CHARACTERIZATION OF DEXTRANS FROM FOUR TYPES OF LEUCONOSTOCMESENTEROIDES

ALLENE JEANES, W. C. HAYNES, AND C. A. WIHAMNorthern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture,

Peoria, Illinois

Received for publication June 27, 1955

Bacteria of the genus Leuconostoc have longbeen known to produce gum or slime in the juicesof isugar beets, sugar cane, and other sucroscontaining fluids (Evans and Hibbert, 1946).The leuconostocs present in Louisiana sugar-canejuiceshave beenstudied intensively by MoCleskeyand co-workers; no comparable observations forany other locality have been reported. Alfordand McCleskey (1942) showed that leuconostocsfound in Louisiana cane juice were heterogeneousand consisted of at least three types on the basisof taxonomy and growth characteristics insucrose-containing media. McCleskey et al. (1947)showed that the most commonly encounteredpecies was Leucomwstoc mewnteroide, and classi-fled over a hundred strains under four types,designated A, B, D, and F, according to colonialcharacteristics. Further evidence that these fourtypes represent relatively distinct entities hasbeen provided by their serological behavior(Leivo-Quiros and McCleskey, 1947), nutritionalrequirements (McCleskey and Barnett, 1949;Bienvenu and McCleskey, 1950), and manner ofsucrose utilization (Giglio and McCleskey, 1953).

Thirty-five strains representative of the fourtypes of McCleskey et al. (1947) were includedamong the 135 strains we have cultured recentlyfor isolation and characterization of dextrans(Jeanes et al., 1954). The polysaccharide productsfrom these colonial types had not been char-acterized previously. It is the purpose of thispaper to amine the relationship between thecolonial types and the chemical and physicalproperties of the dextrans. Consideration isgiven also to the question of whether dextransfrom strains of L. mesenteroie8 originating inlocalities and habitats other than Louisianasugar-cane juices have properties comparablewith those from McCleskey's four types.

MATERIALS AND METHODS

Bacterial strains. From the 35 strains givenus by Dr. C. S. McCleskey as representative ofthe four colonial types, only the 23 dextrans

reported here appeared to be individual and freeof duplication. The type-D strains were isolatedseveral months before being used for dextranproduction; all the others had been isolated about4 years previously.

Unless explicitly stated otherwise, all strainsreferred to in this paper are designated by theirnumber in the culture collection of the NorthernUtilization Research Branch. However, to avoidrepetition, the designation "NRRL" is omittedfrom strain numbers after their first introductioninto the text.Dextrans are identified by the number of the

strain which produced them.Production of dextrans. The conditions for

dextran production were standardized so thatthey might not be held responsible for differencesin the chemical, physical or biological propertiesof the dextrans formed by different strains. Thetemperature of incubation was 25 C and thecultures were of 1-L volume. In a few cases, wherethe yield was low, cultures of 2 or more L wereset up. The medium for all stages in the inoculumbuild-up and for the production was basically thesame: Tryptone, 0.25 per cent; KHPO4, 0.5per cent; yeast extract, 0.5 per cent; distilledwater, pH 7.4. The sucrose concentration was 0.5per cent in the first stage of the inoculumbuild-up, 1 per cent in stage 2, and 10 per centin production flasks. The sugar concentrationwas kept low in the inoculum build-up in anattempt to prevent development of an aciditydetrimental to the microorganisms and toexpedite transfer of the inoculum by keeping theviscosity low. Deep culture conditions were en-couraged by use of 10 ml of medium in 16- by150-mm test tubes inthe first stage of the build-upand 90 ml in large test tubes in stage 2. Produc-tion flask were 1-L Erlenmeyers charged with900 ml of medium. Inoculation was at the rate of10 per cent. Incubation was continued for 5 daysor, in a few cases, until such later time as theobserved viscosity suggested a satisfactory yield.

Isolatoon and purification of dextrans. The167

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JEANES, HAYNES AND WILHAM

procedures for isolation and purification of thedextrans have already been described (Jeaneset al., 1954). However, in order to facilitate under-standing of the data presented in this paper, it isnecessary to outline the method for separatingthe products from the cultures.

Cultures containing primarily soluble dextranproducts were diluted with water, made to 35 per

cent with ethanol, and supercentrifuged toremove bacterial cells and other insoluble matter.In many cases the major product, and the onlyone isolated, was precipitated from the 35 percent ethanolic supercentrifugate by increasingthe ethanol concentration to 45 or 50 per cent.Such dextran products are designated merely bythe number of the strain which produced them.However, when produced by other strains, sig-nificant amounts of the total product differedin solubility from this main fraction and were

isolated separately. Thus, from cultures of strainsNRRL B-1433 and NRRRL B-1438, an appreci-able part of the product was insoluble in theculture, and was isolated separately to givefractions designated B-1433-I and B-1438-I.From cultures of strains B-1498 and B-1501,appreciable amounts of the culture productwere sedimented by supercentrifugation of thediluted culture at 35 per cent ethanol concentra-tion, giving fractions B-1498-A and B-1501-A.Likewise, from B-1498 and B-1501, the fraction

insoluble in 35 to 45 or 35 to 50 per cent ethanolwas found to be separable still further, givingtwo distinct components: "L" the less solubleand "S" the more soluble. A complete discusionof molecular heterogeneity in dextrans and themethods of fractionation has been published byWilham et al. (1955).

Classes of dextrans. Dextrans from 96 strainsof bacteria, including those reported here, havebeen classified on the basis of the percentage of1,3-like linked anhydroglucopyranose units(AGU) present as determined by periodate oxida-tion (Rankin and Jeanes, 1954) as follows:

CklsABC

AGU linked 1,3-like, %0-23-6>6

Dextran products from six strains could not beassigned to one of these classes because theywere shown to be separable into componentsbelonging in two or more of the classes. Suchproducts were assigned to a group designated"Heterogeneous dextrans" (Jeanes et al., 1954).

RESULTS AND DISCUSSION

Charactristics of colonial types vs dextran prop-erties. The characteristics reported by McCleskeyet al. (1947) for their four types of L. mesenteroidegare summarized in table 1, and the characteristics

TABLE 1Summary of characteristics of four types of Leuconostoc mesenteroides*

Medium: Raw Sugar Aar Medi: Raw Sugar Broth

Colony Apparent or--anmUniformitysoug strns |Ropey" FlCoonalyprctritisder of quan- -_Ropey____Fluorescence____Colonial charactristis tity of gum culture

producedt Viscosity production Fermentation reactions

A Smooth, semi- 3 Uniform (low) Uniform _transparent

B Rugose, opaque 2 Diverse Relatively uni- _form

D Smooth, trans- 1 Diverseparent

Di Relatively uni- - +form (high)

D2 Diverse 41 Smooth, opaque 2 Relatively uni- Relatively uni- -

form (high) form

* Condensed from data of McCleskey et al. (1947).t Estimated from height and diameter of colonies. In order to decreasing quantity.

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TABLE 2Characteristics of dextrans produced by the four type8 of Leuconostoc mesenteroides*

McCleskey'sStrain Dextran Dx- AGU, Linked, % a IntrinsicIdentification from Viscosity Yield Fluor- Solu-

nialStrain

Frc- (Water, %t es- bility Nature of ProductiCol- B-RR tion 25 C)cence Water?nial Number B- 1,s 1,4 36- HCONH 1 NKOHtyp .,6ielkeHOE

10661 1951 5

+2100 1.360213 1.569¶

1.45812.60512.514¶3.107¶

0.521

1610187

84

917610

11

0.760Q 23

262129

241716181618

20751437

75

+

+

+

++

++

+

+ P+ P+ P

++P

Soft, stringySirupy, stringyPastey, shortSirupy, stringy

Tough, crumblyFloc. ppt. to roughgum

Floc. ppt.Tough, crumblyTough, crumblyFloe. ppt.

Floc. ppt. to crum-bly gum

Floc. ppt. to shortgum

Rough, shortCrumbly, shortTough, cohesive,

stringCrumbly, shortCrumbly, shortSmooth, shortSmooth, shortRough, shortCrumbly, short

Tough, shortRough, shortTough, shortShortFine ppt.Floc. ppt. to shortgum

Short gumFine ppt.

* These data have been assembled from the publication of Jeanes et al. (1954) to provide the necessarybasis for comparisons in the present paper.

t Based on the weight of sucrose in culture. The percentage yield X 2.11 = per cent of theory basedon dextrose moiety of sucrose.

$ At room temperature. "p" indicates soluble if precautions are observed (Jeanes et al., 1954).§ Products are gums unless stated otherwise. Observed when precipitated from aqueous solution by

ethanol at 45 to 50 per cent concentration.1¶ Viscosity was measured in 1 N potassium hydroxide solution.

19561 169

1442143914431425

94818074

66108

0131018

1.0190.4750.4181.105

1429 861438 81

5 106 13

1438143314331431

I

I

79636362

7303029

A

B

D

F

735A730A1020A151A

158B719B

706B

700B

8

548

49F246F46F

IOIF

+214°221220222

215

215

214

213214

215

212213227211

14779

0

0

300

000200

0500272

020

94

949396

959595959493

908791946280

213217217217

204208

206216

1198

120512141064

121012081211120912121204

150015021498149814981501

15011501

6

374

555367

10896

1118

715

0.865

0.887

0.6980.6280.8430.693

0.846

0.8011.0431.1561.0960.3291.004

1.0540.412

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JEANES, HAYNES AND WILHAM

of the dextrans produced by these type culturesare given in table 2.The characteristics of the bacterial colonies

appear to be related to the solubility of thedextrans and to the appearance of their very con-centrated solutions. Dextrans from colonial typesA, D, and F were water-soluble in contrast tothose from type B, which were of low solubilityor insoluble. The type B colonies were describedas rugose; the others were described as smooth.Concentrated solutions (20 to 40 per cent, suchas might be present in plate cultures) of dextransfrom type A and D strains were brilliantly clear.Those from types B and F were turbid, althoughto differing degrees. The corresponding colonieswere described as transparent or opaque,respectively.The height and diameter reported for the

colonies is in general agreement with the yieldsof highly purified dextrans obtained. Thus, theaverage percentage yields (based on the weightof sucrose in the medium) from cultures of typesA, B, D, and F were 13, 13.5, 22, and 17, respec-tively (table 2).The relationship between the viscosity of cul-

tures and that of the purified dextrans obtainedtherefrom is complex. Under favorable culturalconditions, the viscosity of cultures depends uponsuch known factors as the yield, structure, molec-ular homogeneity, molecular weight, and molec-ular-weight distribution of the dextran product.It appears that the viscosity of dextran-contain-ing cultures may be influenced also by physicalorganization of the dextran and by interactionbetween dextran and other constituents of themedium. Thus, it is not surprising that consistentcorrelation cannot be shown between the qualita-tive viscosity characteristics for cultures asstated in table 1 and the intrinsic viscosities ofthe purified dextrans shown in table 2. Thesecorrelations are discussed more specifically in thenext section.

It would be of unusual interest if relationshipcould be established between the fermentationreactions shown by strains of a colonial type andthe structure of the corresponding dextrans.However, no consistent relationship appearsbetween the uniformity of fermentation reactionsof strains of the colonial types and the uniformityof structure of the corresponding dextrans.Thus, although type A strains showed uniformfermentation reactions, the dextrans showed

diversity in percentage and type of linkages, inviscosity, and in gum properties. Type D strainsshowed diverse fermentation reactions, while thedextrans showed homogeneity of linkage type andcontent, but diversity of gum characteristics.Therefore, our results do not indicate a closerelationship between the pattern of fermentationreactions of the strain and the nature of itsdextran-synthesizing enzyme system as revealedby the structure and properties of its dextran.Proies of dextrans from the colonii types.

Dextrans from the type A strains examined hadcontents of 1,6-linked anhydroglucopyranoseunits in the range from high to rather low. Onthe basis of their contents of 1,3-like linked units,these dextrans are classified in structural clasesA (B-1442) and C (B-1439, B-1443, and B-1425)(Jeanes et al., 1954). The gum of one of thesewas short (B-1443); the others were stringy andof either soft or sirupy consistency. The dextranshaving stringy gums showed high as well as lowviscosity. This scattering of dextran charac-teristics correlates poorly with the uniformityreported for viscosity production and for fermen-tation reactions by type A strains.

Dextrans from type B strains showed a rangein 1,6-linkage content from medium to low, buttheir 1 ,3-like linkage content placed them all inthe structural class C. Products from two ofthese strains (B-1438 and B-1433) were separatedinto two fractions on the basis of solubility duringisolation; for both of these strains, there waslittle difference in the other properties of thefractions. The dextrans from all the type B strainswere tough, crumbly gums or flocculent precipi-tates. Their low solubility in water necessitatedmeasuring the viscosity of most of them in dilutealkali. The viscosities showed a range in valuesbut ranked among the highest of all those we haveobserved for dextrans. Thus, the dextrans fromtype B cultures constitute a somewhat morehomogeneous group than those from type A.The insolubility of the dextrans in the culturefluids may have influenced the observation ofMcCleskey et al. that type B cultures showeddiverse viscosities. The fermentation reactionsof type B cultures were relatively uniform(table 1).The dextrans from type D strains were prob-

ably more uniform in the measured characteristicsthan those from type B strains, but showedgreater diversity in gum characteristics. They

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had high percentages of 1,6-links in a narrowrange, and all except one (B-1205) were of struc-tural class A. Their viscosities were in the inter-mediate range, although our cultures, as well asthose previously reported (table 1) for many ofthese strains, were unusually viscous. Culturesof B-1064 set almost solid.

Cultures of four of the type D strains showedyellow-green or blue-green colors in ordinarylight, an effect first observed by Alford andMcCleskey (1942) and later by McCleskey et al.(1947) and designated "fluorescent." All theother cultures showed only the normal colorof the media. This fluorescence persisted indense solutions of the purified dextrans fromthese four strains (B-1066, B-1198, B-1205, andB-1214). This effect appears to result from somebacterial product other than dextran. Dextransfrom the fluorescent- and non-fluorescent-pro-ducing strains did not differ significantly in pro-portion and type of linkages or in viscosity.

Dextrans from type D strains had short,rough gums, with three exceptions as follows:dextrans B-1209 and B-1211 had smooth gums,and B-1064 had tough, cohesive and stringygum (that is, under gentle tension is pulled intoa thread). Strain B-1064 was the only one whosedextran-contaning culture appared "ropey."Although our yields of B-1064 dextran from cul-tures on 10 per cent sucrose broth were high,they were not in agreement with those reportedby Gigio and McCleskey (1953) from cultureson 20 per cent sucrose broth. As these investiga-tors themselves suggested, their experimentalprocedures made possible the precipitition ofpolymeric products other than dextran, and theweighing of products which retained solvents.The observations we have made on B-1064dextran do not support Giglio and McCleskey'shypothesis of its structural uniqueness.

Dextrans from two of the five type F culturesexamined were separable into several fractions.In each case one fraction had low percentage of1,6-links and high 1,3-like, and very low vis-cosity. However, the average viscosity of thesedextrans before fractionation would have beenhigh, and thus in reasonable agreement with theobservations reported for the cultures (table 1).Likewise, there is sufficient uniformity in thestructural and gum properties of these dextransfor reasonable correlations with the relatively

uniform fermentation reactions observed forthe cultures.The specific rotations of the dextrans, shown

in table 2, are a further characterization but donot differentiate among the dextrans from thefour colonial types. Dextrans showing no 1,3-like links by periodate oxidation usually havespecific rotations of +214 14- 20 in formamide orabout +202 i 2° in 1 N potassium hydroxide.The presence of 1,3-like links results in propor-tionate increases in the specific rotation (Jeaneset al., 1954).The observations reported here are in keeping

with the previously established fact that thecharacteristics of dextrans are determined by thestrain which produced them (Jeanes et al., 1954).There is variation in properties of the dextranswithin each colonial-type group, yet there isdefinite correlation between these properties andthe colonial type. The dextrans within eachgroup have a distinctive combination of proper-ties. Dextran B-1443, which does not fit wellinto any group, is the main exception. These ob-servations make possible the selection of a type-strain from its natural habitat for productionof a dextran having characteristics within certainrather definite limits.

Other characterizon of the dextrane. In addi-tion to the characterizations shown in table 2,representative dextrans from colonial types A,D, and F (from strains B-1442 and B-1443;B-1205, B-1064, and B-1212; and B-1500, re-spectively) have been examined for molecularheterogeneity by the technique of analyticalfractional precipitation (William et al., 1955).The low solubility in water of dextrans from typeB strains prevented their inclusion. The weightdistribution of a dextran on the basis of sedi-mentation under the influence of a non-solventsuch as ethanol and centrifugal force appears todepend on both molecular structure and molec-ular weight. Differences in structure of fractionshave been established by preparative fractiona-tion of dextrans B-1498 and B-1501, as is shownin table 2, and differences in both structure andapparent molecular weight have been shown forfractions from other dextrans (Wilham et al.,1955).

Analytical fractionation curves, shown in figure1, for dextrans B-1205, B-1064, B-1212, andB-1500, indicate normal, high molecular weightdextrans of high degree of homogeneity within

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3536 37 383940363637 3636W37N33M

ETHANOL CONCENTRATION, %

Figure 1. Fractional precipitation curves for dex-trans from strains of tvpes D and F.

the limits of detection of this method. DextransB-1064 and B-1500 appear to be less homogeneousthan those from B-1205 and B-1212. The curvefor dextran B-1442, as is shown in figure 2, issimilar to these others but indicates greaterpolydispersity. Approximately 25 per cent ofB-1442 dextran appears to be of smaller molec-ular weight or to have a higher proportion ofnon-i,6-links than the remainder, as indicatedby its precipitation at ethanol concentrationsgreater than that required to precipitate mostother dextrans (38 per cent).The curve for dextran B-1443, shown in figure

2, indicates still greater polydispersity, withpossibly one main component and minor amountsof differently constituted materials of lesser andgreater solubility, respectively. Among all thedextrans examined, only one other (B-1142) hadso large a proportion of the total culture productprecipitated at so high an ethanol concentration.The fact that the major component of B-1443dextran required a higher concentration ofethanol for precipitation than did that fromB-1442, apparently is not due to lower molecularweight. Molecular weight values obtained bylight scattering techniques for these two dextranswere 20 X 106 and 8 X 10., respectively. DextranB-1443 appears comparable with the more solublecomponent of dextrans B-742, B-1355, B-1498and B-1501 but, unlike these products, it con-tained a low proportion of less soluble fraction(Wilham et al., 1955).

Relation of other dextranm to those from the fourbacterial types. The question arises as to whetherMcCleskey's four bacterial types encompass allvarieties of L. mesenteroides regardles of theirnatural habitat or origin. In other words, do all

dextrans from L. mesenteroides have propertiescorresponding to those shown for the four colonialtypes in table 2? Partial answer to this latterquestion is provided through characterizationfor the dextrans from 55 other strains of thisspecies (Jeanes et al., 1954).There is not sufficient agreement between

all the observed properties of many of thesedextrans and those shown in table 2 to establishcomparability with one of the four types. How-ever, approximately half of these dextransshowed properties apparently in agreement withthose from one of the four types. Thus, thosefrom strains B-1424 and B-1525 appeared to haveproperties comparable with those from the type-Astrains B-1442, B-1439, and B-1425. No dextranseems comparable with that from B-1443. Dex-trans apparently comparable with those fromtype-B strains are B-523, B-1118, B-1120, B-1121, B-1144, and B-1149, all of which wereessentially water-insoluble. Dexan from B-1308,as well as possibly those from B-1392, B-1407,and B-1417, was closely comparable with thosefrom the "fluorescent" D strains. However, noneof these cultures showed as pronounced fluores-cence as those of D strains. The observed proper-ties of dextrans from B-1400 and B-1401 wouldnot differentiate them from the non-fluorescentdextrans from type D strains.Gum characteristics alone do not differentiate

between dextrans from McCleskey's type-F andnon-fluorescent D strains. However, as comparedwith dextrans from D-type strains, those ex-amined from the F-type had lower contents of1,6-linkages, usually higher viscosity, and boththeir dilute and concentrated aqueous solutionswere turbid. On the basis of these criteria,dextrans comparable with those from type-F

50r

R 40o'd

1 30<.a-w

X 20z49

XI-

C'

8-1442

A 8-1443"II

I

II

8

11 ^8I_ ---P

34 36 38 40 42 46 46ETHANOL CONCENTRATION, %

50

FigureS. Fractional precipitation curves for dex-trans from type A strains.

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strains were obtained from strains B-1192, B-1196, B-1383, B-1385, B-1390, and B-1411. Anumber of other dextrans constitute borderlinecases (B-1382, B-1384, B-1396, and B-1397).None of the dextrans examined as representa-

tive of the four colonial types had the soft,long gums which are characteristic of those fromL. mesenteroides strains B-512, B-641, B-1377,and B-1412. These dextrans are similar to thatfrom the type-A strain, B-1442, but their gumstended to flow in considerable masses rather thanmerely in strings or threads, and culture liquorscontaining them were much more viscous. Nonehad as low contents of 1,6-links as B-1425 dextrannor as low viscosity as B-1439. Furthermore, ahigh degree of molecular homogeneity seemedcharacteristic of dextrans B-512, B-641, B-1377,and B-1412, in contrast to the appreciable hetero-geneity of dextrans B-1442 and B-1443.The remainder of these 55 dextrans could not

be classified in one of the four categories solelyon the basis of properties observed. This is duein part to the facts that the combination ofproperties of many individual dextrans does notprovide a sharp characterization, and that theindividual properties show incremental variationsbetween dextrans. Furthermore, description ofsome properties still is in qualitative terms. These55 strains have not yet been examined in rela-tion to criteria for the four culture types ofMcCleskey et al.

ACKNOWLEDGMENTSWe should like to acknowledge our indebted-

ness for assistance as foliows: Ralph W. Kuehneand Lenora J. Rhodes in culturing the strains;Dr. Henry M. Tsuchiya in producing some of thedextrans, and Marjorie J. Austin and Jay E.Pittsley in isolating and characterizing them;and Robert Tobin for light scattering meas-urements.

Relationship has been sought between thechemical and physical properties of the dextransfrom 23 strains of Leuconostoc mesenteroidesrepresentative of the colonial types A, B, D,and F of McCleskey et al., and the colonial typeof the strains, respectively. The combination ofcharacteristics was found to be distinctive for thedextrans from each of the colonial types, thussubstantiating the microbiological observationsof McCleskey et al. that these four types arerelatively distinct entities. The data reported

here permit prediction, within certain limits, ofthe chemical nature of the dextran to be expectedfrom a given strain on the basis of the colonialand cultural characteristics of the strain.

Correlation has been shown between thedextran properties and some, but not all, of thecolonial characteristics of the strains.Approximately half of the dextrans prepared

from 55 strains originating elsewhere than inLouisiana sugar cane appeared comparable withthe dextrans derived from the cultures of typesA, B, D, and F, respectively. However, dextranssuch as that from the strain NRRL B-512, havenot yet been found from cultures of McCleskey'stypes.

REFERENCESALFORD, J. A. AND MCCLESKEY, C. S. 1942

Some observations on bacteria causing slimein cane juice. Proc. Louisiana Acad Sci.,6, 36-42.

BIENVENU, R. AND MCCLESKEY, C. S. 1950 Theamino acid requirements of Leuconostocrmsenteroides isolated from sugar cane juice.Proc. Louisiana Acad. Sci., 13, 13-18.

EVANS, T. H. AND HIBBERT, H. 1946 Bacterialpolysaccharides. In Advances in carbohy-drate chemistry, 2, 203-233. Academic Press,Inc., New York, N. Y.

GIGLIO, D. M. AND MCCLESKEY, C. S. 1953The fermentation of sucrose by Leuconostocmesenteroides. J. Bacteriol., 65, 75-78.

JEANES, ALLENE, HAYNES, W. C., WILHAM,C. A., et al. 1954 Characterization andclassification of dextrans from ninety-sixstrains of bacteria. J. Am. Chem. Soc., 76,5041-5052.

LEIVO-QUIROs, A. AND MCCLESKEY, C. S. 1947The application of bacteriophage and serologyin the differentiation of strains of Leuconostocmesenteroides. J. Bacteriol., 54, 709-712.

MCCIESKEY, C. S. AND BARNETT, R. 0. 1949The nutritional requirements of Leuconostocmesenteroides. Proc. Louisiana Acad. Sci.,12, 38-46.

MCCLESKEY, C. S., FAVILLE, L. W., AND BARNETT,R. 0. 1947 Characteristics of Leuconostocmesenteroides from cane juice. J. Bacteriol.,54, 697-708.

RANKIN, J. C. AND JEANES, ALLENE. 1954 Eval-uation of the periodate oxidation method forstructural analysis of dextrans. J. Am.Chem. Soc., 76, 4435-4441.

WILHAm, C. S., ALEXANDER, B. H., AND JEANES,ALLENE. 1955 Heterogeneity in dextranpreparations. Arch. Biochem. and Bio-phys., 59, 61-75.

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