concentration 8 h. The sample was extracted by heat- ing on the steam bath for half an hour. The ethanol extract was decanted into a liter beaker, and the extraction repeated using 300 ml. fresh 8%? ethanol. The extracts were combined. The residue from the preliminary ethanol extraction was dried, first over night at 500 C., then on the following day for eight hours in a vacuum oven at 80° C. The dry residue was ground in a 'Wiley cutting mill, Yo 8-338A, with a capacity of 1725 revolutions per minute, and fitted with a No 20 mesh sieve. The ground residue was transferred to a paper extraction thimble (33 x 94 mm.) which in turn was placed in a Soxhlet extraction apparatus. Two hundred ml. of 805 ethanol and several glassbeads were placed in the extrac- tion flask, and the Soxhlet extraction proceeded until the extract was colorless, 8-10 hours. The extract was combined with the one previously obtained. The combined alcoholic extracts were evapor- ated on a steam bath where the air was brought in circulation with an electric fan. The extract left after the ethanol had been evaporated off was filtered into a 500 ml. volumetric flask, the resi- due washed and the washingS'were added to the filtrate. Five ml. of saturated neutral lead acetate were added to the filtrate which was then cooled and brought up to volume. Excess lead acetate was re- moved with anhydrous potassium oxalate. The solution was filtered and Toluene was added. The alcohol was evaporated off the residue left after the alcoholic extraction. The residue was then extracted three times by digestion in a boiling water bath with 100 ml. of boiling water each -10- time. The extracts were decanted through a fluted 16.5 cm. filter into a 500 m1. volumetric flask, and after the third extraction the residue was added to the filter and washed with boiling water until the volume was nearly up to mark. Four m1. of neutral lead-acetate were then added; the extract cooled and brought up to volume. Excess lead was removed by solid anhydrous potassium oxalate, and the precipitate was filtered off. Toluene was added. Aliquots for fur- ther sugar determinations were taken from.these solutions. Part B. The grass for part B was dried for two hours in an oven at 100° C. The oven was then turned off and the grass remained in there over night. The grass was then ground in an electrical Wiley cutting mill, Ho 8-337, with a capacity of 1725 revolutions per minute and fitted with a 2 mm. mesh sieve. The ground grass was kept in a glass jar with screw top and kept out of sunlight. When sugars were to be determined, four 10 g. samples were weighed out, two for aqueous extraction and two for alcoholic extraction. The moisture was deter- mined in the Brabender Toisture Tester, as already described. The extractions were carried on as described in the following paragraphs. Water extraction. Ten grams of the dried material and 1 g. of CaCC3 were trans— ferred to a 600 ml. beaker and extracted with three 200 ml. portions of boiling water for half an hour each time. The water was decanted -11.. through a fluted 18.5 cm. ho. 12 filter into a one 1. volumetric flask. After the third extraction the residue was transferred to the filter and washed with 300 m1. boiling water. From this stage on, the extract was cooled, lead acetate etc., was added as described in Part A. Ethanol extraction. The ground grass was transferred to 33 x 94 mm. ether extract- ion thimbles. Preliminary extractions removing most of the colored material were made by adding successively three 100 ml. portions of boiling 80$ ethanol into the thimbles. The thimbles were kept in 160 ml. narrow lipless beakers, and each extraction was continued for 30 minutes on the steam bath. The extracts were combined into a 1. beaker. The grass was transferred to Soxhlet extractors and extracted until a colorless extract was obtained, that is from 3 to 6 hours. This extract was combined with the preceeding ethanol extracts; the alcohol was evaporated off and the procedure followed as described under Part A. The subsequent extraction following the ethanol extraction was also followed as described under Part A. Part C. The samples were dried for three days in a dark room at tempera- ture varying from 42° - 500 C., averaging 450 C. The grass was ground and extracted as described in Part B. -12.. The grass in Part D was dried in the laboratory by spreading the grass out on a table top. It was placed just inside a window towards the west and got only the evening sun. After fourteen days it was ground and handled as in Part B. Part E. The grass in E was dried outside in a bundle for seven days. It was placed in a tree towards the south wall of the chemistry laboratory where it was exposed to the sun, and had little protection from rain. After one week it was taken inside and dried at room tem- perature for seven more days. It was then handled like that in Part B. Carbohydrate determination. Total extractable and hydrolyzable carbohydrates were determined by the same procedure for all samples. In order to obtain these carbohydrates, 100 m1. aliquots of the respective solutions were either hydrolyzed with acid or in a few cases hydrolysis was accomp- lished with invertase as the catalyst. (13). The invertase was a commercial product made by Difco Laboratories, Detroit. Five ml. of the invertase solution would complete inversion of sucrose within one hour at room temperature, at a p3 4.4 to 4.6. For each sample analyzed 0.5 m1. invertase solution was used. Its activity was first tested by its ability to hydrolyse a standard solution of sucrose. The hydrolytic ability was also tested subse- quently in recovery tests where 25 ml. of standard sucrose solution were added to 50 ml. aliquots of the extracted carbohydrate solution, as shown in Table III. The recovery tests showed too low values for the sucrose present, whereas analysis of the standard solution gave , + the same amount sucrose as added - .25 mg. -14.. |€ 3‘3 r,” VJ H E: H H 14 "‘7“ 1'" " n :‘ -‘ r1 ' .' ’ ffl'fiffir"! '1; WIT ‘I Y "1 ‘, “ ,‘, , CITY I‘; . 7‘ ’V " ‘r‘rr "r \‘VT. P ”H" “ ‘_" 41-; er-MT‘JLD .J JLCLI I: DY 111:) 1. L11-) (4..“ bin. rgrxl‘) u)’.'C:-‘.CSsz bk‘L-VI PlC'l‘. , 11' D .111! -‘- . " ‘_.1;i:(’;<:rrsn' Tiisfs . ° Aliquot equivalent to Aliquot containing orig. carbohydrates - 12.5 mg. sucrose. hr. ‘, ‘ "’."‘ 1 dL) 7.1;“). SL'xk/i CS "'3. 5......— ._-.~.‘. .— -—— o-—-q —— -—.-..‘ ml. “aranfl 7?. Lu. Sucrose m1. fa 3,0 m1.VanS 0. Cu.red. Sucrose used. 2 4 ’ reiuced. content used. 2 used 4 2 6 after present found. when no sucrose in aliq. sucrose add. found. added. 7.79 51.44 24.7 16.94 13.02 24.96 11.9 25.2 10.01 6.22 23.36 11.1 m 0 C) (D (.31 m 0 U1 O 10.0 5.69 24.00 11.4 0.1044? Ya,§203 used for the titrations. 4 . -15— The final reducing sugar determination was made on 50 ml. aliquots according to the method of Vunson and Walker (13), and the volumetric Sodium thiosulfate titration (13). Data obtained in these experiments are given in Tables IV - VIII. The data are shown graphically in Figures 2-7. In Figure l, the treatment of the plant material is shown graphically. Two alcoholic extractions and two water extractions were made from each sample. According to Figure 1, the first data in the first column in Tables IV - VIII represents the average titration values for l-TaZSZC3 used in the determination of reduced copper in aliquots a and a' (Figure l). 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H.me mwm.HH m¢.ow «0.0H m.¢mH m5m.@ em.om mam.o H35 0 o m.0mH mm©.5 mHH mon.¢H v.5mH moo m.wHH mm¢.5 mwaH o o m.mmH oH.m oHH mo.vH c.mmH 1¢N.w H.wHH mmm.5 5Hno mm.HH mn5. m.m¢H mum.m o.¢mH www.mH o.mmH ma5. e Q:H mmm.5 mme 5m.m mm. w.an mm.m w.¢mH www.mH ¢.m¢H am. e m.0mH am.5 Hum ¢¢.mH ¢N.H* H.w¢H ma. 5¢.mw mm.m 0.05H mm5 m.mHH mmm.5 mon HH.Hm m¢.H* ¢.mvH mm.m* 5¢.m¢ mm.m m.55H m5 5.moH mmm.5 5H m H m H m H m H m H .fiomAHopwhs «How use empoahpxo ampaa .mm.o op.am.eLL< .m L op.em.eLHa .m mm.c op.em.eLH« .mm.o op.em.eLL< mpao m%~ osvpmom wom5Hoaw53 wHo< vom5Hopu5g.co: :0 m5~wpw5clwpofi vothopu5g no: quHoEdw .mxbhdmmm:mc .20 OML Edw QMH mwmmo m.Lo¢s ho wmeH mHm¢e ma 9. Bmflewro E33 -21.. I" Firure 14 Treatment of Sanole during the Ahalytical Procedure . DeterninatiOL of carbohydrates: a ted. carbohydr. 'd o .L 3 g a' Red. carbohydr. 7H 4,.) b hydrolgsis b1 Total carbohydr. l . b' hydrolysis b'1 Total carbohydr, .p g a Reducing carbohydr. H 8 H4 a' Reducing carbohydr. O ,c O Q 51 <1 h hydrolysis bl Total carbohydr. Zesidue b' hgirolgsis h'I Total carbohydr. Eater extr. following the ethanol 0 h;drolg:is C. Total carbohydr. . I extraction. Cl hgdrolgsis c'l Tctal carhohydr. a, a' etc. are aliquots from the same extract. ~ r.fi "V’T'If1_’ " V" 3.1.:)( "“ '1: (T Samolingx Jhen working with biological material the selection of representa- tive samples which show significant values for the whole is always a problem. In order to obtain such samples, the grass was taken in scat- tered places over a relative large field, chopped up, and mixed. Other procedures have also been recommended such as selecting a small lot, (one square meter), in the field and cutting all the grass on that lot and mixing it. Samples taken from that mixture would then be assumed to represent the whole field (14). The first procedure was chosen be- cause it was considered the more practical. The grass had to be taken many times during the summer at different growth stages and if a smaller lot were used, a new one would have to be selected and measured out each time. fihen the grass was taken from a large lot, however, the same lot could be used the whole summer. Effects caused by local soil and moisture conditions might also be more correctly eliminated when the sampling field is large. There are definite relations, for example, between nitrogen fertilization of the soil and carbohydrate content in plants as studied by Kraus and Kraybill (15). Archbcld (6) has found similar results for fructosans in studies of these compounds in barley, whereas Russel (8) has shown that potassium and phosphorus also affect the fructosan content in barley. As the composition of plants have daily fluctuations varying with light and temperature (2), the grass was -25- always picked under as constant climatic conditions as possible. Data showing climatic conditions were obtained from the U. S. Department of Commerce, Weather Rureau at East lansing and are shown in Table IX. -24- TddLE IX. CLI"‘TATIC COV'DITICITS 1.17753” SA"PLL‘JS TM’EV. Sampling Temperature 'Wind Percentage Average Date Direction of possible cloudiness Wax. Win. Aver. 2 P.M. sunshine Scale 1—10 5-17-48 67 49 58 62 If 49 8 6-1-48 81 48 64 76 NW 100 5 6-17-48 73 4B 60 70 N 72 5 7-1-48 73 48 60 65 PM 98 1 8-15—48 82 54 68 79 I?! 100 4 Vixing and Drying of Samples. Other problems are also involved when handling plant material for quantitative analyses. Cne difficulty when using fresh material is to obtain a well mixed sample so that a small sample is represen- tative. As the material is succulent, it can not be chopped very fine. When samples of mature grass are taken many variations arise because tissue from relative few plants will be analysed. As a rule, however, when similar samples were taken the variations between the samples were not too great. It is often more convenient to have the material for analyses in a dry state. The samples can then be kept stored for a long period of time before being analysed, and it can be very finely ground which permits a thorough mixing, thus giving more homogenous result when analysed later. A series of analyses were done on grass dried at 1000 C. in order to inactivate most of the enzymes as recommended by Yorman (14). Drying methods in which the conditions for enzymatic activity were 0 o favorable were also employed, such as drying at 45 -50 C. Drying under conditions similar to those sometimes used for the preservation of hay was also carried out. The data found for the different drying methods are given in Tables IV-TIII. Enzymatic activity and extraction. The main difficulty which occurs when fresh material and aqueous exteactions are used is caused by enzymatic activity which constantly changes the composition of the material. In order to get a true picture of the original composition either (a) the analysis should be done so quickly that the enzymes will not have time to alter the composition of the tissue to any extent -- this RRS done when fresh material was used for the analysis -- or (b) the enzymes must be des- troyed before they have time to act -- as was done when the material lriel at 10:0 r. \ 7.1: U. U) .Jher ,x‘lircwi et al. (1) Lxrbiined fits hici;est valites in.ifi:eir cama- bchyirate determinations, they used aqueous extraction for fresh tissue. It is obvious that enzymes within the plant extract will easily be able to act in such a solution, and it is difficult to get reliable results. Browne (16) has recommended the use of ice cold water for the extrac- tion, and the maintenance of the extract in an ice bath in order to inhibit enzymatic activity. Vollroy (17), has, however, recommended hot water extraction as the most satisfactory method. Research workers at Iowa State College (12) when determining fructosans of plants ex- tracted the carbohydrates with boiling water in a boiling water bath. Forman (14) found that for rye grass one hour extraction with hot water, gave approximately the same values as 48 hours of cold aqueous extraction. The determinations done at Iowa State College and by Norman were all done on dried material. In the present work, however, hot water was employed also for fresh material. Boiling the tissues as done in the present experiment would des- troy some enzymes, but probably not sucrase (invertase) which accord- ing to Platolenko (18) needs high temperature for destruction. -2 7.. The latter worker found that the sucrase activity of geranium leaves was highest after drying at 850 C., and complete destruction only occurred after drying at 150° C. The main reason for boiling the material as done in the present work was, therefore, not to destroy the enzymes, but to get a quick extraction and also to make the tis- sues easier to handle in the flaring Blendor. waldron et al. (1) did not boil the tissues previous to the blending, therefore the method reported in this thesis is thus slightly different. When filtering the extract after the blending those authors (1) used filter aid in order to get the extract through the filter; when paper pulp was used instead of filter paper in this work the filter aid could be eliminated. It was inconvenient to add any enzyme inhibitors such as heavy metal ions e.g. Ag+, Cu+ +, or Vg+ +, since these ions would have to be removed before determination of the carbohydrates, or they would interfere with the results. lead ions, which usually "poison" enzymes, were added, but they probably had li tle inhibitory effect on the invertase as lead acetate is commonly used to precipitate other proteins when invertase is prepared. Bryan et al. (19) employed 0.2% "fTaZCO3 solution when extracting plant tissue, and claimed that the enzymatic hydrolysis was inhibited. The CaCO3 that was added to the first extraction water in the present work might have had a similar effect, because the extract was neltralized and invertase acts best in a medium at a pE-I about 4.5. When aqueous extraction was employed it was necessary to know whether or not the extraction was complete using the procedure des- cribed previously under Experimental Procedure. The residues of two extracted samples were tested for reducing and easily hydrolyzable carbohydrates. Since no carbohydrates were found to be present the rest of the samples were extracted under the same conditions without further testing. When alcoholic extraction was used, the extraction was continued until the extract was colorless. The alcohol soluble chlorophyll was thereby extracted and the alcohol soluble carbohydrates were supposedly extracted as well (13). Separation of alcohol soluble and water soluble carbohydrates. Both alcohol extraction and water extraction were employed in an attempt to separate the alcohol soluble carbohydrates from the fructo- sans which are soluble in water but only slightly in ethanol. The separation was not very successful, however, because some of the fructosans are so soluble in water that they will dissolve even in a solution containing EOfi of ethanol. The alcohol extract was sup- posed not to contain any fructosans, but when hydrolysed with dilute acid, the amount of reducing sugars obtained was larger than when invertase solution was used for hydrolysis. This showed that the alcoholic extract contained some of the fructosans not very suscept- ible to enzymatic hydrolysis. These facts are evidenced by the data in Table X. Some separation was possible, however. It was interesting to notice that the part that would easily dissolve in water but not in 80% ethanol, gave no reducing sugars upon hydrolysis with inver- tase. The amount obtained by aqueous extraction following the alcoholic extraction kept very constant during the summer, whereas the total amount of carbohydrates would undergo rather large changes. When the grass had been dried slowly before extraction, so that the enzymes had converted most of the soluble carbohydrates to their reducing form, the sanples usually contained approximately the same amount of alcohol insoluble but water soluble carbohydrates in the non reducing form as had been found for fresh material. In a few cases, when the grass had been stored and had a high moisture content, the alcohol insoluble and water soluble carbohydrates also had been hydrolysed. Determination of fructosans. According to Archbold, the quantitative determination of fructo- sans resolves itself into the estimation of the fructose obtained by hydrolysis with weak acid, not stronger than N 5. Barnell (7) used N/E, and Official and Tentative Vethods of Analysis (13) recom- mends Y HCl. The procedure followed in this work is that described in Cfficial and Tentative ”ethods of Analysis. It is obvious that whether 7/5 or N acid is used the hydrolysis will also include the hydrolysis of the sucrose present in the extract, since it has not been possible to make a complete separation of the two groups. The true amount of fructosans will not be obtained if it is calculated -30- on the basis of difference between reducing power before and after hydrolysis. A possibly better way to estimate the two fractions is to use the separate action of invertase, to hydrolyse the sucrose, and to use acid for hydrolysis of sucrose and fructosans. The amount of fructosans can then be calculated from these data. Data as an example of this method are shown in Table X. Fven this separa- tion is not entirely complete, since some of the fructosans are slowly hydrolyzable by invertase action (3), and thus the amount of sucrose would be over estimated, whereas the amount of fructosan would be underestimated. ? wever, this is a method that has been widely used by French workers (5). -31- dEDWCIVG CAAECVYIZATES 2?le"33 CT ACID TYURCLYSIS, CC"PARED TC TUE AVC”“TS CBTAITED WIT? ET?Y”3 KYlifilYSIS. 1) 2 :7) Sampling Drying and Date Extraction Acid Tydrol Invertase Hrdrol m1. 0.1044? mg Cu per ml. 0.1044K mg Cu per Fa93903 g dry grass Nags 03 g dry grass usedu reduced used reduced 7-1-49 Drying 45° c 12.54 122.5 7.55 111. alc. extr. 12.25 178.5 8.59 125.2 7-1-45 Drying 10000 19 226.5 5.04 140.5 7-1-45 Drying 10000 14.155 137.0 7.745 97.9 alc. extr. 13.06 04.9 -——— _--_ 8-15-11p iresh 2.61 208.3 7.71 127.5 HZ“ extr. 12.78 211.2 7.77 12r.4 E-l5-4E Fresh 23.21 16 .2 13.02 107.5 alc. extr. 23.77 123.9 12.42 132.6 6-15-46 Drying 49° C 6.00 192.6 3.60 103.7 H20 extr. 6.60 162.8 4.04 112.9 8-15-46 Drying 49° C 10 20 142.4 7.06 98.79 alc. extr. 9.59 133.9 7.28 101.6 8-15-48 Drying 100°C 6.55 185.4 2.95 63.47 $20 extr. 6.36 160.0 2.f0 70 40 4-15-49 Drying 10000 9.55 136.2 5.22 86.22 alc. extr. 9.07 141.1 5.60 70.11 l) The aliq. for the water ext. rep. a sample twice that of the aliq. for the alc. extr. (\5 \J Each data in these columns is the average for two aliquots. Another way of differentiating between fructosans and sucrose is to separately determine the increase in glucose and fructose due to hydrolysis. Sucrose can be calculated as the double amount of glucose set free, and fructosans as the difference in increase be- tween fructose and glucose. Glucose may, however, occur without being derived from the sucrose and thus errors be irtroduced. The interference of glucose might be eliminated by use of the colorimetric ex determination of fructosans in plant materials as described by .cxary Carbohydrate determination. I 3 In the determination of carbohydrates in plants VcIlroy (1?) recommends the ‘Unson and Walker method for macro determinations, whereas a modified method of Shaffer-Somogyi (17) is described for micro analysis. The Cu-reducing method of Vunson and Walker, follow- ed by thiosulfate titration which was used in this work has proved to be satisfactory and well suited for plant material by several workers. Some recent results obtained by Waldron et a1. (1) with this method as ‘ compared to other commonly used procedures are shown in Table XI. The Tunson and walker method is not ideal, but as in all sugar veter- minations an emperical method must be used. The principle of this procedure is the reduction of Cull to Cu* in an alkaline medium. Due to the complex reactions that occur when sugars are oxidized in alka— line solution, the amount of Cu reduced will not be in stoichiometric proportions to the amount of sugars present. Thus the first ten milligrams of sugars will have greater reducing power than the next ten, and these will reduce more than the following ten etc. Other factors t‘lat have to be taken into consideration according to inves- tigations made by many workers (21) are: concentration of alkali, oxidation reduction potential, temperature time, shape of vessel, etc., which all will influence the amount 01 I5 ()0 I)" H VVVVV of reduced Cu. She 'unson and alker n.ethod employed specific condi- tions as con stant as possible. Determinations both with standard sugar solutions and aliquots from the samples showed that the results could be .uplicated u.ith in a sma all range of error. One should recog- nize, however, that when working with extracts the results obtained might be influenced by compoundso other than sugars present in the solution. When strongly colored extracts were used in this experiment there was a tendency for some of the Cu to be reduced to some slightly orange colored compounds instead of red CuZC. It is possible that in this case some of the copper was filtered through the funnel in a col- loida 1 suspension, thus resulting in lower values. It is also pos— sible that the extract contained copper reducing compounds other than sugars that had not seen removed when the proteins were precipitated with lead acetate. ”any workers have been aware of the fact that lead acetate probably does not remove all copper reducing compounds (other tna n sugars) completely. Various substances have been suggested as a substitute for lea acetate. ”cllroy (17)'recomnends a mixture of Cadmium sulfate solution and VaCH solution as a clarifying agent to be superior to lead acetate. ‘ 0 h 1 H 11 ireift .— ‘.~.‘ "19“, J- ,4.“ hr . ”ethod es repres ained by fialdron et "2 J- v“ T.’A"lT f1" A31 ‘ff‘,TT"‘{! '..J‘_ #13 -A.LJ‘. Li‘ ‘J m ' "~ 1"] v‘I I ‘ n 1‘ I lip.) :14 4.. v‘ " ,\ y --_‘ '1 i 4 L V. k 1'; .L x; f“ v.‘ ‘7‘ \J .g.‘ ’ _ ’3 '-LJ-). 91. (1) using four commonly employed enting percentage carbohydrate found per 3. sample, ht). “urson Halker Tunson Walker Shaffer Hassid 'Yolumetric dartnann Ceric Cravimetric thiosulfate Sulfate A —‘-7— In -— rm In A In A E [\5 O [\5 }_l 1...] o ‘4 [\3 1.66 1.82 1.47 1.13 1. 77 1.47 'l.24 1.70 1.35 I = II : III: acid Hydrolysis. invertase Hydrolysis. Marin: Haring Soxhlet extraction, EOfl ethanol as solvent. Tlendor , I ‘1' 1. .L 20 as solvent. Blendor SOS ethanol as solvent. Determination of cuprous copper. .-.-_ The amount of copper reduced can be calculated from the titra- tion values obtained and normality of TaZSZCS used in the titrations as shown in the following formula: 1) ml ?aSO used x f x 63.57 : mg copper reduced in the aliquot II) The number of milligrams of copper reduced per gram of dry material is calculated as follows: mg Cu x V _ x 17199 - vi, milligrams copper reduced per gram sample. 100 mg copper : amount of copper reduced, calculated according to formula I) V = volute of solution from which aliquot is taken. weight of sample used. (. . 'l “ A = volume of aliquot used. T : moisture percentage of sample used. Example: Fresh sample picked 5-17-48 contained 79.4f moisture and gave the titration value 4.56 for non hydrolysed water extract. The amount oi copper reduced per gram of dry weight is: I) 4.56 x 0.1o39 x 63.57 = 50.11 mg. cu. T (W f‘f‘ - . . 1+) 5J‘11 x liio = 58.45 mg Cu reduced per gram dry weight. 53 x 50 x (loo—79.4) 100 This method of calculations is a convenience but introduces an error ‘ o o 1 1 . l as Siown 1n the follow1ng paragraphs. In tne example cnosen to illustrate the de ermination of carbohydrates 50.11 m: Cu has been re- duced by the sugars in the aliquot solution. Expressed as Cu reduced L -30- per gram dry material the value 58.45 mg Cu is found. If these *values were to be expressed in terms of e.g. invert sugar, 15.1 mg invert sugar would correspond to 30.11 mg, Cu - (found in Sugar Table). In terms of sugar per gram dry material that would be: 15'1 x 1000 : 29.32 mg invert sugar. 50 x 50 x (100 - 79.47 100 Whereas to the value 58.45 mg Cu as obtained according to the calcu- lation in equation II - 29.43 m invert sugar would correspond. The sugar determined according to the calculations used in the experi- ment in this case thus gives a value 0.13 mg too high i.e. nearly 0.5fl too high. This is due to the fact that the COpper reduced will not be in stoichionetric proportions to the sugar present - as has already been discussed. If the reduced Cu were converted in terms of other sugars the error would be slightly different. The main factor determining the error is, however, the factor by which the Cu obtained according to equation I) has to be multiplied in order to be converted in terms of copper per gram of dry material. The error for the hydrolyzed sample will be the double of that obtained for non hydrolyzed material, because in the first case one is dealing with aliquots representing only half as large samples as in the latter case. In the present work the error would not as a rule exceed lfi. _37_ Analytical errors. The average difference between the titration values of two 4 , aliquot samples was 1.4 , or the deviation from the average value was i 0.7fl. The average deviation was below 0.55 when a feW'values with exceptional great deviation from the average were discarded. If the deviation exceeded 1 31 (except for the first sample) the analyses were repeated on two n w aliquots. fihen dealing with fresh material, the titration values for two different extracts of the same sample might show rather great varia- tions. The difference between the two first data in column 1, Table 17 is thus 0.54 ml. or 15.53. The data can however be considered as having value because the amount of carbohydrates present in the sample is finally expressed in terms of grams of copper reduced per hundred grams of dry material ( or per hundred grams of fresh mater- ial). When these values are calculated (see calculations elsewhere) the difference between the data in this case is only 1.1% 2.6? for fresh material). when dry material was used the values came much closer to each other. Discussion of data and graphs. Whether one prefers to calculate the reduced copper on the basis of dry weight or on the basis of fresh weight sample; is of minor significance. The curves will be different according to the Easis chosen, but both ways can be used for a comparison of the different samples. When analysing for reducing carbohydrates the values ob— tained expressed in terms of glucoseo or in terms of invert sugar - ’3 ’ L.. CA] r‘vy when analysing for total hydrolyzable carbohydrates has commonly been used. When dealing with brome grass it is known that part of the non reducing carbohydrates, and probably a small part of the water soluble reducing carbohydrates are fructosans, giving fructose on hydrolysis. Invert sugar gives on hydrolysis equal amounts of fructose and glucose, which do not have the same reducing powers. It is then obvious that converting the whole amount of reduced copper in terms of invert sugar would not give the correct values. It must also be considered that a certain amount of the carbohy- drate content is glucose. Therefore, calculating the whole as fruc- tose would also give incorrect values. This is the reason why re- duced copper has been chosen as the basis for a comparison between the carbohydrate contents in the different samples. The total carbohydrate content for fresh material has been plot- ted as copper reduced per hundred grams of fresh material in Figure ('1 4, and as copper reduced per hundred grams dry material in Figure 5. The first curve shows an increase in total carbohydrate during the period in which the samples were taken. whereas in Figure 3, the amount of copper reduced has been plotted as copper reduced per hundred grams of dry material, the graph obtained shows a quite dif- ferent picture. In this case both the total carbohydrates and the reducing carbohydrates reach a definite peak in the beginning of June. The difference between water soluble and alcohol soluble carbohydrates is also largest in this period. Felval (32) has analysed wheat and rye and found the fructosan content to be highest 0 1n the beginning of June. According to the data obtained in the present experiment this may also be the case with brome grass. One should, however, be careful not to draw too definite conclusions as to the seasonal variations after one crop of one season. Environ- mental conditions may be the cause of many variations in the plant material. It is interesting to compare the data given for the grass ‘Q sampled on ay 17th, with the ones obtained for the grass of second growth taken on August 15th. These samples were of about same length and growth stage, but the total carbohydrate content is much higher ‘ I in the sample taken in August than in the one gathered late in the spring. There is also a great di'ference in moisture content; the sample taken on the 17th of ”ay contains nearly 2] mire wcistgre t :n ‘ e s Mle ccllecbel on the lfith of august. .31 .ne analytical data obtained JCT all samples confirm the find— ings of fialdron et a]. (1) that the water extract gives more carbo- hydrates than the ethanol extract. Waldron et al. (1) determined only total carbohydrates. The present work shows the same for reduc- ing carbohydrates. As a rule the difference is small when dealing with reducing sugars, but as the alcoholic extract consistently gives lower values than the water extract, the difference can not be re- garded as a purely analytical error. The difference between reducing power in aqueous extract and alcoholic extract before hydrolysis is -4r- very constant for all the samples both when they are analysed as fresh material and as material which has been dried at 1000 C. Fructosans are usually considered non reducing. The data obtained in this experiment show that the water soluble carbohydrates have larger reducing power than the alcohol soluble carbohydrates. It might be that some endgroups of the fructosan molecules might have some reducing power. The effect of di°ferent drying methods. When comparing the values found for water soluble and alcohol soluble carbohydrates in fresh grass with those found for material subjected to different drying methods the data obtained were expres- sed graphically in Figures 3-7. The sampling dates were plotted along the horizontal axis, and copper reduced per 100 g. of dry material taken on the respective days was plotted along the vertical axis. Drying samples at 100° C. previous to the analysis has often been recomnended when an aqueous extract is desired. The heated and dried material is supposed to give the same values as the fresh sample. Lhe heating is supposed only to inactivate the enzymes in the plant tissue. According to the data obtained in this experiment the results obtained for dried grass are not equivalent to those obtained for fresh grass. The values both for total carbohydrates and for reducing carbohydrates present were found lower when the material had previously been dried at 1000 C. than when the fresh O material was used. This indicates transformation to non reducing compounds e.g. cellulose or destruction of the carbohydrates. This latter was the reason for the extremely low values found for samples taken on the 17th of June when part of the grass was charred in an overheated oven. But, even when no charring occurred the values were d (D finitely lower. It is shown, however, that the hydrolytic activity of the enzynes is stopped, as the values of reducing sugars do not increase in comparison to the ones found in fresh material. Reduc- ing sugars are on the other hand constantly lower, also indicating transformation to other compounds. The values obtained for the samples dried at 45° C. gave an interesting '0 icture. This temperature was chosen because it was con- sidered as being favorable for enzymatic activity. Also it is approxi- mately the same temperature as often used for artificially drying grass. The values obtained here for total carbohydrate showed defin- itely lower values than those obtained with fresh material and material dried at 1000 C. The difference between water extracted material and alcoholic extract is relatively great. It is also in- teresting to study the curve (Figure 5) for reducing material. The .... values for the alcohol extracted sample indicate very little hydroly- CO H o 0‘} of the soluble non reducing carbohydrates which are present in about the same amount as in fresh material. The values for water .‘2 ‘ ' ‘ " . «‘0 1‘~"‘ Lw"" " I :— I n a ‘1 - . u ' ‘v . ‘ solahle FGahClUw c iOOm'dFleGS in cases, howev;r, were very high thus showing a possible hydrolysis of the fructosans down to a smaller molecular size, but not snell enough to be dissolved in the ethanol. \ obtained from the grass dried inside (Eig. e and 7). In both a high degree of hydrolysis of non reducing carbohydrates to reducing carbohydrates occurred as well as a large conversion of total carbo- hydrates to compounds not having reducing power upon hydrolysis. The grass dried outside showed a very low value for total soluble carbohydrates. Forman (14) suggests that fructosans may be broken down to fructose, and this sugar be used in the plant respiration where it is converted to C02 and water. 1) 2) FP- v 5) SVVTAEY A79 CCTCIVSICfs --' mater extracted material from ?romus inermis will give higher values for total soluble carbohydrates and reducing carbohy- drates than will material extracted with BOZ ethanol. Some fractionation of the Carbohydrate was possible when ex- tracting the material udth 805 ethanol, followed by hot water extraction. Part of the carbohydrates extracted in an alcoholic solution was not hydrylyzed by invertase, whereas they were easily hydro- lyzed in an acid solution. The carbohydrate fraction extracted by water following the alcoholic extraction was very constant during the season in which the material was sampled. This fraction was easily hydrolyzed by acid, but very slightly attacked by enzyme hydro— lysis. The four different drying methods employed for the preservation of the samples gave all lower values for total soluble carbohy- drates than did the sample analysed when in fresh condition. 'flhen the grass had been dried at low temperatures an increase in reducing sugars was found as compared to the reducing sugars found in the fresh material. A high degree of enzymatic hydro- lysis had probably occurred during the drying period. We increase in reducing sugars was shown when the samples had been dried at 100° C., this temperature thus probably destroys the hvdrolytic en ymes in T‘rome grass. (I -44- It is to be sugpested that when quantitative carbohyd ate determinations are to be done on Brome grass (and very likely on most other plant materials) the analyses should be done on fresh material as soon as possible after the sampling. If dried material has to be used the same drying method should be used for all samples in order that a fair comparison between the samples can be made. Drying at high temperature (1000 C.) is to be recommended as the changes occurring in the plant material is lowest at this temperature, but even then one must expect to find lower values than when fresh tissue is analysed. u. reduced per grad fresh eelpht 1‘ V FIG. 2 FRESH SAMPLE Ieter extrect hydrolyzed. I} Ethanol extract hydrolyzed Ethenol extrect non hydrolyzed leter extreot non hydrolyzed " . . . - - ° '° -" ' ' ' Ieter extr. following ethanol extr.. hydrolyzed 5-" Col C-l? T-I Sempllng detee - month end dey 28 F16.35 FRESH SAMPLE water extract kwdrolyzed. ,A Ethanol extract Mdrolyzed. Cu reduced per gran dry weight. 5-!7 G-I 6-l7 Ethanol extract non hydrolyzed. Water extract non hydrolyzed. Water extr. following ethanol extr. hydl’Olyifid. 7-l Supling dates - month and dqx 24 ‘Iater extract hydrolyzed. FIG- 4 22 SAMPLE DRIED AT 00'!) ' Ethanol extract marolyted. I no ’ P " " I6 \ M ‘v’ l2 IO .3 fi '6 E e 3 h 3 o y Water extract nm midrolyzed. g ‘ “' ~ -. Ethanol extract nm hydrolyzed. :1 o ’N .' later extr. following ethanol extr. 2 no...“ ..' hydrolyzed. 1., I. o . o e '. .'.'. S-IT S-l G-IT 7-! Sampling detee - month and day Cu reduced per gram dry weight. FIG. 5 n SAMPLE omen AT45'C Voter extrect hydrolyzed. to IO 1’ Ethanol extract tudrolyzed. IO hater extract nan hydrolyzed. C Q I ‘- - - "‘ ~ ‘ ~ + Ethanol extract nut hydrolyzed. ’ g... g e‘e. ..e 0.... eeee ..oO°eeO.. ' 0e.-;oe e. "W W. {011mg etma “tr. "V hydrolyzed. 0 5- I? M M? M Sampling dates - nmth and day FIG. 6 SAMPLE DRIED INSIDE to later extract m'drolyled. I. Ethanol extract mound. leter ctrect nan march-led. ‘. Ethanol extract non turdrolyecd. Go reduced pu- gra- dry weight- \ 2 * ’ "0...... a. later extr. following! ethanol extr. ..*°e. gee. ”drab“. o ...°..e'.. 3-” e-: H" 74 Sampling dates - month and day Out-ethaoedperp-udryweighte EIGJ SAMPLE DRIED OUTSIDE 20 II IF, let.- extrect Weed. MK \\ , tthenol atrect Wde \ O T I I I I leter extrect nan Ivar-Med. ‘, a. tunnel extract non Ivor-chum 0’ o ”N...” ,,,,.. . Weter extr. tollaing ethenol extr. WWe .5." C‘I “7 7" Senpllng detee - month end an 1. CD 10. BIBLIOGr‘.nPHY Yaldron, Dorothy R.; Ball, C.u:.; Killer, 3.3.; Donne, E.F.: A Study of Methods for the eternihatien of Sugar in CrOp Plants. (In print--Of. Assoc. Cffic. Apr. Cheaists.) Killer, E.C.: Plant Physiology, wit11-eference to the Txr rec n Plant. IcGra7-Eill Book Company, Inc., Les York and nondon. 3nd id. 953 Archbold, H.K.: Fructosans in the Monocotyledons. A ievio". h w Phytologist. Vol. 59, 135 (1910) -1311 inor ’ S .21: Q = II“ CA.“ orth, ‘3‘". .1: . (IA :1 1d I‘Zilfl St ’ E .L . : Carbohydrates of Grass. lsolati n of a Polysaco harid me of the Laevan Tyi. e. of Chem. Soc. -CO (1951b) Ieyer, A.: Uber die Assimilations grosse bei Zuckcr und QC]: btrirke B attern. Botan. Zentr. Vol. 15 oo Archbold, H.K.: Physiolo ical Studies in Plant Lu utrition. VII The role of fruct osans in the carbohydrate metabolism of the barley plant. 1. Katerials used and methods of sugar analysis employed 2. Seasonal changes in the carbohydrates, with a note on the effect of nitrogen deficiencv. Ann. Botany (N.S.) 2, 185, 135 ( 955) B nell, H.R.: Distribution of Carbohydrates Between Component Parts of the Wheat Plant at Various Times During the Season. cw Phytologist, 57, 85 (1058) Russel, 3.8.: Physiological Studies in Plant Hutrition. IX Th effect of mineral deficiency on the fructosan etabolism of the barley plant. Ann. Botany (h.3.) 11, 865 (1958) is ylor, H., Evans and Hibbert, harold: Bacterial Poly- saccha.rides. Advances in Carbohydrate Chem. Vol. 2 (1916) Hestrins, 5., and Avincri-Shapiro, s.: The Iechanism of Polysea accharide Production from Sucrose. Biochem. of. 58,21911 ivineri~312piro 8., and Iestrin 5.: The Icchanism of Polysaccaride Production from Sucrose 2. Biochem. of. 39, 167 (1915) 11. 12. 15. 16. 17. 18. 19. 00 5.1“]. . Importance de Cugnac, Ade: Les Glucides des Gr 3 7 ol. 15, 25 (1951) Fructoholosid es. Bull. soc. 011m. b‘ Norman, A.G., Silsie, C.P., and Gaessler r, K .G.: The Fructosan Content of Some Grasses Adapted to Iowa. A preliminary survey. Iowa St te Coll. of Sci., 15, 501 (1941) Off101an :nd Tentative Iethods of Lnalyses of the Associati n of Official Agricul ural Chexzists. Publ. Assoc. Cffic. Agr. Chemist, Jashington 3.3. 6th Ed. 1945 ”-0 horman, A.G.: The Compo.s itio n of P0 orage Ciops. I Rye Crass. Biochen of. 50, 1651 Kraus, E.F., and Praybill, H.R.: Vegetation and 3e- pro oduction xith ‘pecial Reference to the Toml -to. Ore egon ngr. Expt. Sta. Bull. Ho. 11—9 (1918 Browne: A Handbook of Sugar Analyses. John hiley and Sons, Inc., New { rk. Chapman and Hall, Limitea. London, lst hd. 1912 1aridcs. Lonamans, v HcIlroy: The Chemistry of the Polysacc Green ana Co , You" 1 r ’iuard Arnold and Co., rn Platolenko, P.I.: Pub. State Inst. Tobacco and hakhorks Ind., Krasnodar, U.5.s.3., Bull., Lo. 118 (1955) Bryan, Given and Str aughn: U.3. Eur. Chem.21nd Soils, Circular 71 KcRary, Hillard L., andeIattery, Iarion .: The Color- fictr Determination of F W1C 03 an in Pla.t Laterial. 11 1c Of. 3101. Chem. 157, 151. (1915) Crowne and Lcrban: Physical and Chemical hcthods of Sugar - wilys is. John “iloy and Sons, Inc., Lew York. Chapman and Hall Limited, London, 5rd. Ed. 1911 Belval, 3.: La Genese de l'flnidon Cans 1es )Cerealcs. 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